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Between pathogenesis and treatment of psoriasis:

a search for discriminating markers and therapeutic

applications

Rosanne G. van Lingen

ISBN:

Print:

Design and Layout:

978-90-9023528-8

PrintPartners Ipskamp, Nijmegen

M.K.P. Poll & R.G. van Lingen

No part of this book may be reproduced in any form without written permission of the au-thor. All published papers are reprinted with credit to their resource.

Financial support of the publication of this thesis was kindly provided by:

Abbott BV, Astellas Pharma BV, Boehringer Ingelheim BV, Dalton Medical BV, EuroTec BV, Fagron BV, Galderma SA Nederland, Janssen-Cilag BV, KCI Medical BV, Leo Pharma BV, Louis Widmer Nederland, Meda Pharma BV, medi Nederland BV, Merck Serono, Novartis Pharma BV, Schering-Plough BV, Wyeth Pharmaceuticals BV

Thesis Radboud University Nijmegen Medical Center, Nijmegen,The Netherlands, with summary in Dutch; 208 p. ©2008

ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus, prof. mr. S.C.J.J. Kortmann, volgens besluit van het College van Decanen in het openbaar te verdedigen opdonderdag 15 januari 2009 om 13.30 uur precies

Een wetenschappelijke proeve op het gebied van deMedische Wetenschappen

DOOR

Rosanne Gwendolyn van Lingengeboren op 26 juli 1979 te ’s-Gravenhage

Between pathogenesis and treatment of psoriasis:

a search for discriminating markers and therapeutic

applications

PROEFSCHRIFT

Promotor:

Copromotores:

Manuscriptcommissie:

Paranimfen:

Prof. dr. dr. P.C.M. van de Kerkhof

Dr. P.E.J. van ErpDr. M.M.B. Seyger

Prof. dr. P.A.B.M. Smits Prof. dr. E.P. Prens (Erasmus MC Rotterdam)

Prof. dr. T.J.M. de Witte

Drs. R.J.B. DriessenDrs. H.M. Kroon

Aan mijn ouders

TABLE OF CONTENTS

List of Abbreviations

Chapter 1 General Introduction

1.1 Epidemiology and clinical presentation of psoriasis 1.1.1 Epidemiology1.1.2 Clinical features and presentations1.1.3 Clinical course and triggering factors1.1.4 Associated diseases

1.2 Histopathological features of the psoriatic plaque

1.3 Cellular aspects of the immunopathogenesis of psoriasis1.3.1 T-cell subsets1.3.2 The cutaneous T-cell response1.3.3 A tangled web of cytokines and chemokines

1.4 CD26 / Dipeptidyl peptidase IV (DPPIV)1.4.1 Structural properties1.4.2 Patterns of CD26 / DPPIV expression1.4.3 Functions of CD26 / DPPIV1.4.4 Protease function and possible substrates1.4.5 CD26 / DPPIV in auto-immune diseases

1.5 Therapies for psoriasis 1.5.1 Topical therapy1.5.2 Systemic therapy: classics1.5.3 Systemic therapy: biologics1.5.4 Lasers and their therapeutic application in psoriasis

1.6 Outline and objectives of this thesis

1.7 Investigational approach1.7.1 Clinical efficacy parameters1.7.2 Laboratory parameters 1.7.3 Quantification methods

11

17

19

23

26

36

44

63

65

Chapter 2 Innovative anti-psoriatic treatments: clinical observations and interference with T-cell subsets

Compartmentalization of T-cell subsets in response to anti-psoriatic therapy2.1 Relevance of compartmentalization of T-cell subsets for clinical improvement in psoriasis: effect of immune-targeted anti- psoriatic therapies2.2 Good clinical response to anti-psoriatic treatment with adalimumab and methotrexate does not inflict a direct effect on compartmentalization of T-cell subsets: A pilot study

Vascular lasers in the treatment of plaque psoriasis2.3 A comparative study on the efficacy of treatment with 585 nm Pulsed Dye Laser and Ultraviolet-B TL01 in plaque type psoriasis2.4 Nd: YAG laser (1064 nm) fails to improve localized plaque type psoriasis: a clinical and immunohistochemical pilot study

Chapter 3 Expression of CD26 / DPPIV in psoriasis

3.1 CD26 / DPPIV in psoriatic skin: Upregulation and topographical changes3.2 Distribution of epidermal dipeptidyl-peptidase IV on psoriatic keratinocytes: a comparison with hyperproliferation and aberrant differentiation markers3.3 Reduced CD26bright expression of peripheral blood CD8+ T-cell subsets in psoriatic patients3.4 Persistent expression of CD26 / DPPIV after treatment with infliximab in psoriasis despite clinical improvement

Chapter 4 General Discussion

4.1 Introduction4.2 AIM I Assessing clinical observations and interference with T-cell subsets of pathogenesis-based therapeutic interventions4.2.1 AIM IA4.2.2 AIM IB4.3 AIM II Defining the expression of CD26 / DPPIV and characterizing it as a potential novel marker in the immunopathogenesis of psoriasis

87

89

97

103

115

123

125

135

143

151

159

161

161

166

4.4 Concluding comments4.5 Future outlooks

Chapter 5 Summary and Conclusions

5.1 Summary and conclusions5.2 Samenvatting en conclusies

List of PublicationsCurriculum Vitae

Dankwoord

Colour Illustrations

169170

177

179181

184186

187

191

11

also known as3-aminopropyltriethoxysilaneadenosine deaminase3-amino-9-ethylcarbazoleantigen(s)anti-nuclear antibodyanalysis of varianceantigen presenting cell(s)body mass index bovine serum albumincalcipotriol/betamethasone dipropionatechemokine (C-C motif ) ligand 2 (MCP-1)chemokine (C-C motif ) ligand 3 (MIP-1α)chemokine (C-C motif ) ligand 4 (MIP-1β)chemokine (C-C motif ) ligand 5 (RANTES)chemokine (C-C motif ) ligand 7 (MCP3)chemokine (C-C motif ) ligand 11 (eotaxin)chemokine (C-C motif ) ligand 14α (HCC-1)chemokine (C-C motif ) ligand 17 (TARC)chemokine (C-C motif ) ligand 20 (MIP-3α/β)chemokine (C-C motif ) ligand 22 (MDC)chemokine (C-C motif ) receptor 5cluster of differentiationcutaneous lymphocyte antigencentimetre ciclosporin Acytotoxic T-lymphocyte antigen 4chemokine (C-X-C motif ) ligand 2 (MIP-2α)chemokine (C-X-C motif ) ligand 9 (MIG-1)chemokine (C-X-C motif ) ligand 10 (IP-10)chemokine (C-X-C motif ) ligand 11 (I-TAC)chemokine (C-X-C motif ) ligand 12 (SDF-1)chemokine (C-X-C motif ) receptor 3 (GPR-9)chemokine (C-X-C motif ) receptor 4 (receptor for CXCL12)diamidino-2-phenylindoleDPP-IV activity and/or structure-homologuesdendritic cell(s)deoxyribonucleic aciddipeptidyl peptidasedipeptidyl peptidase IV

a.k.a.AASADAAECAg(s)ANAANOVAAPC(s)BMIBSACBDCCL2CCL3CCL4CCL5CCL7CCL11CCL14αCCL17CCL20CCL22CCR5CDCLAcmCsACTLA-4CXCL2CXCL9CXCL10CXCL11CXCL12CXCR3CXCR4DAPIDASHDC(s)DNADPPDPPIV

LIST OF ABBREVIATIONS

12

e.g.EMEAFCMFDAFoxp3gGATA-3GLP-1/2HCC-1HEHEVHIVHLAHLA-Cw6HLA-DRHRPi.e.IBDICAM-1IFN-γIg(G)ILIP-10I-TACJK16kDakgKi67

LASERLFA-1LFA-3LFA3-TIP

MCP1MCP3MDCMEDmgMHCMIG-1MIP-1α/β

exempli gratiaeuropean medicines agencyflow cytometerfood and drug administrationtranscription factor forkhead box p3gramGATA binding protein 3glucagon-like peptide 1 and/or 2hepatocellular carcinoma protein 1hematoxylin and eosinhigh endothelial venuleshuman immunodeficiency virushuman leukocyte antigenhuman leukocyte antigen Cw6human leukocyte antigen DRhorse radish peroxidaseid estinflammatory bowel diseaseintercellular adhesion molecule 1 (CD54)interferon gamma immunoglobulininterleukininterferon gamma inducible protein 10 (CXCL10)interferon-inducible T-cell alpha chemoattractantjoulekeratin 16kilo daltonkilogrammarker for increased recruitment of keratinocytes becom-ing cycliclight amplification by stimulated emission of radiationlymphocyte-function associated antigen 1lymphocyte-function associated antigen 3fusion protein that blocks the interaction between LFA3 and CD2monocyte chemotactic protein 1monocyte chemotactic protein 3 (CCL7)macrophage-derived chemokineminimal erythemal dosemilligrammajor histocompatibility complexmonokine induced by interferon gamma 1macrophage inflammatory protein 1α/β

13

MIP-2αMIP-3α/βmmmRNAMSMTXNd:YAGNFNIDDMNK(T)nmNoTxP3NPPASIPASI50

PASI75

PASI90

PBMCPBSPDLPGAPsAPUVAqPCRRARANTESRARRNAROIsAGsCD26SCIDSDF-1SEMSLESTAT4STAT6SUMTARCT-bet

macrophage inflammatory protein 2αmacrophage inflammatory protein 3α/βmillimetremessenger ribonucleic acidmultiple sclerosismethotrexateneodymium-doped yttrium aluminium garnetnuclear factor kβnon-insulin dependent diabetes mellitusnatural killer (T-)cellnanometreno treatmentamino terminal type III procollagen peptide psoriasis area and severity indexpercentage of patients achieving 50% or more reduction in PASIpercentage of patients achieving 75% or more reduction in PASIpercentage of patients achieving 90% or more reduction in PASIperipheral blood mononuclear cellsphosphate buffered salinepulsed dye laserPhysician’s Global Assessmentpsoriatic arthritispsoralen + ultra violet Aquantitative polymerase chain reactionrheumatoid arthritisregulated on activation normal T-cell expressed and secretedretinoic acid receptorribonucleic acidregion of interestsoluble antigensoluble CD26severe combined immunodeficientstromal cell-derived factor 1standard error of the meansystemic lupus erythematosussignal transducer and activator of transcription 4signal transducer and activator of transcription 6local psoriatic plaque severity scorethymus and activation-regulated chemokineTh1 specific T box transcription factor

14

TCRTGF-βTh1Th17Th2TNF (α/β)TregTRTUNKUSUVUVBVASVCAM-1VLA-4Wµm

T cell receptortransforming growth factor βT helper 1T helper 17T helper 2tumor necrosis factor (α/β)regulatory T-cellsthermal relaxation timeunknownUnited Statesultravioletultraviolet Bvisual analogue scalevascular adhesion molecule 1very late antigen 4wattmicrometre

17

General Introduction

Chapter 1


19

1.1 Epidemiology and clinical presentation of psoriasis

1.1.1 Epidemiology

Psoriasis vulgaris (also known as (a.k.a.) psoriasis when mentioned in the rest of this thesis) was erroneously recognized and described as a variant of leprosy in ancient times. However in 1841 Ferdinand von Hebra definedpsoriasis as it is known nowadays.1 Psoriasis is a common, inflammatory,chronic, relapsing skin disorder affecting 1-3 % of the worldwide Cauca-sian population.2-4 Despite this roughly estimated prevalence of psoriasis in the general Caucasian population, ethnic factors and geographical re-gions seem to influence the prevalence as significant differences are ob-served among Japanese, West-Africans, Samoans, North American blacks and Native Americans.5-7 Men and women are equally affected.7 Psoriasis can present at any age. Nevertheless a bimodal age of onset has been rec-ognized in several large studies distinguishing a mean age of onset on or before the age of 40 years (type I psoriasis) or beginning after the age of 40 years with a peak occurring at 55-60 years (type II psoriasis).8-10 Type I dis-ease accounts for more than 75% of cases and demonstrates a strong asso-ciation with HLA-Cw6 as a genetic marker. Patients with late-onset psoriasis often have less severe skin involvement.8;9;11 In general, patients frequently report a family history of psoriasis.Genetic disposition appears to play an important role in the susceptibility to develop psoriasis. Population studies have shown that the incidence of psoriasis is greater in first and second degree relatives of patients than inthe general population. Moreover, as briefly mentioned above, several HLA-types are associated with psoriasis.12 There is evidence that multiple genes are involved and this has led to the identification of different psoriasis-sus-ceptibility loci. The exact locations of the genes involved however, remain to be definitely identified.3;13

1.1.2 Clinical features and presentations

Psoriasis is a skin disease with variable distribution, severity, course and morphology as the psoriatic phenotype has a changing spectrum of dis-ease expression. The commonest type of psoriasis is psoriasis vulgaris, also designated as chronic plaque psoriasis, and accounts for approximately 90% of all cases. It is a papulosquamous disease in which the lesions have a red or salmon pink colour covered by dry white or silvery scales and are well-delineated from surrounding normal skin (figure 1). The amount of scaling and indu-ration of plaques varies among patients and at different sites on a patient.

20

The lesions may initially begin as erythematous macules or papules and coalesce to form plaques of one to several centimeters in diameter. Fre-quently, lesions are slightly raised and most active at the edge as they are known to expand centrifugally. The scales are only lightly attached and can be easily peeled. Upon peeling several layers at once, point bleeding can occur in the dermal papillae named “Auspitz’ sign”.The distribution is typical as the lesions are predominantly located sym-metrically on the extensor side of the extremities, the lumbosacral region, umbilicus and the scalp. This thesis focuses on this type of psoriasis.Psoriasis guttata, from the Greek word ‘gutta’ meaning a droplet, describes the acute onset of these droplet-like lesions disseminated over the body. Classically, guttate psoriasis occurs shortly after an acute group B haemo-lytic streptococcal infection or viral infection of the pharynx or tonsils14-16 and can be the presenting episode of psoriasis, mainly in children.11 In adults guttate flares may complicate chronic plaque type psoriasis, some-times triggered by a sudden cessation of an anti-psoriatic treatment. Gut-tate psoriasis is known to be self-limiting, resolving within 3-4 months of onset.12

Psoriasis inversa (inverse psoriasis) appears as shiny, red plaques in inter-triginous areas, particularly infra- and submammary, perineal and axillary, where scaling is minimal (figure 2). Occasionally these lesions are com-bined with candidal, intertrigo and dermatophyte infections. In psoriatic erythroderma, the whole body surface is affected by psoriasis,which can lead to hypothermia, hypoalbuminaemia and high output car-diac failure eventually rendering it a possible life-threatening condition. On one hand chronic plaque psoriasis can progress as plaques become conflu-ent and extensive, on the other hand erythroderma may be a manifestation of unstable psoriasis precipitated by infection, drugs or withdrawal of anti-psoriatic therapies.Generalized pustular psoriasis, also known as Von Zumbusch psoriasis, is an acute rare form in which small, monomorphic sterile pustules develop in painful inflamed skin and can coalesce to form large affected areas filledwith pus. Precipitants include intercurrent infection and acute withdrawal of systemic and, on occasion, ultrapotent topical corticosteroids.17 Local-ized forms include palmoplantar pustulosis (a.k.a morbus Andrews-Barber) and acrodermatitis continua of Hallopeau, consisting of yellow-brown, ster-ile pustules on a background of erythema and scaling affecting the palmsand / or soles (figure 2). Approximately 25% of cases are associated with classic psoriasis vulgaris but genetic analysis has implied they can have dif-ferent causes.18 The demographics are markedly different from those of theclassic variant in that it more commonly affects women (9:1), onset occursin the 4th or 5th decade and has a strong association with smoking either current or past.19

Chapter 1


21

Psoriasis is not only limited to the skin. Extracutaneous manifestations can affect patients considerably in their daily activities. About 10-50% of psori-atic patients have distinctive nail changes related to psoriasis. Fingernails are more commonly affected than toenails.20;21 The commonest findings innail psoriasis, or psoriasis unguium, are small pits in the nail plate, nail plate separation at its distal or lateral attachments (onycholysis), orange-yellow sub-ungual discolouration (oil spots) and thickening of a dystrophic nail (figure 2). In addition, yellow, keratinous material may collect under the nail plate and is known as subungual hyperkeratosis.12;20;21

Psoriatic arthritis (PsA) is a seronegative inflammatory arthritis that can oc-cur in the presence of psoriasis.12 The presence of inflammatory arthritisin patients with psoriasis varies between 5% and 42%.22-24 Around 15% of patients with PsA have an onset of arthritis before the onset of psoriasis. Classification Criteria for Psoriatic Arhritis25 have recently been developed reflecting a diverse range of clinical patterns that can change by time andby the influence of therapy. In general the classical psoriatic arthritis con-sists of oligoarthritis, distal interphalangeal joint involvement, dactylitis and calcaneal enthesitis.26 A classical radiological hallmark of PsA are mar-ginal erosions with adjacent proliferation of bone, joint-space narrowing and periostitis.27 No serologic markers exist for the diagnosis. There are conflicting data addressing the question whether PsA is as destructive asrheumatoid arthritis. Nevertheless, the quality of life and functionality are significantly reduced in PsA patients.24 Moreover, psoriasis deeply affects well-being and has emotional and rela-tional consequences that go far beyond the skin.28;29 The impact of psoriasis on quality of life can even be considered similar as compared to other ma-jor medical diseases such as cancer, depression, heart disease, diabetes and hypertension.30

Figure 1 Typical clinical presentation of psoriasis vulgaris with sharply delineated ery-thematous plaques covered by silvery white scales.

22

1.1.3 Clinical course and triggering factors

The course of psoriasis is as fluctuating as its distribution and morphology.Every patient has a unique typical disease history, although usually char-acterized by exacerbations and remissions. On the one hand, the disease process can remain changeless over years, on the other hand worsening and flares can surprise the patient and dermatologist adventitiously. Thefluctuating course can be influenced by triggering factors. For many pa-tients, the symptoms of psoriasis are subject to environmental factors as they frequently improve in the summer and worsen in the winter. Physi-cal trauma may trigger psoriatic lesions at sites of injury, a.k.a. Koebner’s phenomenon.6 A positive Koebner reaction predicts subsequent disease activity.31 Drug-induced flares are well-known to induce exacerbations, e.g.

Chapter 1

Figure 2 Clinical pictures of patients with psoriasis unguium, psoriasis pustulosis (palmo)plantaris and psoriasis inversa.


23

β-receptor blockers, lithium, chloroquine, non-steroidal anti-inflammatoryagents, tetracyclines and interferons.14;32 In addition, psychogenic stress has been reported to be a triggering factor in patients with psoriasis. As men-tioned before, infections, in particular streptococcal infections of the upper respiratory tract, have been recognized as triggers of psoriasis as well.14-16

1.1.4 Associated diseases

Awareness is increasing that psoriasis as a disease is more than skin deep.33 Psoriasis can be associated with other diseases, which have a major impact on patients. Not only psoriatic arthritis is included herein, but more recently epidemiological investigations have documented that diabetes, hyperten-sion, hyperlipidemia, alcohol consumption, smoking and increased BMI are associated with both mild and severe psoriasis.34-38 These findings sug-gest that psoriasis is associated with the complex disorder of metabolic syndrome which incorporates hypertension, dyslipidemia, central obesity and impaired glucose tolerance.36;39;40 This association is stronger in patients with severe psoriasis compared with those with mild psoriasis. The meta-bolic syndrome is a strong predictor of cardiovascular diseases, diabetes and stroke.41-43 Even severe, but not mild, psoriasis was recently associated with an increased risk of death.44

Other associated disorders include Crohn’s disease45;46, depression40 and cancer. For the latter it is unclear whether cancers, particularly lymphoma47 and skin cancer48, are related to psoriasis or to its treatment.12

1.2 Histopathological features of the psoriatic plaque

In order to understand the histopathology of psoriatic skin, an understand-ing of the basic normal histology of the skin is essential. The skin can be divided in two seemingly separate layers: the epidermis and dermis. The epidermal layer is composed primarily of keratinocytes with minority pop-ulations of Langerhans cells, melanocytes, neuroendocrine cells and un-myelinated axons. As the keratinocytes differentiate into horny cells, theyare arranged in four layers: the basal cell layer (stratum basalis), the sq-uamous cell layer (stratum spinosum), the granular layer (stratum granu-losum) and the horny layer (stratum corneum). An additional layer, the stratum lucidum, can be recognized in areas that have a thick stratum gran-ulosum and corneum forming the lowest portion of the stratum corneum, especially on the palms and soles.49 The undersurface of the epidermis is undulant with downward invaginations termed rete, and interdigitating mesenchymal cones termed dermal papillae. The epidermis is separated

24

from the dermis by the basement membrane zone. The dermis consists of endothelial and neural cells and supporting elements, fibroblasts, mono-cytes and macrophages and mast cells enveloped within a matrix of col-lagen and glycosaminoglycan. Adnexa extend from the epidermis into the dermis and consist of specialized cells for hair growth, epithelial renewal and temperature regulation. The subcutis is an underlying cushion formed by cells engorged with lipid and cultivated by vessels that grow within thin intervening septa.49 The diagnosis of psoriasis relies almost entirely on the reviewed characteristic clinical features, and skin biopsy for histopathologic review is usually not required. However, the histopathology is characteristi-cally confirmative and of additional value to correlate the clinical signs andsymptoms with histopathologic changes.As a consequence of the course of remissions and exacerbations of psoria-sis, the histopathologic findings can vary with the age of the lesions.50-52 In general, psoriasis has three main histopathological features (figure 3):

1. epidermal hyperplasia with epidermal thickening 2. dilated, elongated and increased prominent superficial capillaries in the dermis 3. a predominantly intra-dermal inflammatory infiltrate of mainly T-cells

Hyperproliferation of keratinocytes in the epidermis is a prominent feature in the psoriatic plaque. Basal keratinocytes maintain expression of Keratin 5 and Keratin 14 (the keratins typical of basal keratinocytes in normal skin), whereas Keratin 1 and Keratin 10 (the keratins typical of suprabasal cells in normal skin) are replaced by so-called hyperproliferation-associated keratins Keratin 6 and Keratin 16, in addition to Keratin 17.53-57 The mitotic activity of basal keratinocytes is increased by a factor 50 in psoriatic skin. As a result, keratinocytes only need 3-5 days in order to move from the basal layer to the cornified layer as opposed to the normal of 28-30 days. This increasedrecruitment of keratinocytes becoming cyclic in the psoriatic epidermis can be visualized by staining the Ki67 nuclear antigen. Ki67 is an representative marker to determine the growth fraction of the keratinocytes.58

Due to the increased proliferation of keratinocytes, epidermal rete ridges become very elongated and form long, thin downward projections into the dermis.59 Consequently, the epidermis that lies immediately above the der-mal papillae is thinned (suprapapillary plate thinning). The significant reduced transition time of keratinocytes is accompaniedby altered differentiation, reflected by the focal reduction or absence ofthe granular layer of the epidermis and parakeratosis.6 The terminally dif-ferentiated keratinocytes fail to stack normally and adhere to each other causing the typical psoriatic scaling. Besides being in a hyperproliferative state, keratinocytes are also described to be highly immunologically active

Chapter 1


25

participating in and modulating immune reactions in the skin e.g. through production of cytokines.Compared to the abnormalities in psoriatic vasculature, in normal skin the superficial micro-vasculature is composed of capillary loops which arisefrom terminal arterioles in the upper horizontal dermal vascular plexus, pass up into the dermal papillae and arch back to connect with the post-capillary venules in the horizontal plexus.60 In psoriasis, each papilla con-tinues to be served by a single capillary loop, but the limbs of the loop are twisted along their axis.61;62 There is a fourfold increase in the endothelium of the superficial micro-vasculature in lesional skin, but not in the deepervasculature indicating that micro-vascular expansion is restricted to the upper plexus.63 These papillary vessel changes are uniformly distributed throughout clinical lesion.64 Blood flow in plaque skin is also substantiallyelevated compared with clinically uninvolved skin and the normal skin.61;65 This increased vascularity in the psoriatic dermis causes the visible redness of psoriatic skin lesions. The vascular abnormalities are probably the most neglected feature in the psoriatic pathogenesis (see also paragraph 1.5.4).The psoriatic infiltrate in the dermis varies in density depending on the ex-tent of activity and involvement of immunecompetent cells. It has a mixed

Figure 3 Histopathological characteristics of psoriasis. Top left: normal skin of healthyvolunteer, Top right: psoriatic skin (both HE-staining). Bottom left: T-cell infiltrate in psoriasis(CD3; red stained), Bottom right: Prominent vasculature in psoriasis (CD31; red stained).

26

composition of cell-types, however the T-cell dominates this composition. In the mid-nineties the T-cells gained ground as primary players in the pathogenesis.66-71(see also paragraph 1.3)This thesis touches upon the above-mentioned three main histopathologi-cal aspects of the pathogenesis of psoriasis in one or the other way. Other prominent histological features of psoriasis comprises the polymorphonu-clear leukocytes / neutrophilic granulocytes, dendritic cells and mast cells. Collections of neutrophils within the stratum corneum, the so-called Mun-ro’s micro abscesses, are present in approximately 75% of the cases. When present in the spinous layer they form very characteristic spongiform micro pustules of Kogoj.52;72;73 In psoriasis, antigen-presenting cells (APCs) are of importance for activation of T-cells by presenting antigens to the CD4+ and CD8+ T-cells (see para-graph 1.3.2). Epidermal Langerhans cells (CD1a+, HLA-DR+), dermal den-dritic cells (CD1a-, HLA-DR+), HLA-DR+ keratinocytes and dermal dendro-cytes are all APCs relevant in the psoriatic pathogenesis.74

Mast cells are described to infiltrate developing psoriatic plaques beforemacrophages, T-lymphocytes or neutrophils. Mast cells can produce large amounts of TNF, IFN-γ, IL-8 and multiple other mediators.75;76

1.3 Cellular aspects of the immunopathogenesis of psoriasis

Hypotheses of the psoriatic pathogenesis have changed over the decades. As the massive epidermal changes have been impressive from the first timeresearchers examined histological sections of involved psoriatic skin, no wonder the investigations were initially concentrated on the keratinocytes.77 Since then numerous groups have studied and revealed the abnormalities in the proliferation and differentiation of keratinocytes in detail. At the endof the 1970s an observation was made that shed a completely new light onto the explanation of the pathogenesis of psoriasis. Physicians observed a clear improvement of psoriasis in patients who were treated with cy-closporin A (CsA) because of a kidney transplantation.78 The clinical efficacywas proved in a double-blind placebo-controlled trial of CsA in psoriasis79;80 and evidence accumulated that the primary mode of action of CsA inhibits the proliferation of T-cells and their cytokine production. For this reason, these observations were at the basis of a shift in pathogenic hypotheses as psoriasis became viewed as an epidermal (keratinocyte) response to im-munocompetent cell-types. To date, many investigations focus on the T-cell mediated pathology and have created an understanding of the biology of many aspects of the T-cells. Presumably, pathologic activation of both innate and adaptive (T-cells) im-

Chapter 1


27

munity pathways in response to a yet unknown trigger, seems to comprise the main principles of the complex immunopathogenesis of psoriasis. Re-cently, studies have involved the role of innate immunity81-86 and IL-17 me-diated immune responses87;88 as novel pathogenic insights. However, it is beyond the scope of this thesis to fully elaborate on these topics. In this thesis, the adaptive immunity response system will be highlighted addressing the basic aspects and components of the immunopathogenesis of psoriasis in the following paragraphs.

1.3.1 T-cell subsets

The range of T-cell subsets can be distinguished by use of the ‘cluster of differ-entiation’ antigens, a.k.a. CD-markers. The nomenclature is used for the iden-tification and investigation of in particular cell surface molecules on leuko-cytes. Cell populations are usually defined using a ‘+’ or ‘-‘ symbol to indicate whether a certain cell fraction expresses or lacks a CD molecule.89-91 In gen-eral T-cells are characterized by expression of CD3, the pan T-cell marker.

The psoriatic lesionIn fully developed psoriatic skin lesions, there is a mixture of innate immune cells (neutrophils, mast cells, dendritic APCs and NKT cells) and adaptive im-mune cells (both CD4+ and CD8+ T-lymphocytes) composing and in pres-ence of the inflammatory infiltrate. Signals derived from immune cells areable to stimulate keratinocyte proliferation and inversely keratinocytes may activate immune cells through surface receptors and cytokines.92 Laborato-ry and clinical evidence has led to the statement that Th1- cytokines (IL-12, IFN-γ, IL-2) and –T cells predominate in psoriatic plaques.93;94(see paragraph 1.3.3)The majority of T-cells that localize to the dermis are of the CD4+ helper type, while those that migrate to the epidermis are predominantly of the CD8+ cytotoxic type.72;95 In fact the memory effector (CD45RO+) T-cells arethe subtype of T-cells which migrate into skin rather than the CD45RA+ (na-ive) population.96 Most are positive for cutaneous lymphocyte-associated antigen (CLA), a marker for skin-homing leukocytes.95 The epidermal CD8+ T-cells express CD103, a β7integrin that characterizes intra-epithelial T-cells and facilitates their migration into the epidermis.97 Subsequently, studies have shown that both CD4+ and CD8+ T-cells have markers of persistent activation such as HLA-DR98 and CD69 surface molecules and the costimu-latory receptor CD2.99;100 Thus, the major populations of T-cells in the psori-atic plaque are mature, skin-homing, activated memory cells.The so-called natural killer (T-)cells (NKT) are part of the innate immune

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system and have been implicated in the pathogenesis of a variety of auto-immune diseases such as multiple sclerosis101, type I diabetes mellitus102, in-flammatory bowel disease103, autoimmune hepatitis104 and systemic lupus erythematosus.105;106 In line, natural killer and natural killer T-cells are part of the cutaneous inflammation in psoriasis.107;108 CD94 receptors are expressed on most NK cells and some T-cells, whereas CD161 is an obligate NKT-cell receptor.74;109 It has been suggested that NKT cells interact with CD1d, ex-pressed on keratinocytes in psoriasis, leading to the production of IFN-γ by these NKT cells.110 In psoriasis, a significant higher number of NKT cells ispresent in the psoriatic lesion in comparison with uninvolved and normal skin.111 Because of the ability to secrete cytokines such as IFN-γ or IL-4/IL-13, NKTcells are thought to be important in immunoregulation of mature Th1- and Th2-lymphocyte mediated immune responses. The exact function in the psoriatic immunopathogenesis of these cells however to date is still unclear. The past few years, there is mounting interest in T-cells with various im-munoregulating functions that have been described as regulatory T-cells (Treg). These cells play a central role in inducing and maintaining immuno-logic tolerance and in the termination of immune responses. Deficiency ordysfunction of these cells may lead to auto-immune conditions or aggra-vated pathogen-induced inflammation.112 Naturally arising Treg are CD4+ T-cells expressing CD25 (the IL-2 receptor α chain) and are associated with the expression of the transcription factor Foxp3.113-116 They have been linked to a number of autoimmune diseases, including psoriasis. It has been sug-gested that these cells have aberrant suppressive capacities in peripheral blood and skin lesions of patients with psoriasis.112;115;116

Peripheral bloodIn line with the data on the local presence of several types of T-cell subsets in the psoriatic lesion, circulating T-cells have been point of study as well in order to investigate abnormalities in the peripheral blood of patients with psoriasis.111;117-119 Baker et al. suggested that circulating T-cells are activated and subsequently recruited from the circulation during the development of psoriatic plaques.98 Several subsets seem to play a primary role in the lesion, e.g. CD4+, CD8+, CD45RO+, CD45RA+ and CD25+. Increased per-centages of these psoriasis-associated T-cell subsets in peripheral blood have been reported.98;118;120 In particular, more severe forms of psoriasis (de-fined by cut-off point of PASI 12, see paragraph 1.7.1) show significantlyincreased counts of CD4+, CD8+, CD45RO+, CD45RA+, CD94+ and CD161+ T-cells compared to mild psoriasis.119 Moreover, CLA+ T-cells were found to be increased in the peripheral blood of patients with acute psoriasis121 in contrast to NKT cells which are decreased in peripheral blood of psoriasis patients.120;122 As far as the Treg are considered, approximately 10% of CD4+

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lymphocytes in peripheral blood are found to be CD25+. Remarkably, simi-lar proportions were found in psoriatic and normal blood115 despite the fact that the Treg cells showed some deficiency in their ability to suppress effec-tor CD4+ T-lymphocyte responses. This might point to a defective immu-noregulatory activity in psoriasis that permits T-lymphocyte activation and cytokine production. The exact role though still needs clarifying.For T-cells to gain access to skin, they must bind to endothelial cells and move out of the peripheral circulation and into the dermis. The correlation between T-cell subsets present in peripheral blood and the skin has been hypothesized in relation to clinical severity and therapeutic effect.74 How-ever, this alleged recompartmentalization of T-cells in psoriasis has not yet been point of focus in studies.

1.3.2 The cutaneous T-cell response3;92;123

In the course of a normal cutaneous immune reaction, antigen-reactiveT-cells enter the skin at sites that harbour residual antigen. Subsequently, the antigen would be eliminated by an immune mechanism appropriate to the inciting antigen. In psoriasis, the cutaneous immune reaction seems overstrained.

Antigen processing and presentation3 Specific T-cell responses require activation of T-cells through antigen-pre-senting cells. At the onset of psoriasis, the initial step comprises the rec-ognition and uptake of (exogenous) antigen (Ag) by tissue-guarding DCs (Langerhans cells), the migration of these cells to the T-cell areas of local draining lymph nodes and the presentation of selected Ag fragments (pep-tides) on the DCs’ cell surface to naïve T-cells. Naïve T-cells are cells that have not previously been activated by antigen in an immune response. After the Ag uptake, the DCs undergo a maturation process and reduce their capac-ity to further absorb Ags. One question that remains crucial to the under-standing of psoriasis refers to the responsible Ag for initial T-cell priming. Disease initiation is widely suggested to be mediated through DCs present-ing foreign antigens or self-antigens to T-lymphocytes. Optionally, one of these antigens may be a self-peptide cross-reactive to streptococci as it is reported that streptococcal infections precede psoriasis in more than 90% of patients with type I psoriasis.15;124

Generation of effector and memory T-cells3

The conversion from naïve to effector memory T-cells in the secondary lym-phatic organs is necessary for T-cells to obtain their functions according to the type of immune response that needs to be generated and their ability

30

to immigrate into tissues. Naïve T-cells constantly circulate between blood and secondary lymphatic organs, such as lymph nodes and tonsils. Naïve T-cells express high levels of L-selectin, allowing them to attach, or tether, and then roll on the surface of the high endothelial venules (HEV) within lymph nodes. After stimulation by an adhesion-triggering chemokine ex-pressed on the luminal surface of the HEV, the rolling T-cells arrest and bind tightly through an integrin (LFA-1). Now, the T-cells can extravasate through the HEV into the lymph node. In T-cell areas of these organs, they congregate with mature DCs.123 At the heart of the antigenic activation of naïve T-cells is the recognition of an antigenic peptide bound to either class I or II major histocompatibility complex (MHC) molecules on dendritic APCs that have migrated from the skin via efferent lymphatics to skin-draininglymph nodes. Antigens are recognized, in turn, by the T-cell receptor (TCR) complex which is present on the surface of all T-cells. A naïve CD4+ T-cell (CD45RA+) whose TCR has a high enough affinity for the respective MHC-IIpeptide complex, attaches to the respective DC and forms a so-called im-munological synapse. The CD8+ T-cell has affinity for MHC class I boundantigen. Subsequently, in order to become activated, the T-cell needs three signals: the first signal has already been delivered by interaction betweenTCR and MHC-I/II peptide-complex. The second signal is given by the so-called co-stimulatory molecules. The third signal for T-cell activation is de-livered by soluble mediators (cytokines).Costimulation can be defined as a stimulus necessary for optimal T-cell acti-vation delivered jointly to T-cells along with the TCR signal.125 The most im-portant co-stimulatory molecules expressed on DCs are: CD86, CD80, and CD40. CD86 and CD80 interact with the T-cell molecules CD28 and CD152 (cytotoxic T-lymphocyte-associated protein 4, CTLA-4). CD86 is a major co-stimulatory molecule in DCs as it is critical for full activation, particularly of naïve T-cells. Engagement of CD152 inhibits TCR- and CD28-mediated sig-nal transduction, increases the threshold for activation of these cells and re-presses the cell cycle. Therefore, it counteracts acute T-cell responses but is also essential for the formation of memory T-cells. The counterpart for CD40 on T-cells is CD40-ligand (CD154). The immunological synapse is stabilized by adhesion molecules expressed between DCs and T-cells such as the in-teraction between intercellular adhesion molecule 1 (ICAM-1; CD54)(DC) and lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) (T-cell) and the interaction between lymphocyte function-associated antigen 3 (LFA-3; CD58) (DC) and CD2 (T-cell).The third signal delivered to the T-cell is from the cytokines and essential-ly influences the mode of action of the effector and memory (CD45RO+)T-cells, which evolve from these activated T-cells. Naïve T-cells can be po-larized into different Th-subsets of which the major classes are Th1, Th2,Th17 and Treg. When IL-12 is present during activation of naïve T-cells, they

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are polarized into Th1 cells through activation of the transcription factors STAT4 and T-bet. The generation of Th1 cells is supported by IFN-γ, which increases the expression of the specific receptor chain for IL-12. When IL-4is present, naïve T-cells are polarized into Th2 cells through activation of

Figure 4 A selected view on the cutaneous T-cell response with its different polari-zation pathways.In order to generate a cutaneous T-cell response, a T-cell must interact with antigen-pre-senting cells (mature Langerhans’ cells) which have taken up and processed auto-antigens. After the processing APCs migrate to the regional lymph nodes where they come in contact with naïve T-lymphocytes. It is in the immunological synapse that various molecular interac-tions ultimately result in T-cell activation. Three sets of signals are required to achieve this. First the APCs present the processed antigen bound to MHC molecules, to the TCR. Second, additional so-called accessory signals are being transmitted through interactions between costimulatory molecules with their corresponding ligands. The immunological synapse is stabilized by adhesive interactions between integrins and their ligands as they transmit ad-ditional signals. Following these activating signals, T-cells differentiate into memory T-cellsunder influence of soluble mediators such as cytokines. Now they are activated they re-enter the peripheral blood circulation and predominantly extravasate at sites of cutaneous inflammation where they can exert their effector functions. In psoriasis, this cutaneous T-cellresponse is chronic of nature. Naïve T-cells can be polarized into different Th-subsets of which the major classes are Th1,Th2, Th17 and Treg. The T-cell lineage is dependent on the cytokine milieu present during the T-cell activation (as explained in text).(Modified from: Schön MP and Henning-Boehncke W. N Engl J Med 2005;352:1899-912 Sabat R, Philipp

S, Höflich C et al. Exp Dermatol 2007;16: 779-798.)

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the transcription factors STAT6 and GATA-3. These T-cells secrete IL-4, IL-5 and IL-13 during repeated activation and have a regulatory role as high concentrations of type 2 cytokines suppress the actions of type 1 effectors.The activation of naïve T-cells with mature DCs in the presence of IL-6 and transforming growth factor β (TGF-β) upregulate the receptor for IL-23 and together with this cytokine induced Th17 cell development. The Th1-cell inducing cytokines IL-12 and IFN-γ actively suppress the development of Th2 and Th17 cells. Conversely, the Th2-cell inducing cytokine IL-4 inhibits the development of Th1 and Th17 cells.Independent of its polarization into a Th1 or Th2 (or Treg / Th17) cell, a suc-cessfully activated naïve T-cell develops into an effector T-cell. The com-munication between dendritic APCs and T-cells should be considered as a fluent ongoing dialogue rather than a brief contact, enhancing the chroniccharacter of the psoriatic immune response. A schematic figure of the cuta-neous T(h1)-cell response is reflected in figure 4.

Skin infiltration of T-cells and other immune cells3

Tissue invasion by activated T-cells from the periphery seems to be essen-tial for the development and maintenance of psoriatic plaques. The pas-sage of leukocytes from blood vessels into tissue occurs in five steps (figure 5). Endothelial cells play a central role in these processes.1. leukocytes roll along the blood vessel wallRolling reduces the flow velocity of the leukocytes and is mediated by theinteraction between P-selectin and E-selectin expressed by endothelial cells and selectin ligands expressed by leukocytes. E-selectin is the endothelial ligand for CLA on memory T-cells. It is normally not expressed on endothe-lial cells in cutaneous micro vessels but is upregulated during cutaneous inflammation.2. recognition of chemokines presented by the endothelial wallsThe rolling immune cells along the vessel wall recognize chemokines pre-sented by endothelial cells and become activated (triggering).3. formation of tight adhesions between endothelial cells and immune cellsThis is achieved by integrins (and adhesion molecules) expressed on im-mune cells and their ligands expressed on endothelial cells. Chemokines induce this integrin-dependent adhesion. LFA-1(CD11a/CD18) seems to be an important integrin for skin-homing as the anti-psoriatic ef-fect of efalizumab has become established, a monoclonal antibody directed (among other things) against LFA-.126-130(see paragraph 1.5.3)4. passage through the endothelial wall (diapedesis)This is probably performed by pores between endothelial cells. 5. migration to the target tissue

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In contrast to the relatively well understood process of lymphocyte extrava-sation, little is known about the exact subsequent migration through the cutaneous extracellular matrix and the processes determining the ultimate localization and arrest of lymphocytes within the (epi)dermal compart-ment. Immigration is apparently initiated by trauma or invasion of a few micro-organisms that leads to the local activation of immune cells. Through the products of these cells and complement components, endothelial cells are activated, which then actively support the infiltration of immune cellsto the skin. Later, the immigrated immune cells activate each other so that the T-cells stimulate monocytes / macrophages and DCs through IFN-γ and these cells then activate the T-cells through IL-23 and IL-6. At the same time, keratinocytes are activated whereby they actively support the infiltration offurther immune cells through the production of chemokines. After migra-tion into the skin, the immune cells may be activated by various types of APC populations or keratinocytes. T-cells may proliferate as a consequence of activation in the skin. As mentioned earlier, CD8+ T-cells primarily home to the epidermis in pso-riasis whereas CD4+ T-cells are mainly present in the dermis. The reasons for this different homing pattern may be caused by the varied expressionof chemokine receptors and integrins. Other immune cells such as NK-cells, monocytes / macrophages and DCs also use a similar mechanism asdescribed above to migrate into the skin. However, different chemotacticfactors are dominant as well as other adhesion molecules. It remains un-clear however, in what way precisely the immigrated T-cells in the skin be-come activated eventually.

Figure 5 Five steps in skin homing of leukocytes (Modified from Sabat R, Philipp S, Höflich C et

al. Exp Dermatol 2007;16: 779-798)

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1.3.3 A tangled web of cytokines and chemokines

Inflammation or tissue damage initiates the production of cyto- andchemokines and the recruitment of leukocytes. Once the leukocytes have crossed the endothelium and the basement membrane, their migration to the focus of inflammation is directed by a constant gradient of cyto- andchemokines.Cytokines are a large family of extracellular protein mediators including but not limited to chemokines, which can be defined as chemoattractant cy-tokines. They have pleiotropic effects. Cytokines and chemokines are themost important facilitators of leukocyte migration. Mosmann et al. were the first to propose the T-helper (Th)1 and Th2 cytokine network theories.131;132 In 1991, the cytokine network was further specified for psoriasis establish-ing a paradigm for understanding these mediators of inflammation in thepsoriatic context.133 As brought up earlier, in the Th1 immune response, IL-2, IFN-γ and TNF represent the key cytokines contributing to a T-cell mediated reaction whereas in the Th2 immune response, IL-3, IL4, IL-5 and IL-10 con-tribute to a humoral or B-cell mediated immune reaction.134 In psoriasis, the described immunocytes communicate with endothelial cells, fibroblastsand keratinocytes via the production of and interaction of Th1 cytokines and chemokines.93;94;100;135 Chemo- and cytokines interact with specific re-ceptors and can influence cells in an autocrine, juxtacrine and paracrinefashion.134 Functionally, cytokines are able to influence the proliferation, dif-ferentiation or secretion of proinflammatory or anti-inflammatory factorsby resident and recruited cell types. The group of proteins referred to as cytokines include lymphokines, monokines, interleukins, interferons and growth factors. Chemokines may overlap the function of cytokines, but are primarily recognized biologically for their ability to modulate the move-ment of cells either by promoting the entry or leaving of migratory (T-) cells.134;136 Beside the function in leukocyte chemotaxis, chemokines have an important function in integrin activation during the multistep process of leukocyte-endothelial cell interaction, stimulation of leukocyte degranula-tion or release of inflammatory mediators and stimulation of angiogenesis.In general, chemokines connect directly to classes of cytokines and effectorleukocytes to orchestrate immune responses.136

Table 1 creates an overview of the cytokines and chemokines upregulated in the psoriatic lesion. Obviously, many cytokines and chemokines contrib-ute to the initiation and maintenance of psoriatic plaques. Considering the wide variety of psoriasis-associated cytokines, it seems worthless to target only a single cytokine for therapeutic success. However, some cytokines seem of primary importance and some of secondary, as the use of high-ly selective biological agents in the treatment of psoriasis has proven to be very effective (anti-TNFα therapy: etanercept137-140, infliximab141-144 and

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adalimumab145-147) (see also paragraph 1.5.3). TNF-α is a key pro-inflamma-tory cytokine taking part in the immune response, that merits some spe-cial attention in this thesis, as three studies have addressed the effect of ananti-TNFα therapy in psoriasis. This cytokine is produced by macrophages, monocytes, mature DC (including Langerhans cells), polymorphonuclear cells, mast cells, NK cells, activated T-cells, keratinocytes and endothelial cells.148-150 High concentrations of TNF-α have been found in psoriatic le-sions, but not in unaffected skin.151;152 Furthermore, serum levels of TNF-α have been shown to correlate with disease severity, i.e. increasing during worsening of psoriasis and decreasing after effective therapy.153 TNF-α has a multitude of pro-inflammatory actions that could account for the signsand symptoms of psoriatic plaques, as it is able to induce a cascade of other cytokines.154;155

In addition, a novel therapeutic agent (targeting cytokines) that recently has led to great clinical responses in psoriasis, is the antibody that recognizes a shared subunit (p40) of the cytokines IL-12 and IL-23.156-161 IL-12 is produced by activated macrophages and dendritic cells and induces a Th1-type dif-ferentiation response with naïve T-cells becoming IFN-γ producing T-cells. IL-23 is also produced by activated DCs leading to memory Th1-type T-cells. Both IL-12 and IL-23 influence the levels of cytokine IL-17 which has led toa new paradigm in the cytokine network highlighted by the dominance of a Th17-type immune response.134;162 The Th17 response is an emerging fieldof interest in psoriasis; for the latest insights and understandings we refer to publications in journals of recent date.

Table 1 Cytokines and chemokines upregulated in psoriatic lesions(From: Lew W, Bowcock Am, Krueger JG.

Trends Immunol 2004; 25(6): 295-305

Gaspari AA. J Am Acad Dermatol 2006;

54: S67-80)

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The exact immunologic mechanisms that trigger the psoriatic phenotype are not proven, but basic elements in the psoriatic immunopathogenesis have been elucidated in the past decades, and touched upon in this para-graph. It seems that a variety of specific and non-specific stimuli lead totriggering of the cutaneous exaggerated adaptive immune response in genetically susceptible individuals to psoriasis. Although it remains open whether the primary defect leading to psoriasis is an inborn metabolic er-ror of keratinocytes or a dysfunction in the innate and/or adaptive immu-nity, in the end it would be difficult to draw a T-cell independent model ofthe psoriatic pathogenesis.

1.4 CD26 / Dipeptidyl peptidase IV (DPPIV)

CD26 / DPPIV is a 110 kDa cell surface glycoprotein which enjoys a great deal of interest in the fields of immunology and inflammation, glucose homeo-stasis, neuroendocrinology and cancerology.163 In particular, it is described to interfere with the pathology of several auto-immune diseases. As a cell surface protease, it is expressed in many tissues where it can truncate sev-eral chemokines, and can influence immune responses via modulation ofcell adhesion and T-cell activation. Consequently, this suggests a potential involvement of CD26 / DPPIV in the psoriatic immune response. Therefore, in this thesis we were triggered to map the expression of CD26 / DPPIV in psoriasis in order to investigate and create more insight into whether the expression of this protein could be of additional value and in the long run could possibly function as a supplementary marker correlating with disease severity in the psoriatic pathogenesis.The following subparagraphs give insight in the known expression and functions of CD26 / DPPIV in the human body, chiefly in the field of immu-nology and inflammation.

1.4.1 Structural properties163

DPPIV was first reported in 1966 as glycyl-prolyl-β-naphthylamide hydro-lyzing enzyme164 and later was renamed as dipeptidyl aminopeptidase IV, post-proline dipeptidyl aminopeptidase and X-Pro dipeptidyl aminopepto-dase. The present name is dipeptidyl peptidase IV. In the context of T-cells however, it is frequently referred to as CD26.165 CD26 / DPPIV is composed of a short cytoplasmatic domain of 6 amino acids, a transmembrane region of 22 amino acids and an extracellular domain of 738 acids. The human CD26 / DPPIV gene has been located on the long arm of chromosome 2 (2q24.3). CD26 / DPPIV belongs to the serine proteases. Proteases are enzymes con-

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ducting proteolysis; the degradation of proteins. According to the character of their catalytic active site and conditions of action they can be classifiedinto four groups: serine, cysteine, aspartic, and metalloproteases. In gener-al, proteases can be involved in a multitude of physiological reactions from simple digestion of food proteins to highly-regulated cascades as in immu-nology and inflammation, which can result in rapid and efficient amplifica-tion of an organism’s response to a physiological signal. By splitting long protein chains into short fragments, (amino)peptidases break the peptide bonds that link amino acid residues. Likewise, CD26 / DPPIV can selectively cleave the N-terminal dipeptide from peptides with proline or, to a lesser extent alanine, at the penultimate position166;167 (indicating the amino acid on the second position counting from the amino terminus).

1.4.2 Patterns of CD26 / DPPIV expression

CD26 / DPPIV has a widespread tissue distribution differing in expressionlevel between tissues depending on cell-type, differentiation and activa-tion status. Immunohistochemical analyses have detected CD26 / DPPIV in human tissue sections of the gastrointestinal tract, biliary tract, exocrine pancreas, kidney, uterus, placenta, prostate, the adrenal, parotid, sweat, salivary and mammary glands and on endothelia of all organs exam-ined including liver, spleen, lungs and brain.168 In human skin, it has been shown that CD26 / DPPIV is expressed on the surface of keratinocytes169 as well as on sebocytes, the lipid-producing cells localized in the sebaceous glands.170;171 Dermal fibroblasts also express the membrane-bound form ofCD26 / DPPIV.172 By contrast, in immunological context the cell surface expression of CD26 on human T-lymphocytes is known to be tightly regulated. As indicated in paragraph 1.3.2, the CD3/TCR complex plays a central role in T-cell activa-tion and function. The significance of CD26 / DPPIV in T-cell function is bestindicated by the enhancement of CD3 and CD2 dependent activation and proliferation by anti-CD26 antibodies.173-179 After T-cell stimulation, the ex-pression levels of CD26 / DPPIV as well as the percentage of CD26+ T-cells are increased which makes it a suitable marker for activated T-cells.180 Further-more, on resting B- and NK-cells in the circulation, CD26 / DPPIV is known to be absent but can be induced on their surface after stimulation.181;182

In general, CD26+ T-helper cells in the peripheral blood are for 76% CD4+ and for 16% CD8+.183 At the resting state in the peripheral blood, CD26 is preferentially expressed on the helper/memory T-cell population(CD4+CD45RO+).184 Moreover, a small subpopulation of T-cells in unstimulated mononuclear cells in the peripheral blood expresses CD26 at high densi-ty on their surface, the so-called bright expression, which encompasses a

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number of defined phenotypic and functional characteristics. This rathersmall CD26bright population belongs to the CD45RO+ T-cells and is re-ported to be necessary for the proliferative response to recall antigens and alloantigen.185 It is also responsible for the induction of cytotoxic T- lym-phocyte activity against alloantigen and the induction of B-cells to synthe-size IgG.184 The specific capacity of CD26+ cells to migrate transendotheli-ally and to infiltrate inflammatory sites186;187, is primarily exhibited by the CD26bright CD4+CD45RO+ T-cells.

The CD26 / DPPIV cell surface expression on CD4+ T-lymphocytes is cor-related with the production of a Th1 like cytokine pattern.188 Th1 hallmarks such as IFN-γ, CCR5, CXCR4 and CXCR3 (IP-10, MIG, I-TAC) can all be proc-essed by CD26 / DPPIV (see paragraph 1.4.4). Subsequently, processing of cyto- and chemokines by CD26 / DPPIV may constitute an important mech-anism to reduce attraction of leukocytes in a Th1 response.183 In the end, CD26 / DPPIV can not be considered a truly specific and selective marker forhuman Th1 cells, yet it can characterize the pathway of this specific immune

Figure 6 Various characteristics of T-cells in peripheral blood expressing high levels of CD26 on their surface, the CD26bright T-cells.(Modified from De Meester I, Korom S, Van

Damme J et al. Immunology Today 1999: 20(8);

367-75.)

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response. Figure 6 reflects an overview of the phenotypic and functionalcharacteristics of CD26bright T-cells.Soluble CD26 / DPPIV (sCD26 / DPPIV) is similar to cell surface CD26 / DPPIV189;190 and can be detected at significant levels in peripheral blood. Itis soluble because it lacks the transmembrane domain. The cellular origin(s) of the sCD26 / DPPIV is unclear. Nevertheless, it is hypothesized that sCD26 / DPPIV may derive from all CD26-expressing cells that contact blood and that it may help to inactivate (locally produced) bioactive substrates and thereby prevent possible systemic effects.168 sCD26 / DPPIV can influenceT-cell proliferation in vitro but the underlying mechanisms are indistinct. Interestingly, altered levels of sCD26 / DPPIV are found in inflammatory dis-ease conditions (see paragraph 1.4.5). A number of other molecules exhibiting DPPIV-like enzyme activity have been introduced and termed “DPP-IV activity and/or structure-homo-logues” (DASH). This group comprises several proteases including attractin, fibroblast activating protein α, DPP 8 and 9.191;192 DASH have potential abili-ties to complement and/or associate to DPPIV on the level of its enzymatic activity. These homologues have been implicated in inflammatory condi-tions as contributing factors193, although the exact contribution and role are still largely unestablished.

1.4.3 Functions of CD26 / DPPIV

A large array of functions has been attributed to CD26 / DPPIV. The two mainstays can be described as its ability to regulate the effect of biologicalfactors through its enzymatic activity and its essential role in human T-cell physiology. Besides its proteolytic function (see paragraph 1.4.4), it has numerous other distinguishing functions as a receptor, a costimulatory protein, as an adhe-sion molecule for collagen and fibronectin and its involvement in apoptosis.Due to this multifunctional character and its widespread expression, CD26 / DPPIV has a so-called moonlighting character referring to the diverse func-tions separately regulated in time and context.183;194 The exact functions of CD26 / DPPIV in vivo however, have not yet been explored to the fullest. In 1993, CD26 / DPPIV was recognized as a receptor, i.e. as the binding pro-tein for adenosine deaminase (ADA).195-198 Adenosine deaminase (ADA) is a soluble enzyme that catalyzes the degradation of adenosine and deoxy-adenosine. It mainly occurs in the cytosol199 (90%), and to a lesser extent onthe cell membrane in many tissues. The highest expression is found on the surface of (T- and B-) lymphocytes.200 Dong et al. found that ADA and CD26 / DPPIV only colocalize on the cell surface and not inside the cell, suggesting that ADA is not transported through the cell membrane by CD26 / DPPIV201

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and the individual enzymatic activities of both molecules remains pre-served. Despite its wide distribution, deficiency of ADA in humans causesprofound lymphopenia and results in severe impairment of cellular and hu-moral immunity (severe combined immunodeficiency disease (SCID)). Thebinding does not occur in rodents which implies interspecies variation.183 Despite this alliance between ADA and CD26 / DPPIV on lymphocytes, no distinctive counter-receptor has been identified on the antigen presentingcell.The costimulatory properties of CD26 / DPPIV have been extensively stud-ied in vitro.180;185;198;202;203 Important to note is that the proteolytic activity of CD26 / DPPIV is not a prerequisite for the costimulatory properties in vitro. In general, the activation of T-cells requires at least two signals. The first is provided by stimulation of the TCR by specific peptide antigens. Thesecond can be delivered by triggering costimulatory molecules which can be provided by a number of accessory molecules expressed on the T-cell surface. The costimulatory characteristics of CD26 / DPPIV comprise that it augments T-cell responses to foreign antigen as it is able to transmit an activating signal to the T-cell180, initiates signal transduction, increases cy-tokine secretion and proliferation, upregulates activation markers such as CD25, CD71 and CD69, induces differentiation into effector cells and en-hances provision of help to B-cells and cytotoxic T-cells.168;204;205 Yet, the spe-cific triggering mechanism for the costimulatory activity of CD26 / DPPIVhas not been defined. Biochemical and immunomodulation studies185 have provided evidence that CD26 as a costimulatory molecule on the T-cell sur-face is able to induce cell proliferation via direct interaction with CD45.206 However, the importance of this association is still to be elucidated. A role for CD26 / DPPIV in cell interactions with the extracellular matrix as an adhesive molecule has been described and specified to involve two ma-jor constituents namely fibronectin and collagen.207-209 Lastly, CD26 / DPPIV is reported to be involved in apoptosis.210 Apoptosis is a form of programmed cell death crucial to ensure correct tissue func-tion and to prevent inappropriate excessive growth. In vitro studies have showed that human keratinocytes derived from psoriatic plaques are resist-ant to induced apoptosis when compared to keratinocytes derived from normal skin.211 Therefore, defects in apoptosis might play an important role in the pathogenesis of psoriasis. In line with this, UVB irradiation has proven to be an effective treatment causing apoptosis in T-lymphocytesand keratinocytes.212;213 When apoptotic mechanisms fail due to defects or mutations in apoptotic proteins (possibly CD26 / DPPIV), pathological mechanisms can take over as in psoriasis.

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1.4.4 Protease function and possible substrates

CD26 / DPPIV has an ecto-peptidase activity that catalyses the cleavage of N-terminal dipeptides from proteins containing proline or, to a lesser extent, alanine at the penultimate position. The rate of the CD26 / DPPIV catalyzed proteolysis is described to be inversely related with the chain length of the protein to be cleaved.214;215 Although the natural substrate(s) for CD26 / DPPIV remain(s) to be identified, CD26 / DPPIV participates in thedegradation of many pro-inflammatory molecules and possible substratesof CD26 / DPPIV include several cytokines, chemokines, hematopoetic growth factors, neuropeptides and hormones which all share the proline (or alanine) at their N-terminus. In particular, as many cytokines and chemo-kines contain DPPIV-susceptible N-terminal sequences, the processing of cytokines and chemokines by CD26 / DPPIV has an important impact on the biological activity of the specific substrates eventually contributing tothe regulation of leukocyte migration. Activity of RANTES (regulated upon expression, normal T-cell expressed and secreted;CCL5) is altered by the en-zymatic cleavage of CD26 / DPPIV as the processed form has a more than 10 times lower chemotactic potency for monocytes and eosinophils. Acti-vation of CCR5 by RANTES activates type 1 T-cells.123;216 In psoriatic lesional skin, RANTES is overexpressed.217

Other important chemokines that appear to be substrates of enzymatic activity of CD26 / DPPIV are CCL11 (eotaxin), CCL22(macrophage-derived chemokine), CXCL10 and CXCL11 (interferon inducible chemokines IP-10 and I-TAC).218 Notably, the chemokines CXCL2 (MIP-2α), CCL2 and CCL7 (MCP3) have penultimate prolines but are not hydrolyzed by DPP IV.168

Table 2 Chemokines as substrates for CD26 / DPPIV (From Boonacker and Van Noorden, 2003, De Meester 1999,

Gorrell 2001)

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Table 2 reflects an overview of the substrates219-221 exhibiting altered chem-otactic activity following CD26 / DPPIV-mediated truncation in inflamma-tory processes. Natural substrates currently attracting interest include glucagon and gluca-gon-like peptide (GLP-1/2) owing to the effects on blood glucose tolerationand insulin productions.222-224 Interestingly, different isoforms of CD26 / DPPIV may have different substrate specificities.225 These aspects of substrate spe-cificity of CD26 / DPPIV make it difficult to predict which natural substratesare eventually processed by which isoforms of CD26 / DPPIV.183;226

1.4.5 CD26 / DPPIV in auto-immune diseases

Aberrant soluble (serum) CD26 / DPPIV activity has been reported for a large array of diseases such as solid tumors, hematological malignancies and in-fectious diseases (e.g. HIV and hepatitis C). In fact, several (auto)immune and inflammatory diseases have been related with altered plasma levelsof soluble CD26 / DPPIV as well (table 3). Reduced serum activity was also observed in patients with inflammatory bowel disease (Crohns disease andulcerative colitis).163;227 Also in psoriasis altered (decreased) levels of soluble CD26 / DPPIV activity in plasma have been found.228 Regarding the levels of the membrane-bound form of CD26 / DPPIV in auto-immune diseases, changed numbers of CD26+ T-cells have been found at inflammation sites as in patients with Graves’ disease and RA where in-creased numbers of CD26+ T-cells in the thyroid and synovial fluids weredescribed.187;229 In addition, enhancement of CD26 expression in these dis-eases may correlate with disease severity.230;231 Referring to the subpopula-tion of the CD26 bright cells, they are found to be increased during active phases of autoimmune diseases and reduced during immunosuppression as reflected in table 3.220 In psoriasis, a significant decrease of CD26 expression on CD8+ T-cells wasfound in the peripheral blood of a cohort of thirty patients when compared to a control population. However, there was no correlation to disease sever-ity as determined by the Psoriasis Area and Severity Index (PASI score).232 With respect to psoriatic dermal inflammation, Novelli et al reported thatCD26 / DPPIV is expressed on human keratinocytes in variable amounts and in a group of patients with heterogeneous inflammatory dermatoses (i.e.erythrodermic psoriasis, spongiotic dermatitis and lichen planus).169 CD26 / DPPIV expression is also found to be significantly increased in fibroblastsderived from patients with psoriasis and lichen planus as compared to their normal counterparts.172 Unfortunately, little is known about the functional relevance of CD26 / DPPIV expression on these skin cells in animal models of psoriasis or normal or pathologic human skin.

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Clinical relevance of CD26 / DPPIV inhibitorsDuring the last decade the blocking effects of CD26 / DPPIV on T-cell ac-tivation and function were examined in several animal models of human auto-immune diseases including RA, MS, inflammatory bowel disease (IBD)and transplantation.233-237 The in vivo observation that inhibitors of CD26 / DPPIV can suppress T-cell functions, suggests a potential use as therapeutic drugs in inflammatory diseases.238 To date no clinical studies have been re-ported using CD26 / DPPIV inhibitors during auto-immune diseases except for non-insulin dependent diabetes mellitus (NIDDM). The CD26-mediated processing of GLP-1 is reported to enhance insulin secretion and improves glucose tolerance. Recently, DPPIV inhibitors have been successfully intro-duced in the treatment of NIDDM.239;240

Concluding remarks on CD26 / DPPIVUnraveling the complex nature of CD26 / DPPIV is continuing as much is still largely unknown about its precise properties and functions. In line, the exact picture of all biological functions of CD26 / DPPIV is difficult to com-pose but explicable due to its moonlighting properties as CD26 / DPPIV exerts its different functions in different cell types and thus in different cel-lular contexts. CD26 / DPPIV expression and activity measurement may be useful for the assessment of the severity of various inflammatory diseases,the response to therapy and the prognosis.Some believe that the psoriatic pathogenesis can be considered an auto-

Table 3 Altered levels of soluble CD26 and number of CD26brightT-cells in inflammatorydiseases (From Boonacker, van Noorden 2003, De Meester 1999, Gorrell 2001)

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immune response against a yet undefined antigen involving the activa-tion of immune cells. CD26 / DPPIV has been shown to be expressed and variably upregulated on the surface of key-pathogenic immune competent cells in various auto-immune conditions. In addition, CD26 / DPPIV can ex-ert a proteolytic function on cyto- and chemokines, potentially influencingthe complex network of interactions in the psoriatic immune cascade. Pos-sessing properties as a T-cell activation molecule and costimulatory pro-tein, CD26 / DPPIV has abilities to interfere with the heart of the psoriatic immune response. Schematically, the outlined position of CD26 / DPPIV in the psoriatic interplay of keratinocytes, T-cells and cytokines is illustrated infigure 7. Targeting CD26 / DPPIV may possibly contribute to the clarifica-tion of the pathophysiology of psoriasis and therapeutic means to treat psoriasis.

1.5 Therapies for psoriasis

In summary, psoriasis can be recognized by a disturbed proliferation and differentiation of keratinocytes accompanied by vascular alterations andepidermal infiltration of activated Th1-type lymphocytes together with alocal Th1-type cytokine immune response. As a consequence of this diver-

Figure 7 Hypothesized position of CD26 / DPPIV in psoriasis

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sity in etiology, the understanding of a multifactorial pathogenesis of pso-riasis has evolved into a remarkable number of therapeutic concepts.Due to diverse presentations of psoriasis and individual needs and coexist-ent factors, the choice of treatment must be individualized for each patient. A holistic approach to treatment, including education and psychological support, is needed for optimal care of psoriatic patients. Patients must un-derstand the genetic, environmental and real-life implications of psoriasis and its treatment. Indications for treatment may arise from local symptoms (itch, pain), cosmetic problems or both.241 The main aim of treatment is to reduce the burden of disease over time by controlling symptoms, helping the patient to cope with the chronic nature of the disease, ultimately lim-iting psychological and relational consequences, and preventing systemic complications and comorbidity.29 Even cost-benefit matters should be in-volved herein. Moreover, an evidence-based approach is essential in makinga choice between the many available treatments. Treatments currently available comprise topical agents, phototherapy and systemic agents in-cluding the injectable biologicals. Other innovative treatments are con-stantly emerging, but often not (yet) registered for psoriasis. This paragraph touches upon the registered treatments and some innovative treatments for psoriasis, with specific reference to the treatments which are the focusof interest in the various manuscripts in this thesis. An overview of the avail-able anti-psoriatic treatments and the corresponding “stepwise approach” is given in figure 8.

1.5.1 Topical therapy

Emollients and KeratolyticsUse of emollients can serve as important complementary therapeutic mo-dality. In general, psoriatic patients should be advised to maintain adequate hydration of their skin with emollients such as lanette, petrolatum or pa-raffine as their barrier function is disturbed. Specifically, they can soften theoften hyperkeratotic surface of the lesional psoriatic skin which creates a less tightening, soothening sometimes cooling sensation on the spot. In this way it can take part in improving the clinical signs and symptoms of psoriasis.242;243

Keratolytics can be considered excellent descaling agents as they promote the removal of the uppermost scaly layer of the epidermis, i.e. the stratum corneum. Salicylic acid is the most widely used and prescribed as an ad-junctive treatment for psoriasis as the combination with a topical cortico-steroid decreases the amount of scale and subsequently enhances the skin penetration of the steroidal agent.244

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Vitamin D3 derivativesThese vitamin D3 derivatives were first introduced in the topical treatmentof psoriasis in the early 1990s in Europe. Nowadays, they represent the first choice of maintenance therapy for mild-to-moderate psoriasis. Theseagents bind with vitamin D-selective receptors in various cell types and have been shown to inhibit the hyperproliferation and abnormal differenti-ation of keratinocytes in psoriasis.245-248 The exact mode of action is not fully elucidated. The majority of available data is on calcipotriol (Daivonex™), in a lesser extent on calcitriol (the active metabolite of vitamin D; Silkis™) and tacalcitol. The latter is not registered in the Netherlands for the treatment of mild-to-moderate psoriasis. The clinical response with these preparations is good although slower than with higher potency corticosteroid agents. Cal-cipotriol is more effective than calcitriol and as effective as potent, but notultrapotent, topical corticosteroids.249-251 The topical vitamin-D3 analogues are generally well-tolerated and practicable for the physician and the pa-tient. It has minimal effects on the calcium metabolism when used in theirrespective dosage regimens and is safe up to 100 g per week. Temporary skin irritation may limit the use, especially on the face or the intertriginous areas.A great benefit of vitamin D3 derivatives is their value in combinationtherapy.252 In the first weeks of treatment, use together with topical cortico-steroids is superior to monotherapy with respect to efficacy and tolerance.253 For moderate-to-severe psoriasis, a combination of topical vitamin D3-ana-logues with UV phototherapy or a systemic therapy (ciclosporine, acitretin) can result in a synergistic effect and therefore be recommended.252

Topical corticosteroidsBesides the group of vitamin D3 derivatives, corticosteroids compose the other cornerstone in the topical treatment for psoriasis. The anti-psoriatic effect of corticosteroids is largely related to their effects on gene transcrip-tion that result in diminishing of inflammatory, proliferative and immuno-logic responses.244;254 They are available in a variety of formulations, with po-tencies categorized in the Stoughton-Cornell classification which assignsclinical effectiveness to corticosteroids on the basis of their ability to inducevasoconstriction, separated into four potency groups (classes I to IV).255 As a general rule, topical corticosteroid therapy is initiated with the lowest po-tency agent expected to be effective. Low potency agents are appropriatefor lesions affecting the face or intertriginous areas and for small children.244 Their potency can be enhanced by occlusion with a plastic dressing.256-258 Care must be taken regarding the development of typical adverse corti-costeroid effects such as skin atrophy, teleangiectases, striae distensae andpurpura in cases of longer application and in particular at sensitive areas. Systemic effects such as iatrogenic Cushing’s syndrome and hypothalamic-pituitary-adrenal axis suppression are also seen with extensive treatment.259

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These side-effects are most likely with high potency corticosteroids. In orderto minimize such complications, the use of (ultra)potent corticosteroids is limited and various treatment regimens are being employed e.g. use in the weekends only, combination with non-steroidal agents and transition to products of weaker potency when initial clinical response is accomplished. In general, topical corticosteroids are not used as the primary treatment when more than 20% of the body surface is affected by psoriatic lesions.

Calcipotriol/betamethasone Interest in the corticosteroid sparing ability of vitamin D3 derivatives has fa-cilitated the development of a new single agent topical therapy for plaque psoriasis. Large well-controlled studies have established the superior effica-cy of a once-daily combination ointment containing calcipotriol 50µg.g-1 and betamethasone dipropionate 0.5 mg.g-1 (class III corticosteroid; Dovobet™) over use of either agent alone.260-263 Side-effects of both active compoundsare minimized in clinical use for up to 1 year by use of this combination.252 In addition, both clinical and immunohistochemical studies have shown that the combination ointment of calcipotriol and betamethasone dipropionate is more effective than the simultaneous use of each of the compounds oncedaily.264;265 Moreover, in terms of quality of life, a once-daily application of

Figure 8 Anti-psoriatic treatment ladder

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this product is favourable when compared with twice-daily application of calcipotriol.266;267

DithranolDithranol (anthralin, cignolin) is one of the oldest topical therapeutics for psoriasis. It induces a cascade of free radicals in the skin, resulting in an-tiproliferative effects and a modulation of inflammation in psoriasis pre-sumably via a direct effect on mitochondria.268;269 Although it is an effec-tive treatment for mild-to-moderate psoriasis, dithranol as monotherapy is less effective than either topical corticosteroids or vitamin D3 derivatives.249 The efficacy can be increased further if either calcipotriol, intermittent highpotency topical corticosteroids or UVB phototherapy is combined with dithranol.252;270

The therapy is safe although skin irritations, burning, erythema and inter-mittent brown discolorations of skin and clothing can frequently be ob-served, which makes it best used in hospital or day care clinics. Coal tarCoal tar is a mixture of thousands of compounds roughly comprising 48% hydrocarbons, 42% carbon and 10% water.268 Like dithranol it has anti-proliferative effects and can modulate inflammatory events in psoriasis.Head-to-head comparative and placebo-controlled studies are sparse with respect to tar treatment. Skin irritation, folliculitis, allergic/phototoxic responses, staining and odour of clothing limit coal tar’s use as a routine outpatient treatment. Carcinogenicity following coal tar applications inpsoriatics remains unconfirmed. Calcineurin inhibitorsThe calcineurin inhibitors tacrolimus and pimecrolimus have both been assessed in the treatment for psoriasis, but were originally approved topical treatments for atopic dermatitis. They are immunosuppressant and inhibitT-cell activation. Unlike corticosteroids, they do not cause skin atrophy. Their efficacy is limited in the treatment of psoriasis because of poorpenetration unless they are used under occlusion or on the thinner skin of the face, intertriginous areas or genitals.252;271-274

Topical retinoids275

Tazarotene is the only topical retinoid (vitamin A derivative) available for the treatment of plaque psoriasis. It has been shown to have antiprolifera-tive and anti-inflammatory effects.Tazarotene (0.05% or 0.1%) is available as gel and cream formulations, al-though not in the Netherlands as it is not registered. It is most effectivewhen used in combination with topical corticosteroids.

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Besides local adverse events such as pruritus, erythema and burning it is important to note that teratogenic aspects are only of concern when more than 20% of the body surface is treated.

1.5.2 Systemic therapy: classics

In psoriatic patients with more than 20% of body surface area involved, topical therapy may be impractical and usually requires some form of pho-totherapy or the use of systemic therapies. All these regimens cause toxic effects in one or the other way, therefore the indication to treatment mustbe considered repeatedly to avoid excessive risk in relation to benefits.

Photo(chemo)therapyPhototherapy is often a good treatment option when the psoriatic lesions are widespread covering a considerable body surface or when the lesions are resistant to various topical therapies. Therefore it can be considered a valuable option “in between” topical and systemic therapy. The interaction between UV radiation and chromophores in the skin induces a multitude of effects such as induction of oxidative stress and activation of transcriptionfactors as well as induction of DNA damage.276 Phototherapy for psoriasis includes broadband UVB (290-320 nm), narrowband UVB (311-313 nm) and psoralen plus ultraviolet A light (PUVA) (320-400 nm plus pretreatment with photosensitizing psoralens). When comparing narrowband to broadband UVB, the most effective wavelength for psoriasis is determined to be 311-313 nm (narrowband range).277 Moreover, use of PUVA has been reduced because narrowband UVB is more convenient and possibly safer for pa-tients.In using UVB phototherapy, the minimal erythema dose (MED) is deter-mined first on the basis of individual patients’ Fitzpatrick skin type I-IV andthe metered output of the UVB unit before beginning treatment.252;278 UVB therapy is initially most common used two to three times a week with cau-tious dose escalation and reduction in correspondence with the clinical response.279 The onset of the clinical effects is usually within 2 weeks.Phototherapy represents a safe and very effective treatment modality forthe treatment of moderate to severe forms of psoriasis. Erythema from overexposure is by far the most common side-effect. With repeated orlong-term application of UV-therapy, the consequences of high cumulative dosages (i.e. premature aging of the skin, carcinogenic risk) must be taken into account.Goeckerman (UVB plus tar) or Ingram (UVB plus anthralin) therapies have been associated with remission times of at least 6 months and more than 1 year for a considerable proportion of psoriatic patients. PUVA therapy is

50

associated with significant and prolonged benefits for psoriasis. Remissionsof more than 1 year have been reported in about 40% of patients treated with a single course of PUVA.280 However, one should bear in mind that the carcinogenic potential of PUVA is higher than of UVB. The most popu-lar combination regimens include UVB (either narrowband or broadband) with vitamin D3 derivatives or systemic treatments.

MethotrexateMethotrexate (MTX) is a folic acid antagonist interfering with the purine synthesis and thereby inhibits DNA synthesis and cell proliferation in the rapidly dividing psoriatic epidermis.281 It also has specific T-cell suppressivecapacities.282 Despite the emergence of novel biologic therapies, it is still an important effective mainstay in the treatment of moderate to severe pso-riasis (and psoriatic arthritis).MTX can be given by the oral, intramuscular or intravenous routes, usually as a single weekly dose, accompanied with folic acid supplementation to minimize side-effects. Following stable full blood counts and liver functiontests, patients can be maintained on a dose of 7,5-25 mg weekly. The most important potential side-effect is bone marrow suppression. Patients needto avoid alcohol intake due to the increased risk of liver fibrosis.281 Prolonged treatment with MTX requires a liver biopsy to detect fibrotic changes, al-though this is now debatable as the measurement of type III procollagen in serum (P3NP) can be used to detect liver fibrosis or cirrhosis.283;284 This has to be further validated in future studies however.

RetinoidsAcitretin is an oral retinoid (vitamin A derivative) approved for the treat-ment of psoriasis. It is a synthetic hormone that binds to nuclear retinoid acid receptors (RAR), thereby altering gene transcription and returning ke-ratinocytes proliferation and differentiation to normal.252 In general, mono-therapy with acitretin is more effective for pustular psoriasis than chronicplaque, guttate or erythrodermic psoriasis which respond more slowly.281;285 For a detailed account on acitretin therapy in psoriasis, the reader is referred to a review article by Yamauchi et al.281

CiclosporinCiclosporin is an effective short-term treatment for moderate-to-severepsoriasis.286 It induces immunosuppression by inhibiting the first phase ofT-cell activation; it may also exert a direct effect on epidermal keratinocytes.Ultimately, the production of cytokines that are important in cell-mediated immunity, such as IL-2 and IFN-γ, is inhibited.252;281 Unlike MTX, ciclosporin is not myelosuppressive, but requires careful monitoring for nephrotoxic-

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ity and hypertension. For a detailed account on ciclosporin the reader is referred to the review article of Griffiths et al.287

Fumaric Acid EstersOral fumaric acid esters (fumarates) have initially been used to treat moder-ate-to-severe psoriasis in Germany as a German chemist demonstrated the benefits of fumarate therapy on his own psoriasis.288 Fumarates are natu-rally occurring molecules that link the urea and citric acid cycles, with their benefit in psoriasis now considered to be due to inhibition of T-cell activity(induction of T-cell apoptosis), a shift to a Th2-type cytokine response and NFkβ inhibition.252;289;290 Nowadays they are effectively used in the treat-ment of psoriasis although approved in a very small number of Northern European countries, excluding the Netherlands.290-293

OthersLess commonly used systemic treatments for psoriasis include mycophenolic mofetil, hydroxyurea, 6-thioguanine, sulfasalazine, azathioprine and tacrolimus.281

1.5.3 Systemic therapy: biologics

Extensive understanding of the immunopathogenesis has provided ration-ale for the development of new strategies targeting the immune cells and molecules that induce and maintain the clinical changes seen in psoriatic plaques. These therapies are named ‘biologics’ or ‘biologicals’ and are used to treat patients with moderate-to-severe plaque type psoriasis in which the classic treatments have failed to a certain extent or are contra-indicated.Biologicals are recombinant molecules that are designed on the basisof genetic sequences from various organisms and that are often similar or identical to proteins produced by human beings. They include fusion proteins, recombinant proteins and monoclonal antibodies each blocking the induction or maintenance of T-cell activation in one or the other way through diverse sites and modes of action (figure 9).Since their introduction, they have provided an effective option to the arrayof anti-psoriatic treatment modalities. Moreover, patients have experienced substantial improvements in their quality of life.

Biologics addressed in this thesis

EfalizumabThe humanized IgG1 monoclonal antibody efalizumab (Raptiva™) is direct-ed against the CD11a subunit of LFA-1. LFA-1 is a T-cell surface molecule important in T-cell activation, T-cell migration to the skin and cytotoxic

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Chapter 1

Sites of action

Modes of action

Interactions between APCs and T-cells are crucial in the pathogenesis of psoriasis. Several immunomodula-tory biological therapies can target the molecules expressed on the surface of both cells.

Alefacept blocks the interaction between LFA-3 on APCs and CD2 on T-lymphocytes. Moreover, alefacept functions as a bridging molecule between CD2 that is upregulated on CD45RO+ T-lymphocytes and the Fc

γ III receptor (CD16) on NK cells which induces apoptosis

of T-lymphocytes through the release of granzyme by NK cells. Efalizumab blocks the inter-action between LFA-1 and ICAM-1 thereby inhibiting the T-cell activation and migration into the skin. Etanercept antagonizes the effects of endogenous TNF by competitively inhibitingits interactions with cell-surface receptors. Infliximab binds to and inhibits the activity oftransmembrane-bound and soluble TNF-α with high specificity, affinity and activity. Finally,adalimumab is directed against TNF-α as well and blocks its interaction with the p55 and p75 cell-surface receptors thereby lysing the cells that express surface TNF-α. (Modified from Gottlieb AB. Nat Rev Drug Discov 2005; 4: 19-34. Kormeili T, Lowe NJ, Yamauchi PS. Br J

Dermatol 2004;151: 3-15.)

Figure 9 Sites and modes of action of major biologics


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T-cell function. Binding of efalizumab to CD11a blocks LFA-1 from binding to ICAM-1, which is upregulated on psoriatic keratinocytes and its partner molecule for adhesion. Subsequently, this inhibits T-cell activation thus preventing cutaneous T-cell trafficking. The blockade is reversible and doesnot deplete T-cells, suggesting that efalizumab can be used as a continous therapy.Efalizumab is available as a subcutaneous preparation that patients self-inject once weekly. Optimal efficacy with efalizumab was seen with weeklydoses of 1 mg/kg (after a first dose of 0.7 mg/kg) as 22-39% of the patientsachieved a 75% reduction in PASI within 12 weeks and 57% a PASI 50.129;130;294 In addition, one third of patients with an outcome between 50% and 75% improvement in PASI reaches at least a 75% reduction after 12 more weeks of treatment (total of 47% reaching PASI75 after 24 weeks).129;295 Clinical improvement was seen as early as two weeks after initiation of treatment. The initial lower dose of efalizumab is lower in order to minimize the tran-sient flu-like symptoms that were observed in the clinical trials at the higherdoses.296 In these large clinical studies, efalizumab was well-tolerated, with the most frequent side effects being transient, mild to moderate flu-likesymptoms including mild headache, fever and chills. Trombocytopenia was observed occasionally thus monitoring should include platelet counts. Efalizumab has not been shown to be effective in treating psoriatic arthri-tis. To date, no evidence of cumulative end-organ toxicity has been found. The risk of malignancy, as with other immunomodulating drugs, has to be determined through additional patient experience. A major concern with efalizumab is the rebound of psoriasis, defined as aworsening of psoriasis by 125% relative to the pretreatment baseline, or the conversion of psoriasis morphology to a more serious subtype such as eryth-rodermic or generalized pustular psoriasis that occurs within 12 weeks of discontinuing treatment. It has been observed in 3% of efalizumab patients during therapy and in 14% of patients following abrupt discontinuation of efalizumab, predominantly in the non-responder group.297 Formulating an appropriate transitional “rescue” therapy strategy before discontinuation of efalizumab may minimize the occurrence of rebound.298;299

EtanerceptAs opposed to targeting T-cells, etanercept (Enbrel™) is a fusion protein comprising domains of the 75-kDa human tumor necrosis factor receptor and human IgG1, which inhibits the activity of soluble TNF-α and slows the inflammatory cycle in psoriasis driven by TNF-α. Etanercept also reduces the levels of several chemokines and cytokines, such as IL-8, CXCL10 and CCL20in psoriatic lesions, leading to a decrease in T-cells, dendritic cells and keratino-cyte hyperplasia.300;301 It is approved in the European Union (EU) and the United States for the treatment of rheumatoid arthritis, juvenile arthritis,

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severe ankylosing spondylitis, psoriatic arthritis and plaque psoriasis.In psoriasis trials, various dosage regimens have been used: self-injected subcutaneously administration of once a week 25 mg, twice 25 mg or 2 x 50 mg per week. The efficacy of etanercept was found to be dose-depend-ent, with a PASI75 response observed in 34% of patients in the 2 x 25 mg weekly group, and in 50% of patients in the 2 x 50 mg group after 12 weeks of treatment. An additional 12 weeks of treatment improved the efficacyup to 59% (PASI75) after 24 weeks at the 2 x 50 mg dose.139 Up to 96 weeks, improvements in PASI of at least 75% have been reported with administra-tion of 50 mg etanercept biweekly.302 When treating patients for the first 12weeks with 50 mg twice weekly, then reducing the dose to 25 mg biweekly for 12 weeks, 77% of the patients maintained the PASI75 response rate after 24 weeks.298 In addition, etanercept is highly effective in psoriatic arthritis,with a reduction in the signs and symptoms of joint disease in 73-87% of patients at 12 weeks of treatment.303 Also in children and adolescents with moderate to severe plaque psoriasis, etanercept significantly reduces thedisease severity.304

Clinical data of both controlled trials and post-marketing surveillance sup-port the safety and tolerability of the use of etanercept over longer peri-ods of treatment (up to 96 weeks302). The main adverse events noted are transient, mild to moderate injection-site reactions (erythema and/or itch-ing, pain, or swelling) which last 3-5 days.305 Infections, mainly of the upper respiratory tract, bronchitis or skin infections have less often been report-ed. In contrast, fatigue and symptoms of depression might be relieved by treatment with etanercept.306;307 Etanercept therapy has rarely been linked to positive ANA and a lupus-like syndrome and increased risk of lympho-ma, demyelinating disorders of the central nervous system and congestive heart failure. Anti-etanercept antibodies have been found up to 18,3 % of patients, but these were found to be non-neutralizing and had no appar-ent effect on the efficacy or safety profiles of etanercept.302 In contrast to efalizumab, rebound of psoriasis is not likely when etanercept therapy is discontinued.295

InfliximabInfliximab (Remicade™) is a chimeric monoclonal antibody that inhibitsboth membrane-bound and soluble forms of TNF-α. It is approved in the EU and US for patients suffering from RA, Crohn’s disease, ankylosing spond-ylitis and plaque psoriasis. Clinical trial results demonstrate that infliximabis a fast-acting agent, highly efficacious for psoriasis. The rapid therapeuticresponse may lie in the observation that infliximab has not only anti-inflam-matory activity but also apoptotic and anti-angiogenetic properties.308;309 Infliximab’s suppression of TNF-α activity not only affects T-cells but alsokeratinocytes and endothelial cells as the expression of K16 and the en-

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dothelial adhesion molecules ICAM-1, E-selectin and VCAM-1 all decreased significantly compared to pretreatment levels.308;310

Infliximab is administered at a dose of 5 mg/kg intravenously over 2 hoursat weeks 0, 2 and 6 and then every 8 weeks afterwards. Large clinical trials have shown that 80-88% of patients reached a 75% reduction in PASI after 10 weeks of which 57% reached PASI90. At 1 year maintenance therapy, 61% of these patients were at PASI75 whereas 45% reached PASI90.144;300;311 Significant improvement is often seen as early as the second infusion.Loss of efficacy however over time appears to correlate with lower serumconcentrations of infliximab and development of antibodies to infliximab(23,3 % of all infliximab-treated patients311) which occurs because 20% of the amino acid sequence is murine.252 Two suggested strategies to avoid the phenomenon of “loss of efficacy over time” comprise on one hand, theperiodic administration of infliximab on a regular basis of 8 weeks interval(in stead of intermittent administration) and on the other hand, the con-comitant use of a small dose of methotrexate. The latter has, so far, been shown helpful in patients with RA and Crohn’s disease298 but has not yet been investigated in patients with psoriasis. As with etanercept, infliximabis also proven to be efficacious, although less, in patients with PsA as signsand symptoms of active arthritis diminish.312;313

Infliximab infusion has been associated with several adverse events. Mostcommon reported adverse event is upper respiratory infection. Other fre-quent reactions include acute infusion reactions with fever, chills, chest pain, hypotension, nausea and dyspnoea. They can be managed by slowing or interrupting the infusion rate. Since anaphylactoid reactions may occur, one should monitor the patients close. Infection is another major concern and multiple reports have indicated reactivation of latent tuberculosis with infliximab.314;315 Screening for tuberculosis is therefore indicated before starting therapy. Another point of focus with the use of infliximab, is thesix-fold greater risk of potential development of lymphomas316 of which the majority represent non-Hodgkin’s lymphomas (81%). Recent data on liver toxicity of infliximab show that 5% of patients have fivefold increases oftheir liver enzymes, indicating that monitoring of the hepatic enzymes is needful.142 Other rare adverse events include demyelinating diseases, wors-ening of cardiac insufficiency, leucopenia, thrombopenia, pancytopeniaand reversible lupus-like syndrome.317

AdalimumabAdalimumab (Humira™) is a fully human IgG1 monoclonal antibody that binds specifically to TNF-α, blocking its interactions at the p55 and p75 TNFreceptors thereby lysing the cells that express TNF. It is self-administered as 40 mg subcutaneously once every other week after an initial dose of 80 mg. Initially, adalimumab was registered for the treatment of psoriatic arthritis.318

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Large studies in psoriasis have shown that at week 12 of treatment, 53% of psoriatic patients reached PASI75. This response was sustained up to 60 weeks of therapy.319 This proportion is intermediate between the results for infliximab and etanercept. Adalimumab was generally well tolerated andas with the other TNF-α agents, primary concerns are infection, malignancy and development of anti-nuclear antibodies. The primary reported side ef-fect was pain at the injection site. Additional long term safety data are not yet available for the indication of psoriasis, but is available from several tri-als in RA. Adalimumab has recently been approved for the indication mod-erate-to-severe psoriasis by EMEA and the FDA.

Others

AlefaceptAlefacept, originally known as LFA-3 T-cell inhibitor protein (LFA-3TIP), re-duces the number of pathogenic T-cells. It is a fusion protein composed of LFA3 with the constant region of human IgG1. Alefacept (Amevive™) binds to CD2 on memory effector T-cells to block costimulation by preventingCD2 from binding LFA-3 on APCs, selectively interfering with the function of APCs and consequently the T-cell activation. Psoriatic lesions exhibita predominance of lymphocytes that are of the memory effector type(CD45RO+). An additional important mechanism of action of alefacept, is apoptosis of memory-effector CD45RO+ T-cells in the skin as natural killercells and macrophages recognize the Fc portion of the alefacept molecule that has bound to CD2+ T-cells.252;320 After a 12-week period of weekly in-tramuscular injections of 15 mg alefacept, 21% of patients achieved a 75% reduction in PASI and 46% a 50% reduction.321 A proportion of responding patients enjoyed long remissions from their psoriasis.322 Clinical improve-ment develops slower than that observed with other biologicals. Alefacept therapy appears to be well-tolerated, albeit the primary concern is T-lym-phocyte depletion. Therefore, patients are monitored with CD4 counts while on the drug. Although alefacept is approved by the FDA for treatment of psoriasis, it is not currently approved by the EMEA in Europe.

Anti-interleukin 12/23 agents160

A fully human IL-12/23 monoclonal antibody has recently been developed which binds with high affinity to the p40 subunit of human IL-12 and -23.This neutralizes the bioactivity of both cytokines, blocking interactions with their associated cell-surface receptors. Preclinical studies have implicated the IL-12 cytokine, a major driver of the Th1-response, in the pathogenesis of psoriasis in addition to its reported overexpression in psoriatic plaques. IL-23, which also shares the p40 subunit and is overexpressed in psoriatic plaques, activates a distinct T-cell lineage that expresses IL-17 which is sug-

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gested to be pathogenically important in the Th17 response. A phase II study demonstrated the great therapeutic efficacy in psoriasis, confirmingthe role of the IL-12 / 23p40 cytokines in the pathophysiology of psoriasis. So far, long-term data of this novel promising biological are not available yet as large clinical trials are currently being conducted.Although biological treatments have been of great advance in the manage-ment of psoriasis, head-to-head trials and comparisons to traditional thera-pies are essential to define their exact place in the hierarchy of systemictherapies.

1.5.4 Lasers and their therapeutic application in psoriasis

The term laser is an acronym for Light Amplification by the Stimulated Emission of Radiation. Although the use of lasers in dermatology began slowly through the six-ties and seventies, the first laser was developed by Maiman in 1959 usinga ruby crystal to produce red light with a 694-nm wavelength. The concept of stimulated light emission however was already introduced in 1917 by Einstein.323 Eventually in 1963, Dr Goldman was the first to introduce theuse of lasers for cutaneous applications by promoting ruby laser treatment for a variety of cutaneous pathologies.324;325 The theory of selective pho-tothermolysis, introduced by Anderson and Parrish in 1981326, opened the door for broad extensive use of lasers in dermatology. Technologic advancesbrought new effective and safe laser systems and a growing range ofapplicable cutaneous lesions manifested itself. As psoriatic lesions encompass histopathological vascular abnormalities such as dilated, elongated and increased prominent superficial capillariesin the dermis (see paragraph 1.2), treatment with vascular lasers form a potential therapeutic option when facing psoriasis. This thesis includes the evaluation of two types of vascular lasers (pulsed dye laser (PDL) andNeodymium-doped Yttrium Aluminium Garnet (Nd:YAG)) in the treatment of psoriasis.

Basic science of lasers327-329

A laser consists of several basic elements (figure 10):• the lasing (gaining) medium, e.g. a gas, liquid, solid or semiconductor• an excitation power source, e.g a flash lamp, a continuous light source• an optical resonator and output coupler (reflective mirrors)

Lasers are named according to the nature of their lasing medium. The thera-peutic action of laser energy is based on the unique properties of laser light itself and complex laser-tissue interactions. To know how the laser light is

58

produced, first the interactions that take place within a laser have to be ad-dressed. Any atom or molecule has a natural tendency to exist in its natural or rest-ing ground state. As the lasing medium is excited, molecules in the ground state are ‘pumped’ to a higher energy state. Once an electron is moved into a higher orbital level, its natural tendency will be to drop back to its resting state. By doing so, a given packet of energy will be released in a random direction as a single photon that will be characteristic for that atom or mol-ecule. This is termed spontaneous emission. If the spontaneously emitted photon travels in an appropriate direction, it strikes one of the mirrors at each end of the resonating cavity and is reflected back into the lasing me-dium. If this photon further strikes an already excited atom, the atom can be stimulated to decay back to the resting state, resulting in the release of a second photon identical to the initial photon. The net result is that two photons are of a single wavelength (monochromatic), are travelling in the same non-divergent direction (collimated) and proceeding in phase. This process which is termed stimulated emission, represents the key features of laser light and makes it the first necessary element for laser light pro-duction. When enough energy is pumped into the system, the majority of the molecules are raised into the excited state which is a prerequisite for the laser to function. Such a condition is called population inversion. Once population inversion occurs and as long as energy is still being pumped into the system, a sufficient cascade or positive feedback reaction produc-ing more and more in-phase photons occurs, that leads to the amplificationof laser light.

Figure 10 Basic laser construction

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59

Properties of lasers330

Each type of laser produces light that is characteristic for that particular laser. Most lasers produce only one wavelength, although some lasers may pro-duce several monochromatic bands. Lasers may differ in the way in whichtheir light is emitted or delivered. Some lasers are capable of the continu-ous discharge of light (continuous wave) whereas others seem to build up power before releasing their pulse of relatively high power allowing more selective tissue damage.327 The pulses delivered by such lasers are usually quite short and are followed by a lag phase before another pulse can be generated. Duration and power of the pulse can be varied. Laser pulses can be delivered by using flash lamps as an excitation energy source, but canalso be produced by Q-switching in which a crystal is placed in front of the reflecting mirror in the resonating chamber. The pulse duration of any laserwill be determined by the thermal relaxation time (TRT) of the intended target (see under ‘Selective photothermolysis’).The output of continuous wave lasers is measured as power in watts (W), for pulsed lasers output is measured as energy in joules (J) taking the pulse duration into account. Irradiance is a measure of power density and refers to laser power per unit area and takes the spot size into account (W cm-2). En-ergy density or fluence is defined as the irradiance multiplied by exposuretime (J cm-2). The spot size of a laser is equivalent to the laser beam cross section. Fluence and irradiance are inversely proportional to the square of the radius of the spot size. When halving the spot size the irradiance or flu-ence increases by a factor of 4.

Laser-tissue interaction330

Whenever light strikes tissues, it is scattered, transmitted, reflected or ab-sorbed, depending on the nature of the tissue and the wavelength of the light. Transmission refers to the passage of light through a tissue without having an effect on that tissue or on the properties of the light. Reflection refers to the repelling of light off the surface of the tissue without entry intothe tissue. Scattering of light occurs after light has entered the tissue and is due to heterogeneous structure of tissue. It spreads out the beam of light within the tissue resulting in radiation of a larger area than anticipated, be-sides it limits the depth of penetration because it can occur both backward an forward. The absorption of the photons of laser light is responsible for its effect on the tissue. The specific wavelengths of light match with cu-taneous absorbing components (chromophores). The main chromophores in tissues are oxyhemoglobin, melanin and water (figure 11). Absorp-tion of energy by a chromophore results in conversion of that energy into thermal energy. Infrared light is primarily absorbed by water, visible light by hemoglobin and melanin and ultraviolet light by melanin. The depth of penetration affects the ability to treat certain chromophores at certain

60

depths with certain wavelengths (figure 11). Oxyhemoglobin has three main absorption peaks (418, 542 and 577 nm). The dominant absorption peak for oxyhemoglobin is at 418 nm but this wavelength only penetrates to the dermal-epidermal junction (100 µm) which limits the use of a laser of this wavelength for cutaneous vascular lesions. As wavelength decreases towards the ultraviolet, the depth of penetration decreases synchronically. Consequently, near infrared light of the Nd:YAG laser has the greatest depth of penetration into biological tissues (figure 12). Beyond this infrared light lasers become ablative of nature. It has to be noted however, that some of the laser energy can be absorbed by epidermal melanin possibly resulting in hypopigmentation, hyperpigmentation or sometimes scarring. A method to limit potential damage is the use of simultaneous protective cooling systems which also have allowed to increase the energy fluenceand improved treatment efficacy.331

Selective photothermolysisThe ultimate aim of use of lasers in dermatology is to direct energy precisely at a specific chromophore without causing a change in adjacent tissues.332 The principle of selective photothermolysis refers to the process whereby appropriate selection of wavelength, exposure time (pulse duration) and energy density results in damage only to the desired target chromophore (and tissue), without significant thermal damage to surrounding tissue(thus minimizing collateral damage). Therefore, the wavelength must be absorbed by the chromophore in the lesion, the fluence must be sufficient-ly high to generate enough energy to thermally alter the target tissue and the pulse duration must be shorter than the TRT of the chromophore to prevent spread of thermal energy beyond the targeted chromophore.330 The TRT is defined as the time needed for the chromophore to cool to halfthe temperature to which it has been heated by laser irradiation and is pro-portional to the square of the size of the chromophore. In general, small objects cool faster than large ones; melanosomes of 0.5 to 1.0 µm have a shorter TRT than capillaries measuring 10 to 100 µm.332 If the pulse width equals or exceeds the thermal relaxation time, unnecessary thermal dam-age occurs due to heat diffusion to surrounding tissues.Lasers emitting visible light were designed on this basis of selective photo-thermolysis. These lasers include the vascular targeted pulsed dye and Nd:YAG lasers, currently addressed in this thesis.

Clinical laser systemsAs new laser devices continue to emerge, an expanding range in versatile laser systems builds up and therefore a wide spectrum of skin lesions can be targeted which presently includes the psoriatic lesion. Table 4 reflectsan overview of lasers together with their corresponding cutaneous applica-

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Figure 11 Absorption spectra of tissue chromophores with wavelengths of medical lasersNd:YAG=neodymium:yttrium-aluminium-garnet; PDL= pulsed dye laser; Er:YAG= erbium;YAG (Modified from Acland KM and Barlow RJ. Br J Dermatol 2000; 143: 244-55.)

Figure 12 Depth of penetration by different medical lasersKTP=potassium titanyl phosphate; Nd= neodymium; PDL= pulsed dye laser; YAG= yttrium-aluminium-garnet (Modified from Textbook ‘Dermatology’, 2nd ed., JL Bolognia et al., Vol. 2, p.2094)

62

tions. In general, cutaneous lasers can target vascular lesions or pigmented lesions, can generate skin resurfacing or can effectuate hair removal.When treating vascular lesions, the chromophore targeted is oxyhemo-globin. Both congenital and acquired vascular lesions can be treated with vascular lasers, including port-wine stains, hemangiomas and facial telangiectasias. In addition, vascular lasers are often used successfully, but not evidence based, for other vascular-dependent but non-vascular indications328;333, such as in psoriasis.

Lasers in psoriasisThe superficial dermal vasculature is a major contributor in the patho-genesis of psoriasis. Yet, this expansion of the dermal microvasculature in psoriatic plaques has not been put a high value on when considering po-tential targets for treatment, as traditional treatments mainly focus on the inhibition of epidermal proliferation, inflammation or both. Literature datais accumulating however, pledging for marked efficacy when the abnormalsupporting vasculature of psoriatic lesions is selectively removed by vas-cular lasers. Studies using the PDL in psoriatic patients vary in design employing differ-ent fluences, pulse durations and spot sizes. An overall clinical improve-ment of 50-80% is reported in plaques after treatment with a proportion

Table 4 Types of lasers and corresponding cutaneous application(s)329

CW=continuous wave; Nd=neodymium; QS=quality-switched;YAG=yttrium-aluminium-garnet

Chapter 1

Laser type

Argon (CW)Argon-pumped tunable dye laser (quasi CW)Copper vapor / bromide (quasiCW)Potassium-titanyl-phosphateNd:YAG, frequency doubledPulsed Dye

Ruby• QS• Normal modeAlexandrite• QS• Normal modeDiodeNd:YAG• QS• Normal mode

Nd:YAG long-pulsedDiode long pulsedErbium: glassErbium: YAG (pulsed)Carbon dioxide (CW)Carbon dioxide (pulsed)Intense pulsed light source

Wavelength (nm)

418 / 514577 / 585510 / 578532532510585-595

694

755

800-8101064

132014501540249010,60010,600515-1200

Application

Vascular lesionsVascular lesionsPigmented lesions, vascular lesionsPigmented lesions, vascular lesionsPigmented lesions, red/orange/yellow tattoosPigmented lesionsVascular lesions, hypertrophic scars, striae, verrucae, non-ablative dermal remodeling

Pigmented lesions, blue/black/green tattoosHair removal

Pigmented lesions, blue/black/green tattoosHair removal, leg veinsHair removal, leg veins

Pigmented lesions, blue/black tattoosHair removal, leg veins, non-ablative dermal remodelingNon-ablative dermal remodelingNon-ablative dermal remodeling, acneNon-ablative dermal remodelingAblative skin resurfacing, epidermal lesionsActinic cheilitis, verrucae, rhinophymaAblative skin resurfacing, epidermal/dermal lesionsSuperficial pigmented lesions, vascular lesions, hair removal, non-ablative dermal remodeling


63

of patients with complete clearance.334-340 Remission after PDL may extend to 15 months.337 As noted, some patients seemed to respond better than others. As PDL-treatment is relatively time-consuming, expensive and painful it is obviously not a leading first choice treatment in psoriasis. The body ofevidence for the use of PDL in psoriasis is still limited as sample sizes were small, no randomization method was used and sporadically comparison with standard therapies for psoriasis was made.There is a need for larger well-designed clinical trials to prove short- and long-term effectiveness and safety profile and to define appropriate pa-tient selection criteria.333 However, for the localized recalcitrant variant of plaque psoriasis, it may be a fair treatment option.In the immunohistochemical evaluation of Hern et al, the effect of PDL waslimited to the superficial capillary bed, with no changes in the micro vesselsof the upper reticular dermis.336 Therefore a vascular laser with a deeper penetration depth, such as the Nd:YAG (wavelength 1064 nm) laser might be able to destruct these deeper micro vessels possibly contributing to a clearance of psoriatic plaques and of potential benefit to the treatment ofpsoriasis. To date, no evaluative study has been done to put the Nd:YAG laser into place in the array of treatment modalities for psoriasis. For a more profound assessment on the use and application of non-vascu-lar lasers in dermatology and specifically in psoriasis, the reader is referredto the publications of Tanzi et al. and Tournas et al.329;341

1.6 Outline and objectives of this thesis

The main point of focus of this thesis encompasses the dynamics of on the one hand well-established psoriasis-associated markers of the T-cell and on the other hand of a novel potential pathogenesis-based marker in psoria-sis (CD26 / DPPIV), in the context of clinical efficacy of established and in-novative treatments. The following aims and questions shed light on this outline.

AIM I Assessing clinical observations and interference with T-cell subsets of pathogenesis-based therapeutic interventions

The last decade innovative treatments have revolutionized the therapy of psoriasis. Pathogenesis-based treatments have become available which permit more precisely targeted therapeutic intervention including the bio-logicals and vascular lasers.

64

A) To define the effect of immune-targeted therapies on well-estab-lished psoriasis relevant T-cell subsets in the cutaneous and peripheral blood compartment, and to address alleged compartmentalization between them in the context of clinical efficacy.A major question is whether innovative pathogenesis-based treatments act on the T-cell as a key player in the pathogenesis of psoriasis, either influenc-ing the peripheral blood T-cells, the lesional T-cells or the shifting of T-cells between the systemic and cutaneous compartment.

Consequently, we defined the following questions:

1a.

1b.

1c.

2.

B) To position vascular laser treatment compared to several estab-lished treatments in the therapeutic array for psoriasis.As clinical studies are sparse with respect to lasers and psoriasis, we ex-panded our studies with some efficacy, safety and immunohistochemicalissues in two explorative studies formulating the following questions:

1.

2.

AIM II Defining the expression of CD26 / DPPIV and characterizing itas a potential novel marker in the immunopathogenesis of psoriasisBased on current insights in the field of auto-immune diseases, evidenceis provided that CD26 / DPPIV can interact with several key players in the pathogenesis of psoriasis. Therefore our objective was to define the expres-sion of CD26 / DPPIV in the immunopathogenesis and treatment of psoriasis.

Are T-cell counts and –subsets affected in the cutaneous and peripheral blood compartment in response to biological therapies such as efalizu-mab and etanercept? Is recompartmentalization of T-cells between peripheral blood and le-sional dermis and epidermis induced by biological treatments in pa-tients with psoriasis and how does such relate to clinical efficacy?Can measurement of T-cell subset counts in blood or skin be used as a prognostic marker for clinical efficacy?What is the effect of a conventional therapy (methotrexate) versus anovel biologic therapy (adalimumab) on T-cell counts and –subsets in the cutaneous and peripheral blood compartment, in the context of clinical efficacy?

Chapter 1

Is treatment with PDL a safe and effective option for plaque type pso-riasis compared to narrowband UVB and does the combination of PDL with narrowband UVB treatment generate a synergistic clinical effect?What is the clinical and immunohistochemical effect of treatment witha Nd:YAG (1064 nm) laser as opposed to the effect of calcipotriol / beta-methasonedipropionate ointment?


65

In order to investigate the expression of CD26 / DPPIV in the cutaneous and systemic compartment, the following questions were addressed:

1.

2.

3.

4.

1.7 Investigational approach

1.7.1 Clinical efficacy parameters

The Psoriasis Area and Severity Index (PASI) is the most frequently used scale to characterize the clinical severity of psoriasis. Equally, several manu-scripts in this thesis employ the PASI as a clinical measuring parameter. The PASI scoring system assesses four body regions: the head (h), the upper ex-tremities (u), the trunk (t) and the lower extremities (l), corresponding to 10%, 20%, 30% and 40% of the total body surface area, respectively. 342 The area of psoriatic involvement for each of the four regions is assigned a nu-merical value (A) of 0-6 corresponding to 0-100% involvement: 0 represents no involvement; 1, <10%; 2, 10<30%; 3, 30<50%; 4, 50<70%; 5, 70<90%; 6, 90-100%. For each region, erythema (E), induration (I) and desquamation (D) are rated according to a five-point scale: 0, no involvement; 1, slight; 2,moderate; 3, marked; 4, very marked. Subsequently, the PASI score is calcu-lated from the following formula: PASI= 0.1A

h (E

h+I

h+D

h) + 0.2A

u (E

u+I

u+D

u) +

0.3At (E

t+I

t+D

t) + 0.4A

l(E

l+I

l+D

l). The PASI score can vary in increments of 0.1

units from 0-72 with higher scores representing a greater severity of psoria-sis. The PASI score is a quick and simple tool used primarily in clinical trials as a means to demonstrate improvement in a standardized manner. Clinical improvement is measured by the percentage change in PASI score which results in commonly used wording of PASI75 and PASI50, respectively re-presenting 75% and 50% improvement in PASI score.Despite its widespread application, use of the PASI score incorporates some drawbacks including its lack of sensitivity. For instance, erythema, desqua-mation and induration are scored with equal weight within each of the four

In what way is CD26 / DPPIV expressed in psoriatic skin? Can it function as a potential novel marker in lesional psoriatic skin compared to nor-mal skin of healthy volunteers?Does upregulation of CD26 / DPPIV in psoriatic skin cohere with kerati-nocyte proliferation- and differentiation markers during developmentof a psoriatic lesion?What are the dynamics of peripheral blood CD26bright T-cells in psori-atic patients compared to healthy conditions?To what extent does treatment with infliximab alter the expression ofCD26 / DPPIV in lesional skin and peripheral blood and how does such relate to clinical efficacy?

66

body regions. Consequently, a reduction in scaling with a concomitant in-crease in skin erythema could be recorded as the same PASI score.343 In ad-dition, although the PASI score ranges from 0-72, most patients have scores ranging between 0-15. PASIs of 40 or more are sporadically seen. Alterna-tive psoriasis severity scores have been proposed but none of them is used as consistently as the PASI.344

After the PASI score, the most often employed score to measure psoriasis severity is the Physician’s Global Assessment (PGA) score (sometimes called Psoriasis or Investigator’s Global Assessment). The PGA has the investiga-tor assign a single estimate of the patient’s overall severity of disease; typi-cally a 6-point scale from clear to severe is used although many variations have been reported. Chiefly, the overall lesions are graded separately forerythema, induration and scaling after which the sum of these three scales will be divided by three to obtain a final PGA score.The PASI and PGA stand for an overall severity index of psoriasis whereas the SUM score reflects the severity of only one designated psoriatic lesion.Although its less widespread use compared to the PASI and PGA, it repre-sents a good tool for a topical impression of lesional severity. Erythema, induration and scaling are scored on a five-point scale as in the PASI score(range 0-4, see above). The total SUM score is defined as the sum of theindividual scores of erythema, induration and scaling reflecting a ‘local PASIscore’. Ensued, this score ranges between 0-12 wherein 0 stands for ‘no pso-riasis’ and 12 for ‘maximal signs of psoriasis’. The PASI, PGA and SUM score each are very practical to couple them with specific laboratory parameters, as was done in several investigations com-posing this thesis.

1.7.2 Laboratory parameters

Besides the clinical parameters, laboratory techniques were performed. In order to investigate the expression and enzyme activity of CD26 / DPPIV in human skin and to evaluate the effect of several anti-psoriatic therapies onT-cell-, epidermal proliferation- & differentiation and endothelial markers,4 mm punch biopsies were taken from patients and healthy volunteers. Sub-sequently, these were snap frozen and ultimately processed for immuno-fluorescent, immunohistochemical and enzymehistochemical stainingsand analyses. Also, epidermal sheets were isolated and used for RNA extrac-tion in order to perform a qPCR to determine the epidermal CD26 / DPPIV expression on mRNA-level. The exact procedures of these techniques are described in the individual manuscripts. The used primary antibodies per technique have been summarized in table 5.In order to assess the T-cell expression of CD26 / DPPIV in peripheral blood

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67

of psoriatic patients and healthy volunteers, flow cytometry was used. Flowcytometry is a method that enables analysis and quantitative measurement of specific cell characteristics. In chapter 3.3 & 3.4, lymphocytes were single,double and triple stained with fluorescent dyes binding to designated sur-face markers on T-cells. Table 5 incorporates the used antibodies.

1.7.3 Quantification methods

A semi quantitative 4-point scoring method was used to evaluate the epi-dermal keratinocyte expression of CD26 / DPPIV on protein level (immuno histochemistry) and enzyme activity level (enzyme histochemistry). For this purpose, sections were analyzed by microscopy at 200x magnification as-sessing the full width of 4 mm. The semi quantitative score could range

Table 5 Primary antibodies used for immunohistochemical staining techniques as well as for flow cytometry.(CD= cluster of differentiation, FITC= fluorescein isothiocynate, PC7= phycoerythrin-Cy7,PC5= phycoerythrin-Cy5, PE= phycoerythrin, BC= Beckman Coulter, BD= Becton Dickinson, IHC= immunohistochemistry, FCM= flow cytometry)

68

from no staining, to slight, to moderate or to pronounced staining. The quantity of CD26+ T-cells was assessed, choosing a region of interest (ROI) on the merged digital images of the sections treated with the immuno-fluorescence staining technique. In this ROI the number of lymphocytesand specifically the CD26+ T-lymphocytes were counted. The digital imageswere taken with an objective magnification of 400x. The proportion of CD26+ T-cell populations in peripheral blood was deter-mined, using specific software for flow cytometric analyses (WinList; VeritySoftware House Inc,USA)In order to quantify T-cell subsets in psoriatic skin sections, microscopy at 200x magnification was used. The number of positive cells for CD3, CD4,CD8, CD25, CD45RO, CD45RA, CD94 and CD161 were manually counted across the whole section in the epidermis from the basement membrane up to the stratum corneum; in the dermis these were counted from the basement membrane down to 100µm beneath. These quantifications wereexpressed as “number of positive cells per mm skin length”. The endothelial (CD31) and proliferation- and differentiation markers (Ki-67and K16) were evaluated on digital images using IP-lab software (Scana-lytics, USA). The CD31, Ki-67 and K16 markers were expressed as “% positive surface area of the ROI”. For the exact methods of quantification, the reader is referred to the cor-responding manuscripts in this thesis.

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1.2.

3.

4.

5.

6.7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

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26.

27.

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29.30.

31.

32.

33.34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.47.

48.

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type III procollagen. Br.J.Dermatol. 2005; 152: 451-8.Lowe NJ, Lazarus V, Matt L. Systemic retinoid therapy for psoriasis. J.Am.Acad.Dermatol. 1988; 19: 186-91.Heydendael VM, Spuls PI, Opmeer BC et al. Methotrexate versus cyclosporine in moder-ate-to-severe chronic plaque psoriasis. N.Engl.J.Med. 2003; 349: 658-65.Griffiths CE, Clark CM, Chalmers RJ et al. A systematic review of treatments for severepsoriasis. Health Technol.Assess. 2000; 4: 1-125.Schwekendiek W. Treatment of psoriasis vulgaris. Med.Monatsschr. 1959; 13: 103-4.Treumer F, Zhu K, Glaser R et al. Dimethylfumarate is a potent inducer of apoptosis in human T cells. J.Invest Dermatol. 2003; 121: 1383-8.Gerdes S, Shakery K, Mrowietz U. Dimethylfumarate inhibits nuclear binding of nuclear factor kappaB but not of nuclear factor of activated T cells and CCAAT/enhancer bind-ing protein beta in activated human T cells. Br.J.Dermatol. 2007; 156: 838-42.Skaria AM, Schmid U. Antipsoriatic effect of fumaric acid derivates. J.Am.Acad.Derma-tol. 1996; 34: 323-4.Altmeyer PJ, Matthes U, Pawlak F et al. Antipsoriatic effect of fumaric acid derivatives.Results of a multicenter double-blind study in 100 patients. J.Am.Acad.Dermatol. 1994; 30: 977-81.Ormerod AD, Mrowietz U. Fumaric acid esters, their place in the treatment of psoriasis. Br.J.Dermatol. 2004; 150: 630-2.Gordon KB, Papp KA, Hamilton TK et al. Efalizumab for patients with moderate to se-vere plaque psoriasis: a randomized controlled trial. JAMA 2003; 290: 3073-80.Boehncke WH, Prinz J, Gottlieb AB. Biologic therapies for psoriasis. A systematic review. J.Rheumatol. 2006; 33: 1447-51.Kormeili T, Lowe NJ, Yamauchi PS. Psoriasis: immunopathogenesis and evolving immu-nomodulators and systemic therapies; U.S. experiences. Br.J.Dermatol. 2004; 151: 3-15.Carey W, Glazer S, Gottlieb AB et al. Relapse, rebound, and psoriasis adverse events: an advisory group report. J.Am.Acad.Dermatol. 2006; 54: S171-S181.Koo J, Khera P. Update on the mechanisms and efficacy of biological therapies for pso-riasis. J.Dermatol.Sci. 2005; 38: 75-87.Golda N, Benham SM, Koo J. Rebound of psoriasis during treatment with efalizumab. J.Drugs Dermatol. 2006; 5: 63-5.Chong BF, Wong HK. Immunobiologics in the treatment of psoriasis. Clin.Immunol. 2007; 123: 129-38.Gottlieb AB, Chamian F, Masud S et al. TNF inhibition rapidly down-regulates multiple proinflammatory pathways in psoriasis plaques. J.Immunol. 2005; 175: 2721-9.Tyring S, Gordon KB, Poulin Y et al. Long-term safety and efficacy of 50 mg of etaner-cept twice weekly in patients with psoriasis. Arch.Dermatol. 2007; 143: 719-26.Mease PJ, Goffe BS, Metz J et al. Etanercept in the treatment of psoriatic arthritis andpsoriasis: a randomised trial. Lancet 2000; 356: 385-90.Paller AS, Siegfried EC, Langley RG et al. Etanercept treatment for children and adoles-cents with plaque psoriasis. N.Engl.J.Med. 2008; 358: 241-51.Goffe B, Cather JC. Etanercept: An overview. J.Am.Acad.Dermatol. 2003; 49: S105-S111.Tyring S, Gottlieb A, Papp K et al. Etanercept and clinical outcomes, fatigue, and depres-sion in psoriasis: double-blind placebo-controlled randomised phase III trial. Lancet 2006; 367: 29-35.Krishnan R, Cella D, Leonardi C et al. Effects of etanercept therapy on fatigue and symp-toms of depression in subjects treated for moderate to severe plaque psoriasis for up to 96 weeks. Br.J.Dermatol. 2007; 157: 1275-7.Goedkoop AY, Kraan MC, Picavet DI et al. Deactivation of endothelium and reduction in angiogenesis in psoriatic skin and synovium by low dose infliximab therapy in com-bination with stable methotrexate therapy: a prospective single-centre study. Arthritis

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RG van Lingen, JEM Körver, PCM van de Kerkhof, MAM Berends, DWA van Rens, AM Langewouters, JBM Boezeman, MMB Seyger, EMGJ de JongRelevance of compartmentalization of T-cell subsets for clinical improvement in psoria-sis: effect of immune targeted anti-psoriatic therapiesBr J Dermatol. 2008;159: 91-96.RG van Lingen, EMGJ de Jong, MAM Berends, MMB Seyger, PEJ van Erp, PCM van de KerkhofGood clinical response to anti-psoriatic treatment with adalimumab and methotrexate does not inflict a direct effect on compartmentalization of T-cell subsets: A pilot studyJ Dermatol Treatment. 2008; Apr 29: 1-6

J de Leeuw, R van Lingen, H Both, B Tank, T Nijsten, M NeumannA comparative study on the efficacy of treatment with 585 nm Pulsed Dye Laser andUltraviolet-B TL01 in plaque type psoriasisDerm Surg. 2008; In Press.RG van Lingen, EMGJ de Jong, PEJ van Erp, WSEC van Meeteren, PCM van de Kerkhof, MMB SeygerNd:YAG laser (1064 nm) fails to improve localized plaque type psoriasis: a clinical and immunohistochemical pilot studyEur J Dermatol. 2008; In Press.

2.1

2.2

2.3

2.4

This Chapter was based on the following publications:

Compartmentalization of T-cell subsets in response to anti-psoriatic therapy

Vascular lasers in the treatment of plaque psoriasis

Innovative anti-psoriatic treatments: clinical observations and interference with T-cell subsets

Chapter 2

88

Chapter 2

RG van Lingen, JEM Körver, PCM van de Kerkhof, MAM Berends, DWA van Rens, AM Langewouters, JBM Boezeman, MMB Seyger, EMGJ de JongRelevance of compartmentalization of T-cell subsets for clinical improvement in psoriasis: effect of immune targeted anti-psoriatic therapiesBr J Dermatol. 2008;159: 91-96.

2.1



89

CLINICAL AND LABORATORY INVESTIGATIONS DOI 10.1111/j .1365-2133.2008.08618.x

Relevance of compartmentalization of T-cell subsetsfor clinical improvement in psoriasis: effect ofimmune-targeted antipsoriatic therapiesR.G. van Lingen, J.E.M. Korver, P.C.M. van de Kerkhof, M.A.M. Berends, D.W.A. van Rens, A.M. Langewouters,J.B.M. Boezeman, M.M.B.Seyger and E.M.G.J. de Jong

Department of Dermatology, Radboud University, Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands

CorrespondenceRosanne G. van Lingen.

E-mail: [email protected]

Accepted for publication5 February 2008

Key wordscompartmentalization, peripheral blood, psoriasis,

skin, T cells

Conflicts of interestProf. Dr P.C.M. van de Kerkhof has received funds

for research, fees for educational events and fees for

consultancy from the following companies: Schering

Plough, Cellgene, Centocor, Almirall, UCB,

Wyeth, Pfizer, Sofinova, Abbott, Actelion,

Galderma, Novartis, Janssen Cilag, Leo Pharma,

Merck Serono, Philips Lighting. Dr E.M.G.J. de

Jong serves as a consultant for Biogen, Serono,

Wyeth, and Abbott. Dr E.M.G.J. de Jong receives

research grants from Schering Plough, Abbott,

Merck Serono, Wyeth, and Centocor.

Summary

Background Therapies targeting the T cell-mediated pathology of psoriasis havebeen found to achieve remarkable clinical improvement and have confirmed thecrucial role of the immune system either in peripheral blood (PB) or in skin. Noanalyses of T-cell counts in both compartments have been conducted in order toconfirm or refute the hypothesized shifts between them.Objectives To gain more insight in the dynamics of compartmentalization of T cellsbetween PB and lesional skin of patients with psoriasis, in response to immune-targeted antipsoriatic therapies.Methods Eighteen patients with psoriasis received either efalizumab (n = 9) oretanercept (n = 9) for 12 weeks. Biopsies were taken for immunohistochemicalanalysis of T-cell subsets and simultaneously T-cell subsets were isolated from PBspecimens by flow cytometry.Results The Psoriasis Area and Severity Index declined significantly after 12 weeksof etanercept, but not for efalizumab. After treatment with efalizumab, a signi-ficantly decreased number of all T-cell subsets was found in the dermis. In theepidermis, CD4+, CD8+, CD25+, CD45RO+ and CD161+ T-cell subsets weresignificantly decreased. With respect to etanercept, few significant changes inT-cell subsets were found. The percentage of lymphocytes in PB was significantlyelevated after efalizumab treatment regardless of responder status.Conclusions Treatment with efalizumab establishes successful recompartmentaliza-tion of T-cell subsets with modest clinical efficacy after 12 weeks, whereas inetanercept-treated patients, a significant clinical response is no guarantee forsignificant changes in T-cell subsets in the different compartments. Reductions inT-cell subsets cannot be used as predictive markers for the clinical response totherapy. Interference with the studied T-cell populations in its own right seemsnot to be responsible for the clinical efficacy of efalizumab and etanercept.

Psoriasis is one of the most common human skin diseases. It

varies greatly in severity. For several decades it has been

suggested to be a T cell-mediated immune disease. Various

biological therapies that target various aspects of this T cell-

mediated pathology have given remarkable improvement in

patients with severe plaque-type psoriasis. These treatments

confirm the crucial role of the immune system in psoriasis,1,2

either at the systemic level and ⁄or in the skin. Several biolo-

gics have been approved by the U.S. Food and Drug Adminis-

tration or the European Medicines Agency and have become a

valuable treatment option for severe psoriasis.

Studies addressing the T cell in psoriasis have been widely

conducted, focusing either on the dermal or the periph-

eral blood (PB) compartment. So far, the hypothesized

dynamic shifts of T cells between these two compart-

ments have remained underexplored as no studies have car-

ried out simultaneous analyses of T-cell counts in both

compartments.

The present study aims at mapping the dynamics of psoria-

sis-associated T-cell subsets in PB and lesional skin of patients

with severe psoriasis during the course of treatment with

immune-targeted therapies. By investigating both systemic and

� 2008 The Authors

Journal Compilation � 2008 British Association of Dermatologists • British Journal of Dermatology 2008 159, pp91–96 91

90

cutaneous T-cell subsets, it is possible to gain more insight in

the recompartmentalization of T cells in response to these

biologics.

Efalizumab is registered for severe psoriasis in the European

Union (EU). It is a humanized monoclonal antibody against

CD11a that blocks leucocyte function antigen-1 interactions

with cellular adhesion molecules 1 and 2, inhibiting T-lym-

phocyte activation and T-cell trafficking. As a result, move-

ment of T cells into dermal and epidermal tissues and their

activation and reactivation are prevented. Accordingly,

efalizumab is a T cell-targeted treatment. The efficacy of

efalizumab in psoriasis has been reported in large clinical

trials.3–5

Etanercept is the other biological agent which has been reg-

istered for several years for psoriasis in the EU. It is a fusion

protein which consists of the p75 portion of the tumour

necrosis factor (TNF)-a receptor linked to an IgG construct. It

binds specifically to TNF which is a proinflammatory cytokine

and thereby blocks its interaction with cell-surface TNF recep-

tors, rendering TNF biologically inactive. In psoriasis, the

inflammatory response is amplified as a result of increased

TNF-a expression in psoriatic plaques. Studies and reports of

daily clinical practice have indicated the efficacy of this bio-

logical agent in psoriasis.6–11

In the current investigation, our objective was to investigate

the dynamics of psoriasis-associated T-cell subsets in PB and

lesional skin of patients with severe plaque-type psoriasis

through the course of treatment with immune-targeted thera-

pies, i.e. efalizumab and etanercept. In particular, we formu-

lated the following questions. (i) Are T-cell counts and

subsets affected in response to biological therapies? (ii) Is

recompartmentalization of T cells between PB and lesional

dermis and lesional epidermis induced by antipsoriatic treat-

ments and how does this relate to clinical efficacy? (iii) Can

T-cell subset counts in blood or skin be used as a prognostic

marker for clinical efficacy?

Therefore, we performed immunohistochemical stainings

for designated psoriasis-associated T-lymphocyte subsets on

psoriatic skin sections and at the same time we isolated

T-lymphocyte subsets from PB specimens by flow cytometry

using fluorochrome-conjugated monoclonal antibodies. In

particular, we quantified the number of T-cell subsets in PB

and the number of T cells in dermis and epidermis of the

psoriatic lesion and have correlated these counts with the

clinical severity of psoriasis.

Materials and methods

Patients

A total of 18 patients with severe plaque-type psoriasis [Psori-

asis Area and Severity Index (PASI) > 10] participated in this

12-week study. At random nine received efalizumab (five men

and four women; mean ± SEM age 51Æ3 ± 4Æ6 years and dis-

ease duration 19Æ8 ± 4Æ1 years) at a dosage regimen of

1 mg kg)1 weekly after an initial dose of 0Æ7 mg kg)1 weekly,

and nine received etanercept (six men and three women;

mean ± SEM age 53Æ7 ± 2Æ8 years and disease duration

22Æ4 ± 4Æ3 years) at a dosage regimen of 2 · 25 or 2 ·50 mg weekly (respectively four and five patients due to

changes in the label of etanercept). Approval of the medical

ethics committee was obtained and all patients had given writ-

ten informed consent prior to any study procedures. Any topi-

cal and systemic antipsoriatic treatment had been suspended

for at least 2 weeks and 4 weeks, respectively, prior to partici-

pation. Furthermore, patients did not use any medication

interfering with disease activity of psoriasis (e.g. systemic cor-

ticosteroids, b-blockers, lithium carbonate, antimalarial medi-

cation and immunosuppressants) nor did they have any other

inflammatory dermatoses. Patients were allowed to use topical

antipsoriatic agents on a maximum of five lesions such as vita-

min D derivatives or low ⁄moderate strength topical steroids

(except on lesions designated for biopsy) in cases when the

effect of efalizumab or etanercept was unsatisfactory (non-

responders).

Biopsies and clinical assessment

One solitary plaque of at least 3 cm in diameter was chosen as

a target lesion. In all patients 4-mm punch biopsies were

taken over time, from representative sites in the target lesion

at least 1 cm from the margin of the psoriatic plaque, after

anaesthesia with 1% xylocaine ⁄adrenaline: one at baseline,

one after 4 weeks of treatment and one after 12 weeks of

treatment. Optionally skin defects were closed with one

suture.

At each visit severity scores (PASI) for overall severity of

psoriasis and SUM scores (for the severity of the target

lesion) were assessed by one single assessor. The SUM score

comprises the total score for erythema (0–4), induration

(0–4) and scaling (0–4); conversely, the PASI encompasses

the total body area involvement as well (range 0–72).

Venepuncture

PB for enumeration of lymphocytes was obtained from all

participating patients in 3-mL vacutainer tubes containing

ethylenediaminetetraacetate at baseline immediately before

administration of any medication, after 4 weeks of treatment

and finally after 12 weeks.

Immunohistochemical staining

Immunohistochemical staining was performed on all obtained

biopsies as previously described.12 In short, biopsies were

embedded in Tissue Tek OCT compound (Miles Scientific,

Napierville, IL, U.S.A.), snap frozen and stored at )80 �C until

use. After blocking endogenous peroxidase, sections were

washed in phosphate-buffered saline. Subsequently, sections

were incubated with the primary antibodies for 1 h. The

following primary antibodies (mouse antihuman) were

used: anti-CD3 (1 : 100; clone UCTH1), anti-CD4 (1 : 200;


Journal Compilation � 2008 British Association of Dermatologists • British Journal of Dermatology 2008 159, pp91–96

92 Compartmentalization of T cells in psoriasis, R.G. van Lingen et al.

Chapter 2


91

cutaneous T-cell subsets, it is possible to gain more insight in

the recompartmentalization of T cells in response to these

biologics.

Efalizumab is registered for severe psoriasis in the European

Union (EU). It is a humanized monoclonal antibody against

CD11a that blocks leucocyte function antigen-1 interactions

with cellular adhesion molecules 1 and 2, inhibiting T-lym-

phocyte activation and T-cell trafficking. As a result, move-

ment of T cells into dermal and epidermal tissues and their

activation and reactivation are prevented. Accordingly,

efalizumab is a T cell-targeted treatment. The efficacy of

efalizumab in psoriasis has been reported in large clinical

trials.3–5

Etanercept is the other biological agent which has been reg-

istered for several years for psoriasis in the EU. It is a fusion

protein which consists of the p75 portion of the tumour

necrosis factor (TNF)-a receptor linked to an IgG construct. It

binds specifically to TNF which is a proinflammatory cytokine

and thereby blocks its interaction with cell-surface TNF recep-

tors, rendering TNF biologically inactive. In psoriasis, the

inflammatory response is amplified as a result of increased

TNF-a expression in psoriatic plaques. Studies and reports of

daily clinical practice have indicated the efficacy of this bio-

logical agent in psoriasis.6–11

In the current investigation, our objective was to investigate

the dynamics of psoriasis-associated T-cell subsets in PB and

lesional skin of patients with severe plaque-type psoriasis

through the course of treatment with immune-targeted thera-

pies, i.e. efalizumab and etanercept. In particular, we formu-

lated the following questions. (i) Are T-cell counts and

subsets affected in response to biological therapies? (ii) Is

recompartmentalization of T cells between PB and lesional

dermis and lesional epidermis induced by antipsoriatic treat-

ments and how does this relate to clinical efficacy? (iii) Can

T-cell subset counts in blood or skin be used as a prognostic

marker for clinical efficacy?

Therefore, we performed immunohistochemical stainings

for designated psoriasis-associated T-lymphocyte subsets on

psoriatic skin sections and at the same time we isolated

T-lymphocyte subsets from PB specimens by flow cytometry

using fluorochrome-conjugated monoclonal antibodies. In

particular, we quantified the number of T-cell subsets in PB

and the number of T cells in dermis and epidermis of the

psoriatic lesion and have correlated these counts with the

clinical severity of psoriasis.


Patients

A total of 18 patients with severe plaque-type psoriasis [Psori-

asis Area and Severity Index (PASI) > 10] participated in this

12-week study. At random nine received efalizumab (five men

and four women; mean ± SEM age 51Æ3 ± 4Æ6 years and dis-

ease duration 19Æ8 ± 4Æ1 years) at a dosage regimen of

1 mg kg)1 weekly after an initial dose of 0Æ7 mg kg)1 weekly,

and nine received etanercept (six men and three women;

mean ± SEM age 53Æ7 ± 2Æ8 years and disease duration

22Æ4 ± 4Æ3 years) at a dosage regimen of 2 · 25 or 2 ·50 mg weekly (respectively four and five patients due to

changes in the label of etanercept). Approval of the medical

ethics committee was obtained and all patients had given writ-

ten informed consent prior to any study procedures. Any topi-

cal and systemic antipsoriatic treatment had been suspended

for at least 2 weeks and 4 weeks, respectively, prior to partici-

pation. Furthermore, patients did not use any medication

interfering with disease activity of psoriasis (e.g. systemic cor-

ticosteroids, b-blockers, lithium carbonate, antimalarial medi-

cation and immunosuppressants) nor did they have any other

inflammatory dermatoses. Patients were allowed to use topical

antipsoriatic agents on a maximum of five lesions such as vita-

min D derivatives or low ⁄moderate strength topical steroids

(except on lesions designated for biopsy) in cases when the

effect of efalizumab or etanercept was unsatisfactory (non-

responders).


One solitary plaque of at least 3 cm in diameter was chosen as

a target lesion. In all patients 4-mm punch biopsies were

taken over time, from representative sites in the target lesion

at least 1 cm from the margin of the psoriatic plaque, after

anaesthesia with 1% xylocaine ⁄adrenaline: one at baseline,

one after 4 weeks of treatment and one after 12 weeks of

treatment. Optionally skin defects were closed with one

suture.

At each visit severity scores (PASI) for overall severity of

psoriasis and SUM scores (for the severity of the target

lesion) were assessed by one single assessor. The SUM score

comprises the total score for erythema (0–4), induration

(0–4) and scaling (0–4); conversely, the PASI encompasses

the total body area involvement as well (range 0–72).

Venepuncture

PB for enumeration of lymphocytes was obtained from all

participating patients in 3-mL vacutainer tubes containing

ethylenediaminetetraacetate at baseline immediately before

administration of any medication, after 4 weeks of treatment

and finally after 12 weeks.


Immunohistochemical staining was performed on all obtained

biopsies as previously described.12 In short, biopsies were

embedded in Tissue Tek OCT compound (Miles Scientific,

Napierville, IL, U.S.A.), snap frozen and stored at )80 �C until

use. After blocking endogenous peroxidase, sections were

washed in phosphate-buffered saline. Subsequently, sections

were incubated with the primary antibodies for 1 h. The

following primary antibodies (mouse antihuman) were

used: anti-CD3 (1 : 100; clone UCTH1), anti-CD4 (1 : 200;




clone MT310), anti-CD8 (1 : 200; clone C8 ⁄144B), anti-CD25(1 : 200; clone ACT-1), anti-CD45RO (1 : 100; clone UCHL1),

anti-CD45RA (1 : 200; clone 4KB5) and anti-CD94 (1 : 100;

clone HP-3D9) all from Dako (Copenhagen, Denmark) and

anti-CD161 (1 : 100; clone 191B8) from Immunotech

(Marseille, France). Secondary IgG-labelled polymer, horse-

radish peroxidase antimouse antibody EnVision+ (Dako), was

added for 30 min. To visualize the staining we applied amino-

ethylcarbazole+ high sensitivity substrate chromogen (Dako)

for 10 min at 37 �C. Counterstaining was performed with

Mayer’s haematoxylin (Sigma-Aldrich, St Louis, MO, U.S.A.).

Subsequently, the sections were mounted in glycerol gelatin

(Sigma-Aldrich).

Furthermore, from each patient we performed a haematoxylin

and eosin staining to verify that the biopsies under study

fulfilled the psoriasis-specific histological criteria.

Quantification of T-cell subsets in psoriatic skin

Quantification of the T-cell subsets in the sections was per-

formed as previously described, with light microscopy at

· 200 magnification.13 Quantitative cell counts of the various

T-cell subsets were expressed as positive cells mm)1 skin

length. The assessor was blinded to treatment and differed

from the one performing the clinical assessments.

Flow cytometry and data analysis

The following monoclonal antibodies were used for flow

cytometry: CD3-FITC (Beckman Coulter, Hileah, FL, U.S.A.),

CD4-PE (Dako), CD8-PECy5 (Beckman Coulter), CD25-PECy5

(Becton Dickinson, Franklin Lakes, NJ, U.S.A.), CD45RO-

ECD (Beckman Coulter), CD45RA-FITC (Becton Dickinson),

CD94-PE (Beckman Coulter) and CD161-PE (Beckman

Coulter).

Before flow cytometric analyses all erythrocytes were

lysed (10 min, in the dark) by means of ice-cold

155 mmol L)1 NH4Cl (pH 7Æ4) according to the lyse ⁄nowash method. Staining of cells was determined in four-col-

our analysis by using an Epics XL flow cytometer (Beckman

Coulter). All measurements were performed on minimally

50 000 cells. Data (collected in list mode) were analysed

blinded to treatment by the same assessor for the immuno-

histochemical quantifications, using the EXPO32 ADC soft-

ware (Beckman Coulter).

Statistical analysis

All analyses were performed using SPSS software, version

14.0 (SPSS, Chicago, IL, U.S.A.). Analysis of variance with

repeated measurement was used to analyse the effect of

treatment and time. Additional contrasts in the time-related

effects of the treatments (between baseline and 4 weeks of

treatment and between 4 and 12 weeks of treatment) were

analysed with paired t-tests. Statistical significance was set

at 0Æ05.

Results

Patient population and clinical severity

Eighteen patients aged at least 18 years participated; their

demographics are presented in Table 1. The mean ± SEM PASI

and SUM score at onset of the various treatments together

with the interval scores (4 weeks) and the percentage change

after 12 weeks of treatment are also presented in Table 1. The

PASI declined significantly after 12 weeks of treatment with

etanercept, but not with efalizumab (P = 0Æ669) which did

not reach a statistically significant improvement. In addition,

the mean SUM score for efalizumab and etanercept decreased

significantly. All biopsies fulfilled the disease-specific histo-

logical criteria for psoriasis.

Compartmentalization of T-cell subsets after 12 weeks

of treatment with efalizumab

Regarding the PB compartment in patients treated with

efalizumab, the CD8+ T lymphocytes were significantly ele-

vated after 12 weeks whereas the CD94+ and CD161+ T-cell

amounts were significantly decreased in this study (Table 2).

Remarkably, the percentage of lymphocytes and the leucocyte

count in PB were both significantly elevated after 12 weeks.

With respect to the dermis we found a significantly decreased

number of all T-cell subsets (CD3+, CD4+, CD8+, CD25+,

CD45RA+, CD45RO+, CD94+ and CD161+ T cells) and

regarding the epidermis, the CD4+, CD8+, CD25+, CD45RO+

and CD161+ T cells were significantly decreased.

Clinically the psoriatic lesions had a significantly lower

SUM score; however, the PASI had not decreased significantly

after 12 weeks of efalizumab. The efalizumab patient group

was split into responders (PASI reduction > 50%) and non-

responders to therapy (PASI reduction £ 50%) in order to

investigate these findings further. Although we found four

Table 1 Demographics and clinical parameters of participatingpatients with psoriasis

Efalizumab Etanercept

Number 9 9Dosage regimen 1Æ0 mg kg)1 2 · 25 ⁄2 · 50 mg

weeklyAge (years) 51Æ3 ± 4Æ6 53Æ7 ± 2Æ8M ⁄F 5 ⁄4 6 ⁄3Baseline PASI 14Æ4 ± 2Æ3 18Æ9 ± 2Æ6Interval PASI (4 weeks) 15Æ1 ± 3Æ4 12Æ9 ± 2Æ2Reduction in PASI EOT (%) 11Æ1 ± 16Æ6 57Æ6 ± 12Æ1(P-value) (0Æ67) (0Æ00)Baseline SUM 6Æ8 ± 0Æ7 7Æ1 ± 0Æ5Interval SUM (4 weeks) 5Æ3 ± 0Æ7 3Æ3 ± 0Æ6Reduction in SUM EOT (%) 35Æ9 ± 10Æ4 69 ± 7Æ8(P-value) (0Æ008) (0Æ00)

PASI, Psoriasis Area and Severity Index; EOT, end of treatment(12 weeks). Results are shown as mean ± SEM.



Compartmentalization of T cells in psoriasis, R.G. van Lingen et al. 93

92

responders and five nonresponders, strikingly in both groups

a significantly increased percentage of PB lymphocytes was

detected (P < 0Æ05).


of treatment with etanercept

The PB compartment showed no significant shifts in percent-

ages of T-cell subsets after etanercept therapy (Table 2). How-

ever, after 12 weeks we found a significantly decreased

amount of CD8+, CD25+, CD45RA+ and CD161+ T cells in

the psoriatic dermis, whereas in the epidermis the CD45RO+

T-cell subset was significantly increased.

The clinical response to etanercept showed a significant

decrease on both lesional (SUM) and total body score (PASI)

(Tables 1, 2). As all patients responded well to etanercept, the

effect on T cells in responders and nonresponders could not

be compared.

Common effects of treatment with efalizumab and

etanercept on the dynamics of T-cell subsets in psoriasis

Both efalizumab and etanercept induced a decrease in the

amount of CD8+, CD25+, CD45RA+ and CD161+ T-cell sub-

sets in the psoriatic dermis, whereas no common effects on

T-cell subsets were found in the epidermis or PB.

Changes in T-cell subset counts in peripheral blood and

psoriatic skin related to clinical severity and extent

during 12 weeks of treatment

In order to determine whether shifts in psoriasis-associated

T-cell subset counts in the circulation and the psoriatic skin

are associated with the clinical severity of psoriasis (reflected

by the PASI), a comparison was made between the shifts in

the first treatment period of 4 weeks and the second period

between 4 and 12 weeks of treatment. Results are shown in

Figure 1.

Efalizumab

Figure 1(a) represents the main significant shifts in T-cell sub-

sets in all compartments and PB lymphocyte percentage during

a 12-week course of efalizumab treatment. The clinical effec-

tiveness of efalizumab, however, was not significant (PASI)

after 12 weeks.

Notably, as depicted in Figure 1(b), the PASI tended to

increase during the first 4 weeks of treatment with efalizumab

whereas the T-cell subsets in the psoriatic (epi)dermis tended

to be dropping in the first 4 weeks of treatment. In the PB,

however, there was a marked lymphocytosis in this first treat-

ment period, which seems mainly attributable to the CD8+

lymphocytes.

In the second treatment period, between 4 and 12 weeks,

the PASI tended to decrease compared with the first 4 weeks

of treatment. In this phase, the percentage of lymphocytes and

specifically of the CD8+ lymphocytes tended to decrease in

contrast to the findings in the first treatment period (Fig. 1c).

Etanercept

Figure 1(d) represents the main shifts in T-cell subsets and PB

lymphocyte percentage which occurred during a 12-week

course of etanercept treatment. In contrast to efalizumab, only

CD8 and CD25 in the dermis showed significant changes due

to the treatment with etanercept. No striking changes in T-cell

Table 2 Dynamics in psoriasis-associated T-cell subset counts during a 12-week therapy with either efalizumab or etanercept in patients withpsoriasis. P-values are given

T-cell subset counts

PB Dermis Epidermis

EFA ETA EFA ETA EFA ETA

CD3 0Æ225 0Æ275 0Æ001 . 0Æ256 0Æ086 0Æ451CD4 0Æ145 ND 0Æ001 . 0Æ078 0Æ005 . 0Æ518CD8 0Æ044 ND 0Æ001 . 0Æ041 . 0Æ011 . 0Æ053CD25 NS NS 0Æ00 . 0Æ034 . 0Æ00 . 0Æ07CD45RO NS NS 0Æ01 . 0Æ34 0Æ004 . 0Æ011CD45RA NS NS 0Æ003 . 0Æ026 . 0Æ136 0Æ275CD94 0Æ042 . ND 0Æ048 . 0Æ085 0Æ094 0Æ139CD161 0Æ042 . ND 0Æ00 . 0Æ024 . 0Æ009 . 0Æ086PASI 0Æ669 0Æ00 .

SUM 0Æ008 . 0Æ00 .

Leucocyte count 0Æ001 0Æ631Lymphocytes (%) 0Æ004 0Æ091

PB, peripheral blood; EFA, efalizumab; ETA, etanercept; PASI, Psoriasis Area and Severity Index; ., decrease during therapy; , increase

during therapy; NS, not shown; ND, not determinable.



94 Compartmentalization of T cells in psoriasis, R.G. van Lingen et al.Chapter 2


93

responders and five nonresponders, strikingly in both groups

a significantly increased percentage of PB lymphocytes was

detected (P < 0Æ05).


of treatment with etanercept

The PB compartment showed no significant shifts in percent-

ages of T-cell subsets after etanercept therapy (Table 2). How-

ever, after 12 weeks we found a significantly decreased

amount of CD8+, CD25+, CD45RA+ and CD161+ T cells in

the psoriatic dermis, whereas in the epidermis the CD45RO+

T-cell subset was significantly increased.

The clinical response to etanercept showed a significant

decrease on both lesional (SUM) and total body score (PASI)

(Tables 1, 2). As all patients responded well to etanercept, the

effect on T cells in responders and nonresponders could not

be compared.

Common effects of treatment with efalizumab and

etanercept on the dynamics of T-cell subsets in psoriasis

Both efalizumab and etanercept induced a decrease in the

amount of CD8+, CD25+, CD45RA+ and CD161+ T-cell sub-

sets in the psoriatic dermis, whereas no common effects on

T-cell subsets were found in the epidermis or PB.

Changes in T-cell subset counts in peripheral blood and

psoriatic skin related to clinical severity and extent

during 12 weeks of treatment

In order to determine whether shifts in psoriasis-associated

T-cell subset counts in the circulation and the psoriatic skin

are associated with the clinical severity of psoriasis (reflected

by the PASI), a comparison was made between the shifts in

the first treatment period of 4 weeks and the second period

between 4 and 12 weeks of treatment. Results are shown in

Figure 1.

Efalizumab

Figure 1(a) represents the main significant shifts in T-cell sub-

sets in all compartments and PB lymphocyte percentage during

a 12-week course of efalizumab treatment. The clinical effec-

tiveness of efalizumab, however, was not significant (PASI)

after 12 weeks.

Notably, as depicted in Figure 1(b), the PASI tended to

increase during the first 4 weeks of treatment with efalizumab

whereas the T-cell subsets in the psoriatic (epi)dermis tended

to be dropping in the first 4 weeks of treatment. In the PB,

however, there was a marked lymphocytosis in this first treat-

ment period, which seems mainly attributable to the CD8+

lymphocytes.

In the second treatment period, between 4 and 12 weeks,

the PASI tended to decrease compared with the first 4 weeks

of treatment. In this phase, the percentage of lymphocytes and

specifically of the CD8+ lymphocytes tended to decrease in

contrast to the findings in the first treatment period (Fig. 1c).

Etanercept

Figure 1(d) represents the main shifts in T-cell subsets and PB

lymphocyte percentage which occurred during a 12-week

course of etanercept treatment. In contrast to efalizumab, only

CD8 and CD25 in the dermis showed significant changes due

to the treatment with etanercept. No striking changes in T-cell

Table 2 Dynamics in psoriasis-associated T-cell subset counts during a 12-week therapy with either efalizumab or etanercept in patients withpsoriasis. P-values are given


PB Dermis Epidermis

EFA ETA EFA ETA EFA ETA

CD3 0Æ225 0Æ275 0Æ001 . 0Æ256 0Æ086 0Æ451CD4 0Æ145 ND 0Æ001 . 0Æ078 0Æ005 . 0Æ518CD8 0Æ044 ND 0Æ001 . 0Æ041 . 0Æ011 . 0Æ053CD25 NS NS 0Æ00 . 0Æ034 . 0Æ00 . 0Æ07CD45RO NS NS 0Æ01 . 0Æ34 0Æ004 . 0Æ011CD45RA NS NS 0Æ003 . 0Æ026 . 0Æ136 0Æ275CD94 0Æ042 . ND 0Æ048 . 0Æ085 0Æ094 0Æ139CD161 0Æ042 . ND 0Æ00 . 0Æ024 . 0Æ009 . 0Æ086PASI 0Æ669 0Æ00 .

SUM 0Æ008 . 0Æ00 .

Leucocyte count 0Æ001 0Æ631Lymphocytes (%) 0Æ004 0Æ091

PB, peripheral blood; EFA, efalizumab; ETA, etanercept; PASI, Psoriasis Area and Severity Index; ., decrease during therapy; , increase

during therapy; NS, not shown; ND, not determinable.




subset counts of the etanercept-treated patients were found in

the first (between onset and 4 weeks of treatment) or second

treatment period (between 4 and 12 weeks of treatment)

which could be related to PASI increase or decrease (data not

shown). In contrast to the modest effects on T cells, etaner-

cept had a robust clinical effect, as shown by the reduction in

PASI, in this patient group.

Discussion

Psoriasis has been suggested to be a T cell-mediated auto-

immune disease. For several decades, studies have focused on

the central role of the T lymphocyte in psoriasis. Some investi-

gators have studied T cells in skin and others in the PB com-

partment. Frequently it is hypothesized that shifting of T cells

between the skin and systemic compartment is relevant to the

pathogenesis of psoriasis. However, the alleged dynamics of

T-cell shifts between the two compartments has never been

studied simultaneously in one investigation. In order to under-

stand the mode of action of antipsoriatic therapies targeting

the immune system, gaining more insight in the compartmen-

talization of psoriasis-associated T-cell subsets during and after

such therapies seems valuable. This study analyses compart-

mentalization of T cells in the cutaneous compartments

(dermis and epidermis) and systemic compartment (PB) in

patients with psoriasis using immune-targeted therapies

(efalizumab, etanercept). Furthermore, the study correlates

T-cell counts in these compartments with clinical efficacy. The

aim was to gain more insight by asking the following ques-

tions. (i) Are T-cell counts and subsets affected in response to

biological therapies? (ii) Is recompartmentalization of T cells

between PB and lesional dermis and lesional epidermis

induced by antipsoriatic treatments and how does this relate

to clinical efficacy? (iii) Can T-cell subset counts in blood or

skin be used as a prognostic marker for clinical efficacy?

Both therapies have a different mode of action, primarily or

secondarily targeting the T cell and ultimately reflected in

some degree of clinical response. In this study the effect on

T-cell subsets of efalizumab and etanercept, the two biologicals

most frequently prescribed in daily practice, proved to be dis-

similar. Efalizumab has been shown to inhibit T-lymphocyte

activation and T-cell trafficking.3–5 This knowledge has been

confirmed and extended by our findings as efalizumab treat-

ment resulted in a significant reduction of psoriasis-relevant

T-cell subsets in the epidermal and dermal compartment while

CD8+ T cells increased in the PB after 12 weeks of therapy.

The effect of efalizumab on T-lymphocyte trafficking and in

particular the shift of T lymphocytes from the cutaneous to

the PB compartment was shown convincingly in the present

study. However, this apparent T-cell trafficking alone does not

comprise the overall mode of action of efalizumab as the

mechanism of blocking T-cell (re)activation with efalizumab

should not be underestimated.14 However, this recompart-

mentalization of T cells was not indicative for the clinical

response to efalizumab therapy as the PASI did not show a

significant decrease after 12 weeks. In order to determine

whether the recompartmentalization of psoriasis-associated

T-cell subset counts in the circulation and the psoriatic skin was

associated with the clinical severity of psoriasis, the dynamics

of changes during 12 weeks was studied by splitting the treat-

ment of 12 weeks into the first 4 weeks and 4–12 weeks.

Between onset of efalizumab treatment and 4 weeks of treat-

ment, the clinical severity of psoriasis of efalizumab-treated

patients had a tendency to increase (Fig. 1b), in contrast to

(b) (c)

(a) (d)

Fig 1. Fluctuations in T-cell subset counts in response to treatment with efalizumab or etanercept. (a) Significant changes in CD markers and

peripheral blood lymphocytes in response to a 12-week treatment with efalizumab and corresponding Psoriasis Area and Severity Index (PASI)

reduction (%). (b) Changes in CD markers and peripheral blood lymphocytes in response to efalizumab through the first 4 weeks of treatment.

(c) Changes in CD markers and peripheral blood lymphocytes in response to efalizumab between 4 and 12 weeks of treatment. (d) Changes in

CD markers and peripheral blood lymphocytes in response to a 12-week treatment with etanercept and corresponding PASI reduction (%). Data

are shown in mean percentages ± SEM. ED, epidermis; D, dermis; BL, peripheral blood; *, significantly changed (P < 0Æ05).



Compartmentalization of T cells in psoriasis, R.G. van Lingen et al. 95

94

the main psoriasis-associated T-cell subset counts in the skin

which tended to decrease and in sharp contrast to the increase

in PB T lymphocytes. Between 4 and 12 weeks T-cell counts

did not change substantially (Fig. 1c); however, the clinical

severity tended to show an initial improvement. We know

from other studies and from experience in daily clinical prac-

tice that prolonged treatment with efalizumab for 24 weeks or

more is needed for considerable clinical efficacy.6,15 There-

fore, we may conclude from the present investigation that

recompartmentalization of subsets precedes clinical effective-

ness in patients treated with efalizumab.

When comparing responders (n = 4) and nonresponders

(n = 5) to efalizumab treatment, both groups showed a com-

parable increase of PB lymphocytes. Also in the psoriatic lesion

no consistent pattern of T-cell subsets exists which may iden-

tify the responder or nonresponder status. Efalizumab induces

a lymphocytosis regardless of clinical effectiveness and reduces

T-cell subsets in the lesion regardless of clinical efficacy. As

responders and nonresponders could not be identified by the

degree of T-cell recompartmentalization, other principles

should be studied in order to understand the entire action of

efalizumab, such as the interaction between antigen-presenting

cells and T cells or the initiation of T-cell activation.

Focusing on the patient group treated with etanercept,

results were different from those of efalizumab as few signifi-

cant reductions in T-cell counts in the various compartments

could be found (Table 2). However, etanercept showed high

clinical efficacy after 12 weeks. Etanercept primarily blocks the

interaction with TNF receptors, making TNF a biologically

inactive cytokine. Secondarily, it affects the T cell by reducing

T-cell activation. The remarkable clinical efficacy of etanercept

after 12 weeks of therapy was not preceded by a recompart-

mentalization of T cells. The discrepancy between the pro-

nounced clinical improvement and the modest reduction of

T-cell subsets suggests that reductions in T-cell subsets after

etanercept therapy are not the precondition for clinical efficacy.

Both antipsoriatic agents in this study are part of the arsenal

of systemic therapies. It is reassuring that only modest changes

of the T-cell subsets in the PB (Table 2) could be detected.

Therefore we may assume that these agents mainly influence

the effector organ, the psoriatic skin.

In conclusion, comparing two major biological therapies in

this current investigation, data show that treatment with

efalizumab establishes successful recompartmentalization of

T-cell subsets with modest clinical efficacy after 12 weeks,

whereas in etanercept-treated patients, a significant clinical

response is no guarantee for significant changes in T-cell sub-

sets in the different compartments of the skin and PB. There-

fore the present study, comparing tendencies of the clinical

efficacy course with the changes in T-cell counts in the vari-

ous compartments, has provided insight in the dynamics of

compartmentalization of psoriasis-associated T-cell subsets.

Subsequently, it revealed that reductions in T-cell subsets in

the various compartments cannot be used as predictive mark-

ers for the clinical response to therapy. As a result, lack of

association between effects on T-cell subsets and clinical

effects suggests that interference with the studied T-cell popu-

lations in its own right is not responsible for the clinical effi-

cacy of efalizumab and etanercept. This undoubtedly reflects

the complexity of psoriasis pathogenesis and the presence of a

multitude of immune pathways.

References

1 Gaspari AA. Innate and adaptive immunity and the pathophysi-

ology of psoriasis. J Am Acad Dermatol 2006; 54:S67–80.2 Lowes MA, Bowcock AM, Krueger JG. Pathogenesis and therapy of

psoriasis. Nature 2007; 445:866–73.3 Gordon KB, Papp KA, Hamilton TK et al. Efalizumab for patients

with moderate to severe plaque psoriasis: a randomized controlledtrial. JAMA 2003; 290:3073–80.

4 Leonardi CL, Papp KA, Gordon KB et al. Extended efalizumab ther-

apy improves chronic plaque psoriasis: results from a randomizedphase III trial. J Am Acad Dermatol 2005; 52:425–33.

5 Papp KA, Bressinck R, Fretzin S et al. Safety of efalizumab in adultswith chronic moderate to severe plaque psoriasis: a phase IIIb,

randomized, controlled trial. Int J Dermatol 2006; 45:605–14.6 Berends MA, Driessen RJ, Langewouters AM et al. Etanercept and

efalizumab treatment for high-need psoriasis. Effects and sideeffects in a prospective cohort study in outpatient clinical practice.

J Dermatolog Treat 2007; 18:76–83.7 Enbrel (etanercept). Full Prescribing Information [Package Insert].

Thousand Oaks, CA: Immunex Corp., 2006.8 Gottlieb AB, Matheson RT, Lowe N et al. A randomized trial of

etanercept as monotherapy for psoriasis. Arch Dermatol 2003;139:1627–32.

9 Kupper TS. Immunologic targets in psoriasis. N Engl J Med 2003;349:1987–90.

10 Leonardi CL, Powers JL, Matheson RT et al. Etanercept as monother-apy in patients with psoriasis. N Engl J Med 2003; 349:2014–22.

11 Papp KA, Tyring S, Lahfa M et al. A global phase III randomizedcontrolled trial of etanercept in psoriasis: safety, efficacy, and effect

of dose reduction. Br J Dermatol 2005; 152:1304–12.12 Vissers WH, Arndtz CH, Muys L et al. Memory effector

(CD45RO+) and cytotoxic (CD8+) T cells appear early in the mar-gin zone of spreading psoriatic lesions in contrast to cells express-

ing natural killer receptors, which appear late. Br J Dermatol 2004;150:852–9.

13 Bovenschen HJ, Seyger MM, van de Kerkhof PC. Plaque psoriasisvs. atopic dermatitis and lichen planus: a comparison for lesional

T-cell subsets, epidermal proliferation and differentiation. Br JDermatol 2005; 153:72–8.

14 Boehncke WH, Ochsendorf FR, Noll S et al. Efficacy of the fullyhuman monoclonal antibody MOR102 (#5) against intercellular

adhesion molecule 1 in the psoriasis–severe combined immuno-deficient mouse model. Br J Dermatol 2005; 153:758–66.

15 Costanzo A, Peris K, Talamonti M et al. Long-term treatment of

plaque psoriasis with efalizumab: an Italian experience. Br J Dermatol2007; 156 (Suppl. 2):17–23.




Chapter 2


95

96

Chapter 2

RG van Lingen, EMGJ de Jong, MAM Berends, MMB Seyger, PEJ van Erp, PCM van de KerkhofGood clinical response to anti-psoriatic treatment with adalimumab and methotrexate does not inflict a direct effect on compartmentalization of T-cell subsets: A pilot studyJ Dermatolog Treat. 2008 Apr 29:1-6


2.2


97

ORIGINAL ARTICLE

Good clinical response to anti-psoriatic treatment with adalimumaband methotrexate does not inflict a direct effect oncompartmentalization of T-cell subsets: A pilot study

ROSANNE G. VAN LINGEN, ELKE M. G. J. DE JONG, MAARTJE A. M. BERENDS,

MARIEKE M. B. SEYGER, PIET E. J. VAN ERP & PETER C. M. VAN DE KERKHOF

Department of Dermatology, University Medical Centre St Radboud, Nijmegen, The Netherlands

AbstractObjectives: The most recently introduced therapeutics for psoriasis are biologicals which can target the T-cell-mediatedpathology of psoriasis in a direct or indirect manner. The present pilot study focuses on and compares the effect of aconventional systemic agent (methotrexate; MTX) with the effect of a TNF-binding biological (adalimumab) on psoriasis-associated T-cell subsets in peripheral blood (PB) and lesional skin. Insight is provided in the hypothesizedcompartmentalization of these T-cell subsets between PB and the cutaneous compartment. Methods: Immuno-histochemical stainings of designated T-cell subsets on psoriatic skin sections were performed and similar subsets wereisolated from PB specimens by flow cytometry. These counts were correlated with clinical severity. Results: Results showedthat adalimumab had a greater clinical effect than MTX treatment after 12 weeks. In the dermis, only the CD3+ T cellswere significantly reduced after 12 weeks of adalimumab therapy, whereas for MTX only CD3+ T cells in the epidermis andCD45RO+ T cells in the dermis reduced significantly. However, PB T-lymphocyte populations did not show significantshifts in quantification of T-cell subsets. Conclusion: Therefore, recompartmentalization of psoriasis-associated T-cellsubsets between PB and lesional skin was not induced in this study as a therapeutic principle. Consequently,recompartmentalization of T-cell subsets does not seem an obligatory event in order to achieve good clinical response.

Key words: Adalimumab, methotrexate, psoriasis, (re)compartmentalization, T cell

Introduction

T cells belong to the key aspects of the psoriatic

pathogenesis. The most recent therapeutics intro-

duced in the treatment of moderate to severe plaque

psoriasis are the biologicals, of which each in its own

way affects the T cell to a certain extent. They have

become a valuable treatment option in the ther-

apeutic arsenal as large clinical trials and daily

practice have shown great clinical efficacy for the

majority of severely affected psoriatic patients. As

biologicals can target the T-cell-mediated pathology

of psoriasis in a direct or indirect manner, it seems

warranted to investigate this effect on the T cells in

psoriasis.

Convincingly, the most widely prescribed conven-

tional systemic agent for several decades for severe

psoriasis is methotrexate (MTX). MTX has potent

anti-inflammatory effects on T-cell-mediated

immune responses as it primarily blocks abnormal

rapid epidermal cell proliferation in psoriasis and

inhibits proliferation or induces apoptosis in acti-

vated T cells (1). Recently registered for the treat-

ment of severe plaque-type psoriasis in the EU is the

biological adalimumab, which is a fully human

immunoglobulin G1 monoclonal antibody binding

with high affinity and specificity to ;TNF (2). Upon

binding to TNF it can modulate biological responses

that are induced or regulated by TNF, subsequently

affecting the T cell, and is reported to be highly

effective and safe in the treatment of severe psoriasis

(3–5).

The current investigation comprised a satellite

study to the Phase III CHAMPION trial (6),

focusing on and comparing the effect of a conven-

tional systemic agent (MTX) with the effect of a

TNF-binding biological (adalimumab) on psoriasis-

associated T-cell subsets in peripheral blood and

lesional skin of patients with moderate to severe

plaque psoriasis. Further light was shed on the T-cell

Journal of Dermatological Treatment jdt150941.3d 19/4/08 16:57:57The Charlesworth Group, Wakefield +44(0)1924 369598 - Rev 7.51n/W (Jan 20 2003) 295701

Correspondence: Rosanne G. van Lingen, Department of Dermatology, University Medical Centre St Radboud, PO Box 9101, NL – 6500 HB Nijmegen,

The Netherlands. Fax: 31 24 3541184. E-mail: [email protected]

(Received 25 January 2008; accepted 29 January 2008)

Journal of Dermatological Treatment. 2008; 000: 1–4

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ISSN 0954-6634 print/ISSN 1471-1753 online # 2008 Informa UK Ltd.

DOI: 10.1080/09546630801955358

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dynamics in the pathogenesis of psoriasis in order

to define whether generally hypothesized shifting

of T cells between the skin and the systemic

compartment is achieved in response to both

therapies. In the end, by comparing these two

treatments in a pilot study, preliminary insight is

provided in the effect of a conventional (MTX) and

a novel (adalimumab) therapy on the T cells in the

cutaneous and systemic compartment in psoriatic

patients and consequently in the hypothesized

compartmentalization of psoriasis-associated T-cell

subsets between peripheral blood and lesional

dermis and epidermis.

Therefore, we performed immunohistochemical

stainings for designated psoriasis-associated

T-lymphocyte subsets on psoriatic skin sections

and at the same time we isolated T-lymphocyte

subsets from peripheral blood specimens by flow

cytometry using fluorochrome-conjugated mono-

clonal antibodies. In particular, we have quantified

the number of T-cell subsets in peripheral blood and

the number of T cells in the dermis and epidermis of

the psoriatic lesion and have correlated these counts

with the clinical severity of psoriasis.

Patients and methods

Patients

A total of eight patients with moderate to severe

plaque-type psoriasis (Psoriasis Area and Severity

Index (PASI) w10) participated in this 12-week

study. Approval of the medical ethics committee was

obtained and each patient provided written informed

consent prior to any study-related procedures. Four

patients received 40 mg adalimumab subcuta-

neously every other week and four received MTX

per os in an increasing dosing regimen from 7.5 mg

up to 25 mg per week. Any topical or systemic

treatment had been suspended for at least 2 weeks

and 4 weeks respectively prior to participation.

Furthermore, patients did not use any medication

interfering with the disease activity of psoriasis

nor were any concomitant anti-psoriatic therapies

allowed during the study. The specific study proto-

col and study design are described elsewhere (6).


One solitary plaque of at least 3 cm in diameter was

chosen as a target lesion. In all patients 4-mm punch

biopsies were taken over time from representative

sites in the target lesion at least 1 cm from the

margin of the psoriatic plaque after anaesthesia with

1% xylocaine/adrenaline: one at baseline (T50) and

one after 12 weeks of treatment (T512). Optionally,

skin defects were closed with one suture. At each

visit severity scores (PASI) for the overall severity of

psoriasis were assessed.

Venapuncture

Peripheral blood for enumeration of lymphocytes

was obtained from all participating patients in 3-ml

vacutainer tubes containing ethylenediaminetetra-

acetate at baseline immediately before any medica-

tion administration (T50) and finally after 12 weeks

of treatment (T512).


Immunohistochemical staining was performed on all

obtained biopsies as previously described (7,8). The

following primary antibodies (mouse anti-human)

were used: anti-CD3 1:100 (pan T-cell marker)

(clone UCTH1), anti-CD4 1:200 (T-helper cells)

(clone MT310), anti-CD8 1:200 (cytotoxic T cells)

(clone C8/144B), anti-CD25 1:200 (activated T

cells) (clone ACT-1), anti-CD45RO 1:100 (memory

T cells) (clone UCHL1), anti-CD45RA 1:200

(naive T cells) (clone 4KB5) and anti-CD94 1:100

(NK-T cells) (clone HP-3D9) all from DAKO

(Copenhagen, Denmark) and anti-CD161 1:100

(NK-T cells) (clone 191B8) from Immunotech

(Marseille, France). Furthermore, we performed a

H&E staining to verify that the biopsies under study

from each patient fulfilled the psoriasis-specific

histological criteria.

Quantification of T-cell subsets in psoriatic skin

Quantification of the T-cell subsets in the sections

was enforced as previously done with light micro-

scopy at 2006 magnification (7,8). Quantitative cell

counts of the various T-cell subsets were expressed

as ‘positive cells per mm skin length’.

Flow cytometry and data analysis

The following monoclonal antibodies were used for

flow cytometry: CD3-FITC (Beckman <Coulter),

CD4-PE (DAKO), CD8-PECy5 (Beckman

Coulter), CD25-PECy5 (Becton Dickinson),

CD45RO-ECD (Beckman Coulter), CD45RA-

FITC (Becton Dickinson), CD94-PE (Beckman

Coulter) and CD161-PE (Beckman Coulter).

Flow cytometric analyses were performed by the

Hematology Laboratory of the Radboud University

Nijmegen Medical Centre which is certified for

analyzing peripheral blood specimens for diagnostic

use. Before flow cytometric analyses, all erythrocytes

were lysed (10 min, in the dark) by means of ice-

cold 155 mmol/l NH4Cl (pH 7.4) according to the

lyse/no-wash method. Staining of cells was deter-

mined in four-colour analysis by using a Beckman

Coulter Epics XL flow cytometer (Beckman

Coulter, Hileah, FL, USA). All measurements were

performed on a minimum of 50 000 cells. Data

(collected in list mode) were analyzed using the

EXPO32 ADC software (Beckman Coulter).


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All analyses were performed using SPSS software,

version 14.0. Analysis of variance with repeated

measurement (ANOVA) was used to analyze the

effect of treatment and time. Statistical significance

was set at 0.05.

Results

Patient population and clinical severity

In total, eight patients of at least 18 years old

participated; for whom the demographics are

expanded in Table I. The mean PASI¡SEM at

onset of the two treatments, together with the

percentage reduction after 12 weeks of treatment

are also given in Table I. Overall, baseline PASI

scores were comparable in range. Although the

patient groups were somewhat small, both therapies

showed to be clinically effective as the PASI scores

reduced significantly after 12 weeks of both thera-

pies, albeit to a different extent. Treatment with

adalimumab evoked a PASI reduction of 84.5%

(pv0.05) in contrast to 48.4% (pv0.05) in the

MTX group. Therefore, statistically significantly

more patients in the adalimumab group than in the

MTX group achieved a remarkable PASI reduction.

All biopsies fulfilled the disease-specific histologi-

cal criteria of psoriasis.

Compartmentalization of T-cell subsets after treatment

with adalimumab or MTX (Table II)

Table II reflects an overview of the changes in the

designated psoriasis-associated T-cell subsets after

12 weeks of therapy. After six injections of adalimu-

mab (every other week), only the CD3+ T cells in

the dermis reduced significantly, whereas for MTX

only the CD3+ T cells in the epidermis and the

CD45RO+ T cells in the dermis reduced signifi-

cantly. Although both adalimumab and MTX

possess a systemic mode of action, in the peripheral

blood neither adalimumab nor MTX induced a

significant change in T cells in general (CD3+) or in

specific T-cell subsets (data not shown).

Discussion

In line with earlier findings (6), the present pilot

study shows that adalimumab therapy results in

greater clinical efficacy than treatment with MTX

after 12 weeks. Namely, a mean PASI reduction of

84.5% was achieved in the patients treated with

adalimumab versus 48.4% in patients treated with

MTX. It should be noted, however, that daily

practice teaches that probably the greatest clinical

response to therapy with MTX is likely to be

achieved after a more lengthy treatment period.

Nevertheless, both PASI reductions showed to be

statistically significant after 12 weeks. However, this

good clinical response to treatment with adalimu-

mab and MTX did not obviously inflict a major

direct effect on compartmentalization of T cells in

the peripheral blood and lesional dermis and

epidermis of these patients. Immunohistochemical

analyses of psoriasis-associated T-lymphocyte sub-

sets on psoriatic skin sections before and after 12

weeks of therapy with either adalimumab or MTX

did reveal several significant reductions in T-cell

subset counts but did not lead to alleged compart-

mentalization. However, flow cytometric analyses in


Table I. Demographics and clinical characteristics of participat-

ing psoriatic patients.

Adalimumab Methotrexate

Number of patients 4 4

Age, years 46.8¡3.5 34.8¡4.6

Female / male 1/3 1/3

Baseline PASI 26.5¡5.5 24.2¡6.4

EOT PASI 4.2¡1.3 13.1¡5

PASI-reduction %

(p-value)

284.5¡3.1 (0.016) 248.4¡9.6 (0.018)

Dosage regimen 40 mg/2 weeks 7.5–25 mg/week

Maximum doses 1 patient: 15 mg/week

1 patient: 20 mg/week

2 patients: 25 mg/week

EOT5end of treatment; PASI5psoriasis area and severity score.

Table II. Dynamics in designated T-cell subset counts in psoriatic patients during 12 weeks of therapy with adalimumab or methotrexate.


Compartment Dermis Epidermis

Therapy ADA MTX ADA MTX

CD3 0.02. 0.328 0.154 0.018.

CD4 0.065 ?0.072 0.259 0.784

CD8 0.377 0.06 0.058 0.051

CD25 0.235 0.618 0.159 0.304

CD45RO 0.457 0.002. 0.066 0.062

CD45RA 0.149 0.397 0.161 0.22

CD94 0.283 0.239 0.182 0.464

CD161 0.064 0.531 0.154 0.499

Data presented as p-values. ADA5adalimumab; MTX5methotrexate; .5significant decrease during therapy.

T-cell response to treatment with adalimumab & MTX 3

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peripheral blood specimens before and after therapy

failed to reveal significant shifts in quantification of

T-cell subsets. Therefore, the present study does not

lend support for a recompartmentalization of T-cell

subsets between the peripheral blood and lesional

skin as a therapeutic principle in psoriasis. Yet, even

though the PASI score in the two patient groups was

shown to reduce significantly, it must be noted that

both patient groups were rather small.

Although the exact mode of action of conventional

therapy with MTX is not fully elucidated, it is well

known that it has potent anti-inflammatory effects

on T-cell-mediated immune responses as it primarily

blocks abnormal rapid epidermal cell proliferation in

psoriasis and inhibits proliferation or induces apop-

tosis in activated T cells (1). Further advances in the

knowledge about the effect of MTX on psoriatic key

players can enrich the understanding of the exact

mechanism of action. Jointly with the significant

clinical response, it may be suggested that MTX

ultimately influences lesional, but not peripheral

blood T cells, as our data show that treatment with

MTX had a significant reducing effect on CD3+ T

cells in the epidermis and memory-effector T cells

(CD45RO+) in the lesional dermis and no signifi-

cant effect on peripheral blood specimens.

In contrast with the findings of the T-cell subset

counts after MTX therapy, only the CD3+T-lymphocyte population in the lesional dermis

was reduced by adalimumab therapy. The peripheral

blood specimens once again did not differ signifi-

cantly after 12 weeks of therapy. It is attractive to

speculate that primarily targeting TNF mainly seems

to influence the CD3+ T cell population in the

psoriatic dermis after 12 weeks of treatment with the

TNF-antagonist adalimumab. Surprisingly, in con-

trast with other effective biologic anti-psoriatic

therapies (9,10), in this study the CD45RO+ T-cell

subset was not significantly affected in both the

cutaneous and systemic compartments by treatment

with adalimumab.

Consequently, as no significant changes could be

found in the peripheral blood after both therapies,

no insight could be provided in the present study

concerning the hypothesized compartmentalization

of psoriasis-associated T-cell subsets between per-

ipheral blood and lesional dermis and epidermis.

To encapsulate, adalimumab and MTX have been

given a place in the treatment of moderate to severe

plaque psoriasis, although acting through a different

mechanism of action indirectly targeting one of the

major pathogenetic key players in psoriasis, the T

cell. Although a relatively small number of patients

could be evaluated, both therapies were shown to

influence several designated T-cell subsets modestly

in the cutaneous compartment. In the peripheral

blood specimens, T-lymphocyte populations were

not significantly altered after both therapies.

Therefore, in this pilot study, alleged recompart-

mentalization of T-cell subsets between the systemic

and cutaneous compartments in response to therapy

with adalimumab and MTX could not be brought to

light in contrast with the remarkable clinical effec-

tiveness. Consequently, compartmentalization of

T-cell subsets does not seem an obligatory event in

order to achieve good clinical response. However,

in the end, the mode of action of MTX and

adalimumab does not imply a direct effect on the

T cell, but the present data show that specific

lesional T-cell subsets can indeed be affected by

these therapies.

References

1. Genestier L, Paillot R, Fournel S, Ferraro C, Miossec P,

Revillard JP. Immunosuppressive properties of methotrexate:

Apoptosis and clonal deletion of activated peripheral T cells. J

Clin Invest. 1998;102:322–8.

2. Calabrese LH. Molecular differences in anticytokine thera-

pies. Clin Exp Rheumatol. 2003;21:241–8.

3. Pitarch G, Sanchez-Carazo JL, Mahiques L, Perez-

Ferriols MA, Fortea JM. Treatment of psoriasis with

adalimumab. Clin Exp Dermatol. 2007;32:18–22.

4. Menter A, Tyring SK, Gordon K =, et al. Adalimumab therapy

for moderate to severe psoriasis: A randomized, controlled

phase III trial. J Am Acad Dermatol. 2007.

5. Papoutsaki M, Chimenti MS, Costanzo A, et al. Adalimumab

for severe psoriasis and psoriatic arthritis: An open-label study

in 30 patients previously treated with other biologics. J Am

Acad Dermatol. 2007;57:269–75.

6. Saurat JH, Stingl G, Dubertret L >, et al. Efficacy and safety

results from the randomized controlled comparative study of

adalimumab vs. methotrexate vs. placebo in patients with

psoriasis (CHAMPION). Br J Dermatol. 2007.

7. Bovenschen HJ, Seyger MM, Van de Kerkhof PC. Plaque

psoriasis vs. atopic dermatitis and lichen planus: A com-

parison for lesional T-cell subsets, epidermal proliferation and

differentiation. Br J Dermatol. 2005;153:72–8.

8. Vissers WH, Arndtz CH, Muys L, Van Erp PE, de Jong EM,

Van de Kerkhof PC. Memory effector (CD45RO+) and

cytotoxic (CD8+) T cells appear early in the margin zone of

spreading psoriatic lesions in contrast to cells expressing

natural killer receptors, which appear late. Br J Dermatol.

2004;150:852–9.

9. Bovenschen HJ, Gerritsen WJ, van Rens DW, Seyger MM, de

Jong EM, Van de Kerkhof PC. Explorative immunohisto-

chemical study to evaluate the addition of a topical

corticosteroid in the early phase of alefacept treatment for

psoriasis. Arch Dermatol Res. 2007;298:457–63.

10. Langewouters AM, Bovenschen JH, de Jong EM, Van

Erp PE, Van de Kerkhof PC. The effect of topical

corticosteroids in combination with alefacept on circulating

T-cell subsets in psoriasis. J Dermatolog Treat. 2007;18:

279–85.


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J de Leeuw, R van Lingen, H Both, B Tank, T Nijsten, M NeumannA comparative study on the efficacy of treatment with 585 nm Pulsed Dye Laser and Ultraviolet-B TL01 in plaque type psoriasisDerm Surg. 2008; In Press.

2.3



103

A Comparative Study on the Efficacy of Treatment with 585 nmPulsed Dye Laser and Ultraviolet B-TL01 in Plaque TypePsoriasis

JAAP DE LEEUW, MD,*§ ROSANNE G. VAN LINGEN,¶ MD, HILDE BOTH,* MD, BHUPENDRA TANK, PHD, *TAMAR NIJSTEN, MD, PHD,* AND H. A. MARTINO NEUMANN, MD, PHD *

BACKGROUND Narrow-band ultraviolet-B and pulsed dye laser (PDL) affect psoriasis but via different pathways.

OBJECTIVE To compare the results of PDL with ultraviolet-B light therapy (UVB) and to look for syner-gism of both therapies in patients with plaque type psoriasis.

METHODS In each eligible individual, four similar target plaques were selected, and halves of these plaques were treated using PDL, UVB, or a combination of PDL and UVB or were not treated. Results were recorded single-blind using the Physician’s Global Assessment score at study enrolment and week 13. Nonparametric, paired statistical tests were used to test for differences within and between therapies.The results were also analyzed after dichotomization of the changes in the Physician’s Global Assessment score into responsive and nonresponsive to treatment.

RESULTS A significant improvement of the psoriasis lesions was noted at Week 13 (P0.001) with eachtherapy. No significant differences were noted between the therapies. Synergism of PDL and UVB was notobserved.

CONCLUSIONS PDL is safe for treating plaque type psoriasis, but its efficacy is limited to a subgroup ofpatients. Combining PDL with UVB has no additional benefit.

The authors have indicated no significant interest with commercial supporters.

an intra-dermal and intra-epidermal inflammatoryinfiltrate composed mainly of T cells, accumulationof neutrophil granulocytes, increased numbers of dendritic cells and mast cells, and expansion of the superficial dermal vasculature histologically charac-terize psoriasis.1 In normal skin, the superficial mi-crovasculature is composed of capillary loops that arise from terminal arterioles in the upper horizontal dermal vascular plexus, pass up into the dermal pa-pillae, and arch back to connect with the postcapil-lary venules in the horizontal plexus.2

Nonlesional skin has short lengths of microvessels

in the superficial, papillary dermis, whereas dilatedand elongated superficial capillaries passing into thedermal papillae, representing exaggerated tortuos-ity and coiling of the apical segment of the capillary loop, characterize lesional psoriatic skin.3,4 The en-dothelium of the superficial microvasculature in le-sional skin is four times thicker than in normal skin but not in the deeper vasculature, indicating that microvascular expansion is restricted to the upper plexus.5 Although present evidence indicates that psoriasis is primarily a T cell–dependent disease, the prominent increase in the dermal microvasculaturein lesional skin indicates that psoriasis is also angio-genesis-dependent.6–8

pidermal hyperproliferation with epidermal thickening, parakeratosis and hyperkeratosis,E

* Department of Dermatology and Venereology, Erasmus MC, University Medical Center, Rotterdam, the Netherlands;§ ZBC MultiCare, outpatient clinic for Dermatology, Hilversum, the Netherlands; ¶ Department of Dermatology andVenereology, University Medical Center St. Radboud, Nijmegen, the Netherlands

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Most traditional therapies for psoriasis have focused on the inhibition of epidermal proliferation and sup-pression of inflammation. Limiting factors are thatnone of these treatments is effective in all patients, each may have serious side effects, and the efficacygenerally decreases over time.9,10 The alterations in the capillaries have been neglected as a possible therapeutic target, although it has been reported that, in a new psoriasis lesion, one of the earliest observ-able changes is an increase in the dermal papillary vasculature.6–8,11,12 During treatment, microvascular improvement precedes clinical improvement.13 This indicates that the expanded superficial microvascu-lar bed in psoriasis skin is an essential component for maintaining clinical lesions.13 These observa-tions have led to the hypothesis that, in psoriasis, se-lective destruction of the dilated papillary vessels by selective photothermolysis may reduce the transmi-gration of inflammatory cells, resulting in clearingof psoriasis plaques.14 Selective photothermolysis, destroying only the dilated papillary vessels, sparing the normal sized vessels and without causing dam-age to the epidermis, became an effective procedure once the pulsed dye laser (PDL) was introduced.15,16 Partial and total clearance of psoriasis after PDL treatment was reported in several studies.14,17–22

Narrow-band ultraviolet-B light therapy (UVB), which is highly efficacious against psoriasis,23–28 ap-peared to be superior to broad-band UVB therapy28,29 and nearly as effective as psoralen ultraviolet-A (PUVA) therapy, and because there are no photo-sensitizerrelated adverse reactions and a possible lower long-term cancer risk, it can be considered to be the standard light therapy.30 UVB inhibits the proliferation of keratinocytes and induces immuno-suppression. 31–34 UVB targets primarily structures in the epidermis, whereas PDL light passes through the epidermis and targets primarily the superficialvessels in the dermis, indicating that UVB and PDL clear psoriasis via different pathways.

The aim of this study was to evaluate the efficacyand the safety of PDL treatment in plaque type pso-

riasis, to compare the results of PDL treatment with the results of the UVB treatment and with those of the PDL+UVB treatment, and to look for a synergis-tic effect of PDL+UVB treatment on psoriasis.

MATERIALS AND METHODS

Subjects

Patients were recruited from the outpatient dermatol-ogy clinic of the Erasmus MC, University Medical Center, Rotterdam. Adult patients with stable plaque psoriasis with at least four psoriasis plaques were considered eligible. Patients who were intolerant to light (toxic or allergic), were using drugs with photo-toxic or photoallergic potency, were younger than 18 years, were pregnant, or had preexisting or manifest skin malignancy were excluded. A washout period of 2 weeks was indicated for topical drugs and of 4 weeks for photo (chemo) -therapy and -systemic drugs, except for emollients. All included patients provided written informed consent to participate in the study. The Medical Ethical Committee of theErasmus MC, University Medical Center, Rotter-dam approved this study. The study was started in October 2004 and ended in March 2006.

Materials

PDL treatment was performed using the V Star PDL (Cynosure Inc., Chelmsford, MA). The active me-dium is a Rhodamine dye that emits yellow light at a wavelength of 585 nm. During treatment, the skin was cooled with cold air from the Smart Cool Cy-nosure machine.35 PDL treatment parameters were wavelength 585 nm, pulse duration 0.450 ms, spot diameter 7 mm, spots overlapping 720%, and flu-ences between 5.5 and 6.5 J/cm2.

Ultraviolet 311-nm treatment was performed using the UVB-TL01 S (wavelength 311 nm) handheld device (Cosmedico Medizintechnik GmbH, Vil-lingen- Schwenningen, Germany). Mean irradiance was 19.4mW/cm2. During treatment, the aperture of


105

the appliance (5.0 x10.0 cm) was held against the skin, the implication being that the distance between the device and the skin was constant in every patient. Minimal erythema dose was determined by shoving a metal cover with 6 test squares (each 1 cm2) on the MultiCare device. The squares allowed the penetra-tion of 100%, 83%, 65%, 49%, 34%, and 17% of UVB-TL01 irradiance of the device (19.4mW/cm2).After 24 hours, the test squares were examined. The square showing just observable erythema was con-sidered as MED. Mean MED was 0.86 J/cm2 (range 0.34–1.2 J/cm2).

Treatment Schedule

In each patient, four plaques were randomly allocat-ed, divided into two halves (Figure 1A), and treated as shown in Figure 2. This paired approach enabled us to assess the effect of UVB, PDL, and UVB+PDL at Week 13 and to compare the effect of UVB and PDL with control sides in plaque b and in plaque c. The sides of plaques a and b that were not treated with UVB were protected with metal foil during ex-posure. Plaque d was treated with the combination of UVB and PDL (PDL+UVB) on both sides, and the effect was compared with that of UVB (plaque b) and PDL (plaque c). Therefore, four plaques were available for blind observation: two plaques treated on both sides (plaque a, plaque d) and two plaques treated on one side (plaque b, plaque c). In some patients, the psoriatic lesions were too small to di-

vide into two halves. In these cases, two separate, comparable, nearby plaques were selected as sub-stitutes (Figures 3–5). The four target plaques were selected, as much as possible, on symmetrical sites of the right arm, the left arm, the right leg, and the left leg. A clockwise rotation schedule of the various above-mentioned treatments, of plaque -a through plaque d, was used in which, in every new included patient, the schedule started on a different body site. The remaining plaques in patients who presented with more than the four study plaques were treated as follows: half of the body with UVB-TL01 and the other half with PDL, following a rotation model according to the sequence of inclusion.

Figure 2. Treatment schedule of 4 plaques in each patient.PDL = Pulsed Dye Laser, UVB = Ultraviolet-B TL01, PDL+UVB = combination therapy, NT = no treatment

Figure 1. One plaque divided into two halves on the left elbow.

A: Week 0 PGA-score III = 4, IV = 4 Treatment : III = PDL, IV = NT

B: Week 13 PGA-score III = 0 after PDL PGA-score IV = 1 after NT

PDL improvement 4 PGA levels NT improvement 3 PGA levels

Note : Considerable improvement of NT side adjacent to PDL side

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Patients were treated with PDL every 3 weeks for 10 weeks - in total four treatments (Table 1). During laser treatment, the patient’s skin was cooled with cool air to reduce the pain and to prevent damage to the melanocytes due to overheating.

UVB treatment parameters were wavelength 311 nm, start dose 70% of the MED, dose increments each treatment 20% of previous treatment until erythema

threshold or clearing of the lesion was reached, three weekly treatments for 11 weeks (in total 30–33 treat-ments) (Table 2).36,37 Mean initial dose was 0.62 J/cm2 (range 0.24–0.86 J/cm2), cumulative dose 84 J/cm2 (range 40–120 J/cm2).

Concomitant topical treatment with salicylic acid 5% in petrolatum was permitted for all plaques to reduce scattering of the laser beam by scales but was discontinued 2 days before PDL treatment.

Clinical Assessment

Apecialized dermatological nurses recorded clini-cal results single-blind using the Physician’s Global Assessment (PGA) score38 (Table 1) at Week 13. This moment was chosen 2 weeks after cessation of the UVB treatment and 3 weeks after the last PDL treatment (ensuring that the postlaser purpura had totally disappeared) (Table 1). Although two PGA scores were recorded for the PDL+UVB–treated le-sions (plaque d), to maintain blind assessment, we restricted the analysis to the right side of the treated plaques.

Definition of Response to Treatment

An improvement of 3 points or more from base-line on the PGA score (maximum score 5) or total

� �

Figure 3. Two, almost confluent plaques on the dorsumof the left hand.A: Week 0 : PGA-score VII = 3, VIII = 3Treatment : VII = PDL, VIII = NT

B: Week 13: PGA-score VII = 1 after PDLPGA-score VIII = 2 after NT

PDL improvement 2 PGA levels, NT improvement 1 PGA levelNote : Some improvement of NT side near PDL side.

Figure 4. Two confluent plaquesright lower leg.

A: Week 0 :PGA-score VII = 4, VIII = 4Treatment : VIII = PDL, VII = UVBB: Week 13: PGA-score VIII = 0 after PDLC: Week 13: PGA-score VII = 1 after UVB

PDL improvement 4 PGA levelsUVB improvement 3 PGA levels

Note: The UVB treated area shows more residual scaling than the PDL treated area. Side effect from both PDL and UVB: hyperpigmentation.

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clearance (PGA score of 0) in plaque sides selected for this study was defined as response to treatment.

Statistical Analyses

The Friedman test was used to test for statistically significant differences in the change of PGA scoresof the UVB-, PDL-, and PDL+UVB–treated plaques at Week 13. We also assessed the difference in the degree of PGA score change fter each of these thera-pies in different plaques within the same patient. For UVB and PDL, change in PGA score was compared with the control side of the same plaque at Week 13. For each of these paired statistical tests, the Wilcox-on signed rank test was used. Good clinical response (change of 3 levels of PGA score or clear at Week 13) between the three therapy regimens was com-pared using the McNemar test.

RESULTS

Study Population

In this single-center, single-blind, prospective, paired randomized controlled study 27 patients (17 men and 10 women) aged 20 to 65 (mean 44.4) with

skin types I to IV and stable plaque psoriasis with at least four psoriasis plaques were included (in total 108 plaques). The mean duration of psoriasis was 14 years (range 4–35 years). The distribution of the baseline PGA scores in each of the treatment groups was comparable (Figure 6).

Direct Paired Comparison Between Base-line and Week 13

The PGA scores after treatment with UVB, PDL, and PDL+UVB were significantly lower at Week 13(P0.001) (Table 3 and Figure 6). For each therapy, in approximately 20% of plaques, no improvement was achieved, more than 60% showed improve-ment of two levels in PGA score, and less than 20% showed improvement of three or more levels in PGAscore (Figure 7). No statistical differences were observed in treatment-induced PGA score changes between these three therapies (Table 3). A compar-ison between PGA scores of the UVB- and PDL-treated halves and the control halves of the psoriasis plaques showed no significant differences (P = .47and .42, respectively). In plaque a, a clearly visible difference between UVB treatment and PDL treat-ment was seen in six patients in favor of UVB and in four patients in favor of PDL (Figure 4).

Analyses of PGA Scores in the Subgroup of Plaques That Responded to Treatment

Good clinical response (improvement of 3 points in PGA score from baseline or clearing of psoriasis)

Figure 5. Two small plaques sep-arated from each other by non-psoriatic skin right upper leg.A: Week 0 : PGA-score V = 3, VI = 3Treatment : V = NT, VI = PDL

B: Week 13: PGA-score V = 3 after NT

C: Week 13:PGA-score VI = 1 after PDL

PDL improvement 2 PGA levelsNT improvement 0 PGA levels

� ��

Note : No improvement of NT side separated from PDL side by non-psoriatic skin. Side-effect from PDL treatment: hyperpigmentation.

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was achieved in seven (25.9%) UVB-treated, nine (33.3%) PDL-treated, five (18.5%) PDL+UVB–treated, and two (7.4%) nontreated halves of the 27 psoriasis plaques.

Total clearance (PGA score of 0) was achieved in the UVB-treated sides of five (18.5%) plaques, inthe PDL-treated sides of five (18.5%) plaques, in thePDL+UVB–treated side of two (7.4%) plaques, and in a nontreated side of one (3.7%) plaque. In one pa-

tient, total clearance was obtained after PDL mono-therapy and UVB monotherapy. Total clearance was achieved after PDL treatment in four plaques and after UVB treatment also in four plaques.

Side Effects of Treatment

Regarding side effects of the PDL therapy, all pa-tients reported transient purpura (occasionally ac-companied by crusts) for 1 to 2 weeks, 20 (74%) re-ported moderate discomfort (which is considerably reduced by cooling the skin during treatment), and 20 (74%) reported evident but transient hyperpigmen-tation after therapy. Although hypopigmentation and atrophic scarring were reported in some studies,16,18 these side effects were neither reported in a recent study22 nor observed in our study. Regarding side effects of the UVB treatment, nine (33%) patients reported pain, 15 (55%) reported photo-dermatitis, and 20 (74%) reported some hyperpigmentation. Regarding side effects of PDL+UVB therapy, all patients reported purpura, 17 (63%) reported pain, 13 (48%) reported photo-dermatitis, and 19 (70%) reported hyperpigmentation.

DISCUSSION

Recent breakthroughs in the treatment of psoriasis have led to better understanding of the pathogen-esis of this disease.34,39,40 Although major advances in the development and use of targeted biologicals for controlling psoriasis have been made, the need to develop safer and more cost-effective cures remains. Potential targets for future drugs are cytokines in-volved in T-cell activation and T-cell trafficking.40 Inhibition of immunocyte trafficking by selectivephotothermolysis of the elongated and dilated papil-lary blood vessels by PDL was demonstrated to be efficacious in plaque type psoriasis.13,16–22 The PDL was developed on the concept of selective absorp-tion of a brief radiation pulse that generates and confines heat to oxyhemoglobin, resulting in the

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haemoglobin to met-haemoglobin, which reduces the 585 nm light absorption and increases that of 595 nm compared with native oxy-haemoglobin, both caused by heating of the blood during irradiation. The remaining small disparity between 585 nm and 595 nm can easily be corrected by augmenting the fluence. Therefore, similar results may be obtainedwith the wavelengths of 585 nm and 595 nm. The purpura level for both wavelengths is a good indica-tor of the proper vascular damage thathas been achieved.43

According to the principles of photothermolysis the optimal pulse-duration to treat dilated capillary vessels in a psoriasis plaque is considered to be in the range of 0.450- to 1.5-msec (less or equal to the Thermal Relaxation Time of the vessels),18 which is short enough to damage the dilated psoriasis vessels and long enough to spare the normal-sized capillary blood vessels.

The optimal spot-diameters for destroying psoriasis vessels are 5- and 7-mm. The 7-mm spot diameter has the advantage that the number of pulses required to treat the clinical area is 35% lower than when treating with the 5-mm spot diameter, reducing the time of treatment, and that the penetration of the la-ser light is slightly deeper.44

The beam profile of the PDL machine has a Gaus-sian-like distribution of energy, resulting in consid-erable more light energy in the centre of the beam compared with the border.45 In order to obtain a uniform pattern of applied energy, overlapping of the spots by about 20% is imperative. From this point of view it is likely that without overlapping, the inhibition of the immunocyte trafficking in pso-riasis is sub-optimal resulting in decreased clinical responses.

In order to have a correct simultaneous compari-son between the results of UVB and PDL, the end-point of the study was chosen to be just after +/- 30 treatments with UVB, which is a standard number of treatments, and 3 weeks after 4 treatments with PDL. A universal evidence-based standardized pro-tocol concerning time-intervals for PDL therapy is not yet available.

The PGA-scoring system was chosen instead of the PASI-scoring system, because in the observation of individual plaques the percentage of the total surface area involvement is not an issue, the PGA-scoring is far less time-consuming than the PASI-scoring, and also because the blinded observers (research nurses) were better trained in scoring the PGA than the PASI. In addition it has been shown, that PGA and PASI


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are highly correlated and share a high overall reliability.38

In the statistical data analyses of all included patients PDL and UVB, as well as the combination therapy PDL+UVB resulted in a significant improvement(P0.0001 for all 3 treatment modalities) in the PGA scores at T13 compared with T0 (Figure 6, Table 3). However, there were no statistically significantdifferences between the three treatment modalities mutually (Table 3). After dichotomising the changes in PGA scores into responsive and non-responsive to treatment, PDL scored better than UVB and UVB better than PDL+UVB, but according to the McNe-mar’s test these differences were not statistically significant. This indicates that in terms of response,PDL was statistically as effective as UVB treatment, but there was no synergistic effect of combining PDL treatment with UVB treatment in the sub-group of patients who responded to treatment. A specula-tive explanation for this lack of synergism may be that the inhibition of vessel-active cytokines pro-duced by keratinocytes and immunocytes by UVB-treatment normalises the diameters of the papillary blood vessels whereby these vessels no longer form a target for PDL. A total clearance of psoriasis af-ter both UVB and PDL was seen in only 1 patient. Complete clearance after UVB, but not after PDL was achieved in 4 patients and after PDL, but not after UVB also in 4 patients, indicating that patients who do not respond to UVB may respond to PDL and vice versa.

The non-treated sides of the study plaques also showed an unexpectedly good response. The differ-ences between the UVB-treated and the nontreated sides, and between the PDL-treated and the non-treated sides were not significant (P = .47 and P = .42,respectively). A putative explanation for this phe-nomenon is that the non-treated sides could have benefited from the UVB- and the PDL treatment ofthe neighbouring sides, although in a previous study it was suggested that there was no carry-over effect between the side treated with PDL and the non-treated side.18 In contrast, the differences between

the improvements reflected in the PGA scores ofthe NT sides seen in the Figures 1, 3 and 5 indi-cate an inverse relationship between improvement and the distance between PDL treated sides and NT sides, indicating an effect of PDL in a limited area around the irradiated site. This observation is sup-ported by Babilas et al., who demonstrated tissue damage and thrombus formation in- and also out-side the irradiated area of hamster skin 24 hours af-ter PDL treatment.46 Although the carry-over effect in PDL warrants further study, our findings indicatethat overlapping spot-sizes may not be necessary in the PDL treatment of psoriasis. This implies mul-tiple benefits such as a reduction in the duration oftreatment, less discomfort to the patients and lower costs. Currently, we are investigating this possibility in a separate immunohistochemical study using skin biopsies obtained from the participating patients in this investigation. Other possible mechanisms by which the non-treated sides could have benefitedare via a systemic effect of the treatment of the re-maining plaques with UVB and PDL, and/or a non-expended good effect of the permitted concomitant treatment with salicylic acid ointment. A spontane-ous remission was not likely, because the majority of the patients (19) were treated during the winter.

Taibjee et al.47 conducted a trial on the treatment oflocalised plaque psoriasis in which the efficacyof the excimer laser was compared with the ef-ficacy of the PDL laser (wavelength 595 nm, spotdiameter 7-mm, pulse width 1.5msec and fluence10 J/cm2). They reported a complete clearance in 41% of the patients in the excimer group after 17 treatments and in 27% of the patients in the PDL group, requiring 3.2 treatments. Many patients in that study found PDL to be a very convenient treat-ment modality because of monthly hospital visits. Side effects caused by the excimer were blistering in 68% and hyperpigmentation in 41% of the pa-tients versus 0% blistering and 9% hyperpigmenta-tion caused by the PDL47 Those results are in good agreement with the results presented here. Dur-ing the laser treatment cooling of the skin is im-perative in order to reduce the pain, to protect the

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melanocytes in the epidermis from damage and to prevent activation of epidermal effector T cells via pericapillary heat diffusion after treatment, which will tend to promote the persistence of clinical lesions.20

Better results of PDL treatments may be obtained by increasing the number of sessions, reducing the time intervals between treatments, increasing the fluenceand the pulse width to respectively 10 J/cm2 and 1.5 msec (Taibjee et al.47), applying mineral oil imme-diately prior to PDL treatment and by concomitant treatment with calcipotriol ointment.48

CONCLUSIONS AND LIMITATIONS

All tested treatment modalities appeared to be effica-cious in the treatment of plaque type psoriasis. There were no statistically significant differences betweenthe efficacy of PDL treatment and UVB treatment.A combination of PDL and UVB treatment had no additional value because of lack of synergism. Surprisingly no statistically significant differenceswere seen between UVB treatment and PDL treat-ment on the one hand and the non-treated sides on the other hand. There is no clear explanation for this, but an unexpected carry-over effect (although a previous study suggested otherwise18) from the neighbouring PDL- and UVB-treated sides is sup-ported by the observation of an inverse relationship between improvement of NT sides on the one hand and the distance between the PDL treated sides and the NT sides on the other hand. Another possibil-ity is a systemic effect of the treatment of remaining the plaques. After all, it seems likely that dividing of plaques is not the best approach to study the efficacyof topical therapies, but if pursued, then the treat-ment has to be strictly restricted to study plaques, without treatment of other remaining plaques. In the present study patients with a complete clearance of the target lesion appeared to be divided into either PDL responders or UVB responders, indicating that patients who do not respond to UVB may respond

to PDL and vice versa. Treatment with PDL resulted only in mild and transient side effects and therefore appeared to be safe. PDL treatment is certainly not the panacea for every psoriasis patient and should not be used in widespread psoriasis or in patients who respond adequately to simple topically ap-plied treatments. PDL treatment should be reserved for patients with persistent plaque type psoriasis, not responding to usual treatments (particularly on ‘‘non-covered body’’ sites), plaque type psoriasis complicated by side effects of medication, psoriasis palmoplantaris, psoriasis inversa, patients not willing to use topical corticosteroids or systemic treatment and patients at risk for skin cancer caused by actinic damage or immunosuppression. Most patients in our study preferred PDL therapy above UVB (20 vs 7 patients) because of the less frequent treatment ses-sions, the subjective improvement in the quality of life and the lack of reports on carcinogenesis due to PDL treatment, whereas longterm side effects in-cluding carcinogenesis caused by excimer UVB and narrow-band UVB remain uncertain. With regard to the patient’s preference and the efficacy, the PDLtreatment should be considered as a serious option in the treatment of plaque type psoriasis.

REFERENCES

1. Creamer D, Sullivan D, Bicknell R, Barker J. Angiogenesis in psoriasis. Angiogenesis 2002;5:231–6.2. Yen A, Braverman IM. Ultrastructure of the human micro-circulation: the horizontal plexus of the papillary dermis. J In-vest Dermatol 1976;66:131–42.3. Braverman IM, Yen A. Ultrastructure of the capillary loops in the dermal papillae of psoriasis. J Invest Dermatol 1977;69:53–60.4. Bull RH, Bates DO, Mortimer PS. Intravital video-capillar-oscopy for the study of the microvascular expansion in active plaque psoriasis. Br J Dermatol 1992;126:436–45.5. Creamer D, Allen MH, Sousa A, et al. Localisation of en-dothelial proliferation and microvascular expansion in active psoriasis. Br J Dermatol 1997;136:859–65.6. Pinkus H, Mehregan AH. The primary histologic lesion of se-borrhoeic dermatitis and psoriasis. J Invest Dermatol 1966;46:109–16.7. Goodfield M, Macdonald D, Hull S, Holland D, et al. In-vestigations of the ‘‘active’’ edge of plaque psoriasis: vascular proliferation precedes changes in epidermal keratin. Br J Der-matol 1994;131:808–13.


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8. Barker JNWN. Pathophysiology of psoriasis. Lancet 1991;338:227–30.9. Lebwohl M. A clinician’s paradigm in the treatment of pso-riasis. J Am Acad Dermatol 2005;53:s59–69.10. Macdonald D, Hull S, Goodfield M, et al. Active and inac-tive edges of psoriatic plaques: identification by tracings andinvestigation by laser-Doppler flowmetry and immunocyto-chemical techniques. J Invest Dermatol 1989;92:782–5.11. Heng MCY, Allen SG, Haberfelde GH, Song MK. Elec-tronmicroscopic and immunohistochemical studies of the se-quence of events in psoriatic plaque formation following tape-stripping. Br J Dermatol 1991;125:548–56.12. Yen A, Braverman IM. Ultrastructure of the human micro-circulation: the horizontal plexus of the papillary dermis. J In-vest Dermatol 1976;66:131–42.13. Hacker S, Rasmussen J. The effect of flash lamp-pulseddye laser on psoriasis. Arch Dermatol 1992;128:853–5.14. Anderson RR, Parrish JA. Microvasculature can be selec-tively damaged using dye lasers: a basic theory and experimen-tal evidence in human skin. Lasers Surg Med 1981;1:263–76.15. Anderson RR, Parrish JA. Selective photothermolysis: pre-cise microsurgery by selective absorption of pulsed radiation. Science 1983;220:524–7.16. Ros AM, Garden JM, Bakus AD, et al. Psoriasis response to the pulsed dye laser. Lasers Surg Med 1996;19:331–5.17. Katugampola GA, Rees AM, Lanigan SW. Laser treatment of psoriasis. Br J Dermatol 1995;133:909–13.18. Zelickson BD, Mehregan DA, Wendelschfer-Crabb, et al. Clinical and histologic evaluation of psoriatic plaques treat-ed with a flashlamp pulsed dye laser. J Am Acad Dermatol1996;35: 64–8.19. Bjerring P, Zachariae H, Sogaard H. The flashlamp-pumpedDye Laser and dermabrasion in Psoriasis:Further studies on the reversed Köbner phenomenon. Acta Dermatol Venereol (Stockholm) 1997;77:59–61.20. Hern S, Allen MH, Sousa AR, et al. Immunohistochemi-cal evaluation of psoriatic plaques following selective pho-tothermolysis of the superficial capillaries. Br J Dermatol2001;145:45–53.21. Hern S, Stanton AWB, Mellor RH, et al. In vivo quanti-fication of the structural abnormalities in psoriatic microves-sels before and after pulsed dye laser treatment. Br J Dermatol 2005;152: 505–11.22. Erceg A, Bovenschen HJ, Kerkhof van der PCM, Seyger MMB. Efficacy of the pulsed dye laser in the treatment of lo-calized recalcitrant plaque type psoriasis: a comparative study. Br J Dermatol 2006;155:110–4.23. Parrish JA, Jaenicke KF. Action spectrum for phototherapy of psoriasis. J Invest Dermatol 1981;76:359–62.24. Van Weelden H, DeLaFaille HB, Young E, et al. A new de-velopment in UVB phototherapy for psoriasis. Br J Dermatol 1988;119:11–9.25. Picot E, Picot-Debeze MC, Meunier L, et al. Narrow-band UVB phototherapy (Philips TL-01) in psoriasis. Ann Dermatol Venereol 1992;119:639–42.26. Green C, Ferguson J, Lakshmipathi T, Johnson BE. 311 nm UVB phototherapyFan effective treatment for psoriasis. Br J Dermatol 1988;119:691–6.27. Ferguson J. The use of narrowband UV-B (Tube Lamp) in the management of skin disease. Arch Dermatol 1999;135:589–90.28. Walter IB, Burack LH, Coven TR, et al. Suberythema-togenic narrow-band UVB is markedly more effective than conventional UVB treatment of psoriasis vulgaris. J Am Acad

Dermatol 1999;40:893–900.29. Coven TR, Burack LH, Gilleandeau R, et al. Narrowband UV-B produces superior clinical and histopathological resolu-tion of moderate-to-severe psoriasis in patients compared with broadband UVB. Arch Dermatol 1997;133: 1514–22.30. Tanew A, Radakovic-Fijan S, Schemper M, Höningsmann H. Narrowband UVB phototherapy vs photochemotherapy in the treatment of chronic plaque type psoriasis. A paired com-parison study. Arch Dermatol 1999;135:519–24.31. Aubin F. Mechanisms involved in ultraviolet light-induced immunosuppression. Eur J Dermatol 2003;13:515–23.32. Ozawa M, Ferenczi K, Kikuchi T, et al. 312-nanometer Ul-traviolet B Light (Narrow-Band UVB) induces apoptosis of T cells within psoriatic lesions. J Exp Med 1999;189:711–8.33. Hofer A, Fink-Puches R, Kerl H, Wolf P. Comparison of phototherapy with near vs. far erythemogenic doses of narrow-band ultraviolet B in patients with psoriasis. Br J Dermatol 1998;138: 96–100.34. Lebwohl M. Psoriasis. Lancet 2003;361:1197–204. 35. Tan OT, Morrison P, Kurban AK. 585nm for the treatment of port-wine stains. Plast Reconst Surg 1990;86:1112–7.36. Dawe RS, Wainwright NJ, Cameron H, Ferguson J. Nar-row-band (TL-01) ultraviolet B phototherapy for chronic plaque psoriasis: three times or five times weekly treatment?Br J Dermatol 1998;138:833–9.37. Wainwright NJ, Dawe RS, Ferguson J. Narrowband ultra-violet B (TL-01) phototherapy for psoriasis: which incremen-tal regimen? Br J Dermatol 1998;139:410–4.38. Langley RG, Ellis CN. Evaluating psoriasis with psoriasis area and severity index, psoriasis global assessment, and latice system physician’s global assessment. J Am Acad Dermatol 2004;51: 563–9.39. Krueger G, Ellis CN. Psoriasis-recent advances in under-standing its pathogenesis and treatment. J Am Acad Dermatol 2005;53:s94–100.40. Nickoloff BJ, Stevens SR. What have we learned in der-matology from the biologic therapies? J Am Acad Dermatol 2006;54: s143–51.41. Kimel S, Svaasand LO, Hammer-Wilson MJ, Stuart Nelson J. Influence of wavelength on response to laser photothermo-lysis of blood vessels: implications for port wine stain laser therapy. Lasers Surg Med 2003;33:288–95.42. van Gemert MJ, Smithies DJ, Verkruysse W, et al. Wave-lengths for port wine stain laser treatment: influence of vesselradius and skin anatomy. Phys Med Biol 1997;42:41.43. Pikkula BM, Chang DW, Stuart Nelson J, Anvari B. Com-parison of 585 and 595 nm laser-induced vascular response of normal in vivo human skin. Lasers Surg Med 2005;36:117–23.44. Goldman MP, Fitzpatrick RE. Cutaneous Laser Surgery. 2nd ed. Saint Louis, MO: Mosby Inc.; 1999. p. 26.45. Goldman MP, Fitzpatrick RE. Cutaneous Laser Surgery. 2nd ed. Saint Louis, MO: Mosby Inc.; 1999. p. 25.46. Babilas P, Shafirstein G, Ba¨umler W, et al. Selective photo-thermolysis of blood vessels following flashlamp-pumped pulseddye laser irradiation: in vivo results and mathematical modelling are in agreement. J Invest Dermatol 2005;125:343–52.47. Taibjee SM, Cheung ST, Laube S, Lanigan SW. Control-led study of excimer and pulsed dye lasers in the treatment of psoriasis. Br J Dermatol 2005;153:960–6.48. Leeuw de J, Tank B, Bjerring J, et al. Concomitant treat-ment of psoriasis of hands and feet with pulsed dye laser andtopical calcipotriol, salicylic acid, or both prospective open study in 41 patients. J Am Acad Dermatol 2006;54:266–71.

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RG van Lingen, EMGJ de Jong, PEJ van Erp, WSEC van Meeteren, PCM van de Kerkhof, MMB SeygerNd:YAG laser (1064 nm) fails to improve localized plaque type psoriasis: a clinical and immunohistochemical pilot studyEur J Dermatol. 2008; In Press.


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Chronic and localized plaque-type-psoriasis is often therapy resistant as a result of which dermatologists are often troubled in finding a suitabletreatment option. Traditional therapies for psoriasis merely focus on the inhibition of epidermal proliferation, inflammation, or both. The earliestchanges however in a novel psoriatic lesion concern abnormal micro-vasculature. The position of lasers in the treatment of psoriatic lesions is debatable as different views exist with respect to efficacy and toler-ability. The current investigation evaluates the clinical and immunohis-tochemical effect of the Nd:YAG(1064 nm)laser in chronic localized psoriasis, as this laser can penetrate up to the deeper abnormal psoriatic vasculature. The effects are compared to treatment with the well-estab-lished calcipotriol/betamethasone dipropionate ointment. The use of the Nd:YAG laser with treatment-intervals of four weeks was found not to be of additional value in the array of treatment modalities for chronic localized plaque-psoriasis. Targeting the more superficial located mi-crovasculature in psoriasis seems of stronger significance in achievingclinical effect than the deeper vasculature targeted by the Nd:YAG laser. Therefore, the present data are of importance in preserving dermatolo-gists from treating psoriatic lesions with a Nd:YAG laser. However, fur-ther studies incorporating changes in methodology, in particular short-ened time-intervals between treatments, are needed in order to refute or confirm this position.

Key words: Nd: YAG laser – psoriasis - therapy- 1064nm

modalities, seems available for these patients to clear also these recalcitrant plaques. In general, these plaques are re-stricted to a small body surface area, which makes the choice for a systemic therapy unattractive. Therefore, an unmet need exists for an effective treatment of such chronic therapy resistant psoriatic lesions. Psoriasis is a chronic inflammatory skin disease in whichepidermal proliferation, angioneogenesis and T-cell expan-sion within the papillary dermis are the most impressive features.[1;2] Traditional therapies for psoriasis realise the inhibition of epidermal proliferation, inflammation, or both.The earliest changes noted in an early psoriatic lesion, how-ever, are elongated, widened and tortuous microvessels.[3-5] The position of lasers in dermatology has increased dur-ing the past two decades. As new laser devices continue to emerge, an expanding range in versatile laser systems devel-ops but also an extremely broad spectrum of potential can-didate disorders has developed which presently includes the psoriatic lesion. As compared to other treatment options for psoriasis, the use of lasers aims at a selective removal of the diseased endothelial target structures whilst the epidermis is particularly protected from heat injury rendering it an excel-lent potential treatment option for localized plaque psoria-

Nd: YAG laser (1064 nm) failsto improve localized plaque type psoriasis:a clinical and immunohistochemical pilot study

Rosanne G VAN LINGENElke MGJ DE JONGPiet EJ VAN ERPWilmy SEC VAN MEETERENPeter CM VAN DE KERKHOFMarieke MB SEYGER

sis. Studies have led to the idea that the selective destruction of the dilated papillary vessels by the principle of selective photothermolysis eliminates the extravasation of inflamma-tory mediators into the interstitium inducing improvement of psoriatic plaques[6-10]. This selectivity can be achieved by choosing the appropriate laser light wavelength and pulse duration responsible for the amount of heat diffusion. As put forward, psoriasis has previously been shown to respond to several types of lasers[7;11-14]. In particular with respect to vascular lasers, psoriatic lesions have shown to improve with pulsed dye laser treatment (PDL, 595 nm) although some debate between different groups exists with respect to the efficacy and tolerability.[15-19] However, the remission isusually transient as studies have shown that the persistent secretion of cytokines in treated psoriatic lesions were re-sponsible for a relapse of endothelial proliferation causing a rebound in vasculature and return of the psoriatic plaque.[1] Infrared light with a wavelength of 1064 nm, like that of the neodymium-doped yttrium aluminium garnet (Nd:YAG) la-ser can penetrate the skin up to a depth of 7 mm.[20] So far, no study has been carried out to put the Nd:YAG laser in to place in the array of treatment modalities for localized psoriasis. The deep penetration could well represent a great advantage of the Nd:YAG laser over the PDL since the tar-get endothelial structures in psoriasis are located up to a few millimeters deep in the psoriatic skin. As the psoriatic blood

O ften some chronic and localized plaque pso-riasis appears to be therapy resistant and no ad-equate solution, in terms of effective treatment

Department of Dermatology, RadboudUniversity Medical Centre, P.O. Box 9101,NL- 6500 HB Nijmegen, The Netherlands

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vessels play an important role in immunocyte-trafficking inpsoriasis, it is credible that selective removing of the deeper abnormal psoriatic vasculature in the dermis by means of the Nd:YAG 1064 nm laser may be of benefit for treatment. Inthe long run, this could possibly result in a more long-lasting therapeutic effect. The current pilot study evaluates the clinical efficacy andtolerability of a Nd:YAG 1064 nm against an active well-es-tablished comparator: calcipotriol/betamethasone dipropion-ate ointment (CBD). An intrapatient, left-right comparative study was conducted wherein patients with localized psoria-sis were treated with both the Nd:YAG laser (1064 nm) and topical therapy with CBD for a period of three months. CBD-treatment was given once daily whereas the number of Nd:YAG treatments was three, with a 4-week interval between treatments. In addition, in order to elucidate the mode of ac-tion, immunohistochemical assessments were performed on lesional skin biopsies. Our goal herein was to provide insight in the effect of the 1064-nm wavelength and CBD-oint-ment on key-phenomena in the psoriatic dermis and epider-mis, e.g. keratinocyte differentiation (K10), T-lymphocytes (CD3+ cells) and capillary expansion (Von Willebrandfactor and CD31).

Patients & MethodsPatientsSix patients aged at least 18 years with stable, localized plaque psoriasis were included in the study after giving writ-ten informed consent. All patients had a washout of systemic treatments or phototherapy for at least four weeks and had not used topical treatment for the last two weeks prior to par-ticipation. Other exclusion criteria were pregnancy, lactation and a history of photosensitivity. The study was approved by the local medical ethics committee.

Study design Two weeks before the start of treatment, 10% salicylic acid in petrolatum album was prescribed in order to reduce desq-uamation of the plaque and enhance light penetration and penetration of the CBD-ointment through the stratum cor-neum, ultimately standardizing and optimizing the pretreat-ment situation for both topical and laser treatment. Since treatment with the Nd:YAG 1064 nm (“Gentle YAG VR”, wavelength 1064 nm, with dynamic cryogen cooling device for minimizing patient discomfort, Candela Corporation) has not been described before, questions remain as to the 1064nm laser’s optimal operating parameters for positively effecting dermal endothelial structures. Therefore, we firsttested for optimal settings in three patients with psoriasis for final use in the main pilot study. At the initial visit, two simi-lar plaques of at least 10 cm2 were selected. These plaques were similar in terms of body localization and clinical sever-ity score (SUM-score). Consequently, one psoriatic plaque was treated varying in spotsizes and fluences all within thespectrum of optimal depth for effective endothelial destruc-tion (table 1). Primary outcome measures which were used after 4 weeks and further throughout the study, comprised the SUM-score (for evaluating the clinical effect) and the oc-currence of adverse events (for evaluating safety). No major

significant differences in clinical effect between the settingscould be observed, nor did any interfering adverse events occur. All fluences were tolerated equally. As no specificpreferable setting in terms of clinical efficacy or safety wasdeducible from the testing, the maximal applied fluence perspotsize (1,5 mm spot, 375 J cm-2; 3 mm spot, 310 J cm-2) from the testing was chosen for application in the main pilot study. Comparing PDL-studies with different time-intervals, the intervals vary from weekly to once every 2 to 3 weeks. Even, sometimes residual crusting resulted in postponement of the laser-treatment. Therefore, we opted for a time-inter-val of four weeks between Nd:YAG treatments.In the main study, at random, one of the two selected SUM-comparable plaques was treated with calcipotriol 50 μg g-1 and betamethasone dipropionate 0.5 mg g-1 ointment (CBD) (Daivobet/Dovobet; Leo Pharma, Ballerup, Denmark) once daily for the duration of three months. The other plaque was symmetrically divided in halves to treat one halve with the 1,5 mm spot (fluence 375 J cm-2) and the other with the 3 mm spot (fluence 310 J cm-2) each by a single pass, once every 4 weeks. In total, the plaque was treated three times. Prior to Nd:YAG treatment, watery oil was applied on the designat-ed psoriatic plaque in order to further reduce the amount of scattering and enhance light penetration through the stratum corneum. After three months of treatment, patients entered a follow-up period of eight weeks in which they did not apply the CBD ointment nor received Nd:YAG laser treatment.

Clinical assessmentsClinical severity of the designated plaques was assessed us-ing the SUM-score at baseline and after 4 and 8 weeks and during the follow-up period. The SUM score is a cumula-tive measure which includes scores for eythema, indura-tion (plaque thickness) and scaling on the following scale: 0, absent; 1, minimal (very light pink, hardly any elevation, rare scale); 2, mild (light red/ pink, slight elevation, poorly defined scale); 3, moderate (red, moderate elevation, definedscales); 4, severe (very red, marked ridge, heavy scaling). Ultimately, a global SUM score (range 0-12) was determined as the sum of all three scores together, reflecting the overallplaque severity. Furthermore, the Visual Analogue Scale (VAS) for pain (range 0-10) was used for patients to report the level of ex-perienced pain during Nd:YAG treatment with the two spot-sizes. A score of 0 represented a total absence of pain and 10 represented maximum pain.

Nd:YAG 1064 nm settings

Fluence J cm-2

Spotsize 1,5 mm -Pulse duration

10 ms

300325350375

Spotsize 3 mm - Pulse duration

20 ms

250270290310

Table 1. Nd:YAG 1064 nm test settings varying in spotsize and fluence.

nm= nanometer, mm= millimeter, cm=centimeter, ms= milliseconds, J= Joule.


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Biopsies Biopsies (4 mm) were taken at a central representative site in the target lesions after 3 months of treatment (week 12): one from the Nd:YAG treated, one from the CBD treated lesion and one from an untreated psoriatic plaque which served as a control. Before the biopsy procedure, local anesthesia was given (xylocaine/adrenaline 1:100.000). Skin defects were optionally closed using one suture.

Immunohistochemical stainingImmunohistochemical staining on paraffin and cryostat sec-tions was performed as previously described.[21;22] The following primary antibodies (mouse anti-human) were used: anti-CD3 (1:100) (cryostat, clone UCTH1), anti-K10 (paraffin, 1:100, clone RKSE60) (Monosan LaboratoriesUden Netherlands), anti-von Willebrand Factor (paraffin,vWF, 1:200, clone F8/86) and anti-CD31 (paraffin, 1:200,clone JC70A) both from DAKO (Copenhagen, Denmark). Furthermore, from each patient we performed a H&E stain-ing to verify that the biopsies under study fulfilled the pso-riasis-specific histological criteria.

Blinded quantification of markersQuantification of K10 and CD3 and determination of thethickness of the epidermis was enforced as previously done[23] using the image processing program ImageJ 1.37 (National Institutes of Health, USA) and IP-lab (Scanalytics, USA). Quantitative cell counts of the T-lymphocytes (CD3) were expressed as ‘Positive cells per mm skin length’. K10 was quantified using the % of a representative region of in-terest (ROI) in the epidermal surface as unit. As the stain-ings of both CD31 and vWF showed a similar pattern, CD31 was chosen as the endothelial marker used for quantification.Quantification of CD31 was enabled creating a division inseveral distinct ROI’s as depicted in figure 1, ultimately ex-

pressing CD31 as positive area in % of the ROI. In order to observe and evaluate the potential depth of penetration of the Nd:YAG laser in the dermis, two separate parts of the dermis were selected on the one hand comprising the inter rete ridges dermis and on the other hand the more lower pap-illary dermis. The choice for this separation was based on the background information that the PDL showed to primarily target the more superficial capillaries in the rete ridges andthe Nd:YAG was theorized to penetrate deeper up to the low-er papillary dermis. All sections were analyzed in a blinded manner as the sections were coded by an independent co-worker before assessment.

Statistical analysesTo compare SUM-scores between different moments in time during treatments, we performed the Kruskal-Wallis test. In addition, we performed matched pair analysis using the Wil-coxon signed Ranks test to compare the SUM-scores over time with the non-treated plaques. To compare the epidermal thickness and immunohistochem-ical expression of K10, CD3 and CD31 between the two treatments (Nd:YAG 3 mm spot and CBD) and the untreated psoriatic biopsy, the Student’s t-test was used. A statistically significant difference was set at p< 0.05. All analyses werecarried out using SPSS software version 14.0.

ResultsIn total, 6 patients participated in this study (4 males, 2 fe-males) with a mean age of 47.7 ± 5.2 years. Two patients had to be excluded one because of refusal of the biopsy pro-cedure, the other because the applied fluences had to be re-duced since the patient reported intolerable pain at the initial fluences.

Figure 1. Quantification method for CD31 positive endothelium. The papillary dermis was divided in the “inter rete ridges dermis”, wherein a region of interest (ROI) was drawn encompassing the dermal area’s between the rete ridges, and in the low papillary dermis wherein the area up to 200 μm below the tips of the rete ridges was incorporated in the ROI. * = 200 μm

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Clinical efficacyThe results for the mean SUM-scores per treatment modality during the study are shown in figure 2A . A quick glance at the figure shows that at baseline all plaques had a compa-rable SUM-score, that the treatment with CBD caused the largest reduction of SUM-score and thus clinical effect and that the two spotsizes used for the Nd:YAG laser did not dif-fer greatly in clinical effect. The mean reductions in SUM-score between baseline and week 12 for Nd:YAG 1,5 mm, Nd:YAG 3mm and the CBD-ointment were 17%, 27%, 62% respectively. With respect to the control plaque, there was a 9% increase in SUM-score after 12 weeks. The reduction in SUM-score induced by CBD-ointment reached statistical significance at week 4, week 8 and week 12 (all p<0.05) as compared to baseline. There was a significant reduction inSUM-score caused by treatment with the Nd:YAG (both 1.5 and 3 mm spotsize) at week 4, respectively 29% and 24 % compared to baseline. However, this effect was not main-tained further throughout the study. Between week 12 and 16 (follow-up period), only the mean SUM-score for the CBD treated plaques augmented significantly to a moderate extent(+33%, p<0.05).

Visual Analogue Scale (VAS)With respect to the level of experienced pain during Nd:YAG

treatment, the mean VAS-score per spotsize at the different time-points is depicted in figure 2B. It can be derived that all patients reported a higher VAS-score when treated with the 3mm spot and corresponding fluence than with the 1,5 mmspot. In addition, the VAS-score was comparable in range during the first, second and third treatment and did not differgreatly except for one patient whom experienced intolerable pain at the initial fluences. Therefore, the fluences for thisparticular patient were reduced by ± 40% and this patient was excluded form further clinical and immunohistochemi-cal analyses.

Morphological and immunohistochemical analysesFigure 3 illustrates the results of the quantities of the epider-mal thickness, the endothelial marker (CD31), keratinocyte differentiation (K10) and CD3-counts after treatment with CBD and the Nd:YAG laser compared to the non-treated control biopsies (NoTx). The number of CD3+-lymphocytes decreased significantly (p<0.05) in the epidermis of theCBD treated group and only a tendency of a decrease af-ter therapy(p<0.1) was observed in the epidermis of the Nd:YAG treated group compared to the amount of CD3+ T-cells in the control biopsies of non-treated psoriatic plaques. With respect to the dermis, in the Nd:YAG treated group the CD3+ T-cells were significantly decreased after therapy (p<0.05)ascompared to the control group of biopsies whereas the CBD group showed no significant reduction in CD3+ T-cells.The epidermal thickness decreased significantly in the CBDgroup of biopsies (-43.2%, p<0,05), whilst for the Nd:YAG group no significant decrease could be observed (-24.9%,p>0,05) compared to the untreated biopsies. The area posi-tive for CD31 in the ROI in both the inter-reteridges dermis and the low papillary dermis, was not significantly differentbetween each of the two treatment groups versus the control group. As for the K10 positive epidermal area, no significantincreased amount of normal differentiating keratinocytes af-ter treatment with CBD or the Nd:YAG laser could be ob-served.

Side-effects Nd:YAG laser Two patients experienced a burning sensation on the Nd:YAG treated lesions 2-6 hrs after therapy. Two patients re-ported transient purpuric discoloration of small areas of the lasers lesions. One patient had several white spots on the Nd:YAG treated psoriatic lesion directly after lasering. Four pa-tients reported development of mild crusts in the week after receiving laser treatment. One patient reported slight fluidleakage from the Nd:YAG treated lesion. Another patient experienced increased itch on the Nd:YAG treated lesion compared to the CBD-treated plaque. Scarring was not seen in any of the patients. With respect to the treatment with the CBD-ointment, no side-effects were observed, in particular no atrophy of the skin.

DiscussionAs psoriasis persists to be a chronic skin disease of which the pathogenesis is not completely understood, the search for new treatments and insights continues. Psoriasis is probably

Figure 2: A) Results for the clinical efficacy of treatment withCBD-ointment and the Nd:YAG laser compared to no treat-ment (No Tx) reflected in mean SUM-scores.B) Results for experienced pain during Nd:YAG treatment with different spotsizes reflected in mean VAS-scores. TheVAS-scores at the different points in time are taken together.Error bars indicate the Standard Error of the Mean. * = significantly decreased compared to baseline (p<0.05).** = significantly increased compared to week 12 (p<0.05).

A

B


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not one single disease entity, as multiple expression pat-terns are observed ranging in severity, extent, aspect of le-sions. Furthermore, individual needs vary among patients. The need and search for adequate and effective special-ized therapies for recalcitrant localized psoriatic lesions persists, as no optimal treatment solution is yet available. In the field of lasers, many have investigated the effectof (laser)light to target diseased psoriatic skin structures. The PDL was shown by some groups to be a good thera-py for recalcitrant localized psoriasis, although the effect was momentary and results can be at variance.[6;19] The present pilot study is the first to evaluate whether the Nd:YAG laser is a potential treatment modality for this kind of psoriatic lesions. It is plausible that when targeting the deeper vasculature more effectively, damage can be caused possibly resulting in longer lasting effect of clearance of psoriatic plaques. Subsequently, when focussing on the histologic changes that ensue after irradiation with the 1064 nm-wavelength, clarification of the relevant micro-scopic events can be obtained. In contrast to this outlined hypothesis however, in this study clinical and well-definedhistological evidence supporting this Nd:YAG-theory was not acquired. In order to enlighten this discrepancy, sev-eral interpretations are thinkable bearing in mind that the present pilot study comprised a minimal sample size. It is necessary for pulse duration to be matched to vessel size; the larger the vessel diameter, the longer pulse dura-tion is required to effectively damage the vessel thermally. Absorption by hemoglobin in the long vivible to near in-frared range appears to become more important for vessels over 0.5 mm and at least 0.5mm below the skin surface.

However, high energies must be used for adequate penetra-tion. The key eventually is to find the optimal parametersfor this laser for positively effecting dermal endothelial structures without excess heat that may cause collateral damage. It can be theorized that if the energy level was below the chromophores threshold and the pulse width was extended beyond the thermal relaxation time, the heat could be absorbed by the chromophore but then dissi-pated to surrounding tissues before reaching a destructive threshold level[24] and an ensued clinical effect. Alterna-tively, it could well be, based on the results of this study, that the overlying diseased psoriatic skin is irresponsive in terms of clearance when deeper lying vasculature is being targeted by a 1064 nm Nd:YAG laser. In addition, as the inflammation in psoriatic plaques is highly vivid, it couldwell be reasonable to treat these plaques more frequently than once a month in order to achieve a clinical effect of the Nd:YAG laser.Although lacking clinical efficacy, Nd:YAG treatment in-stigated a substantially reduced number of CD3+ T cells in the dermis in the present pilot study. The present study is at variance with findings of Bovenschen et al.[6] however, who showed a reduction in CD3+ T-cells in the psoriatic skin after PDL therapy but also a marked clinical effect of the PDL in psoriasis. One could question therefore whether changes in the amount of CD3+ T-lymphocytes after laser therapy alone can lead to clinically improved psoriatic le-sions. Some have found that peak epidermal temperature can have an important influence on the histologic changesafter nonablative laser therapy.[25] Therefore, a possible diffuse thermal effect on the T-cell after Nd:YAG therapy

Figure 3: Overview of the results of the mean changes in CD3-counts (A), epidermal thickness (B), the endothelial marker (CD31) (C) and keratinocyte differentiation (K10) (D) in response to the treatment with CBD-ointment and the Nd:YAG laser compared to no treatment (No Tx).

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could lie at the basis of changes in amount of CD3+ T-cells as previously touched upon by Ruiz-Esparza et al.[12] Three anecdotal reports on psoriatic plaques responding to low-en-ergy Nd:YAG (1320 nm) irradiance were described, corre-sponding with a diffuse thermal effect even though the exact mechanism of this therapy remained unknown. In order to minimize patient discomfort and epidermal spar-ing, cooling through direct contact was used throughout the present study, optimally practising minimal pressure against the skin not to compress the target vessel with excessive hand pressure. Trained staff is requisite to garantee proper and consistent use of the laser. It is important to notice that scatter and reflection are two key aspects influencing thepenetration of the laser light to the targeted depth. Although patients were pretreated with 10% salicylic acid in petrola-tum album and watery oil was applied in order to enhance an optimal penetration, in the end psoriatic plaques have a different texture compared to non-psoriatic skin presumably influencing the ultimate depth of penetration.With respect to the active comparator (CBD-ointment), con-sistent with earlier findings [6] the clinical severity of thetreated plaques reduced significantly likewise the epidermalthickness. Although effectively reducing the thickness of the epidermis and corresponding reduction in SUM-score, the CBD-ointment does not tend to have an effect on the amount of abnormal differentiating keratinocytes and on the exces-sive microvascular expansion in psoriasis.To summarize, in the present pilot investigation the Nd:YAG 1064 nm laser was found not to be of additional value in the array of treatment modalities for chronic localized plaque psoriasis since no clinical and histological evidence came across to support this. It is evident that the superficial dermalvasculature remains a major contributor to the pathogenesis of this disease and targeting this vascular expansion is a good manner to try to tackle the influx of immunocytes to the siteof inflammation. However, this study brings to light that theamount of CD31 positive endothelium in the papillary der-mis of psoriatic plaques was not significantly altered by theeffect of the Nd:YAG 1064 nm laser. Dermatologists in the field of lasers should take the present findings to heart and bevigilant when considering the Nd:YAG laser as a potential treatment option for chronic therapy resistant psoriatic le-sions. Targeting more superficial located vascular expansionin psoriasis is likely to be of stronger importance in achieving clinical effect than the deeper vasculature. However, further studies incorporating changes in methodology, in particular shortened time-intervals between treatments, are needed in order to refute or confirm this position.

Acknowledgements: We would like to acknowledge Mr Niels Tanja of Dalton Medical BV for providing us the laser equip-ment to carry out this study. No funding financial sources todeclare.Prof. Dr. PCM van de Kerkhof has consultancy services for Schering-Plough, Centocor, Allmirall, UCB, Wyeth, Pfizer,Abbott, Actelion, Galderma, Novartis, Janssen-Cilag and Leo Pharma.

References

1. Karsai S, Roos S, Hammes S et al. Pulsed dye laser: what’s new in non-vascular lesions? J Eur Acad Dermatol Venereol 2007;21:877-90.2. Barker J. Skin diseases with high public health impact. Psoriasis. Eur J Dermatol 2007;17:563-4.3. Braverman IM, Yen A. Ultrastructure of the capillary loops in the dermal papillae of psoriasis. J Invest Dermatol 1977;68:53-60.4. Goodfield M, Hull SM, Holland D et al. Investigations of the ‘ac-tive’ edge of plaque psoriasis: vascular proliferation precedes changes in epidermal keratin. Br J Dermatol 1994;131:808-13.5. Pinkus H, Mehregan AH. The primary histologic lesion of sebor-rheic dermatitis and psoriasis. J Invest Dermatol 1966;46:109-16.6. Bovenschen HJ, Erceg A, Van Vlijmen-Willems I et al. Pulsed dye laser versus treatment with calcipotriol/betamethasone dipropion-ate for localized refractory plaque psoriasis: effects on T-cell infiltra-tion, epidermal proliferation and keratinization. J Dermatolog Treat 2007;18:32-9.7. de Leeuw J. Tank B, Bjerring PJ et al. Concomitant treatment of pso-riasis of the hands and feet with pulsed dye laser and topical calcipo-triol, salicylic acid, or both: a prospective open study in 41 patients. J Am Acad Dermatol 2006;54:266-71.8. Hern S, Stanton AW, Mellor RH et al. Blood flow in psoriatic plaquesbefore and after selective treatment of the superficial capillaries. Br JDermatol 2005;152:60-5.9. Hern S, Stanton AW, Mellor RH et al. In vivo quantification of thestructural abnormalities in psoriatic microvessels before and after pulsed dye laser treatment. Br J Dermatol 2005;152:505-11.10. Yen A, Braverman IM. Ultrastructure of the human dermal micro-circulation: the horizontal plexus of the papillary dermis. J Invest Der-matol 1976;66:131-42.11. Alora MB, Anderson RR, Quinn TR et al. CO2 laser resurfacing of psoriatic plaques: a pilot study. Lasers Surg Med 1998;22:165-70.12. Ruiz-Esparza J. Clinical response of psoriasis to low-energy irradi-ance with the Nd:YAG laser at 1320 nm report of an observation in three cases. Dermatol Surg 1999;25:403-7.13. Taibjee SM, Cheung ST, Laube S et al. Controlled study of exci-mer and pulsed dye lasers in the treatment of psoriasis. Br J Dermatol 2005;153:960-6.14. Trott J, Gerber W, Hammes S et al. The effectiveness of PUVA treatment in severe psoriasis is significantly increased by additionalUV 308-nm excimer laser sessions. Eur J Dermatol 2008;18:55-60.15. Katugampola GA, Rees AM, Lanigan SW. Laser treatment of pso-riasis. Br J Dermatol 1995;133:909-13.16. Ros AM, Garden JM, Bakus AD et al. Psoriasis response to the pulsed dye laser. Lasers Surg Med 1996;19:331-5.17. Zelickson BD, Mehregan DA, Wendelschfer-Crabb G et al. Clini-cal and histologic evaluation of psoriatic plaques treated with a flash-lamp pulsed dye laser. J Am Acad Dermatol 1996;35:64-8.18. Bjerring P, Zachariae H, Sogaard H. The flashlamp-pumped dyelaser and dermabrasion in psoriasis--further studies on the reversed Kobner phenomenon. Acta Derm Venereol 1997;77:59-61.19. Erceg A, Bovenschen HJ, van de Kerkhof PC et al. Efficacy of thepulsed dye laser in the treatment of localized recalcitrant plaque psoria-sis: a comparative study. Br J Dermatol 2006;155:110-4.20. Landthaler M, Haina D, Brunner R et al. Effects of argon, dye, and Nd:YAG lasers on epidermis, dermis, and venous vessels. Lasers Surg Med 1986;6:87-93.21. Zeeuwen PL, van Vlijmen-Willems IM, Egami H et al. Cystatin M / E expression in inflammatory and neoplastic skin disorders. Br JDermatol 2002;147:87-94.22. Vissers WH, Berends M, Muys L et al. The effect of the combina-tion of calcipotriol and betamethasone dipropionate versus both mono-therapies on epidermal proliferation, keratinization and T-cell subsets in chronic plaque psoriasis. Exp Dermatol 2004;13:106-12.23. Bovenschen HJ, Seyger MM, van de Kerkhof PC. Plaque psoriasis vs. atopic dermatitis and lichen planus: a comparison for lesional T-cell subsets, epidermal proliferation and differentiation. Br J Dermatol 2005;153:72-8.24. Dayan S, Damrose JF, Bhattacharyya TK et al. Histological evalua-tions following 1,064-nm Nd:YAG laser resurfacing. Lasers Surg Med 2003;33:126-31.25. Alam M, Hsu TS, Dover JS et al. Nonablative laser and light treatments: histology and tissue effects--a review. Lasers Surg Med 2003;33:30-9.


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RG van Lingen, PCM van de Kerkhof, MMB Seyger, EMGJ de Jong, DWA van Rens, MKP Poll, PLJM Zeeuwen, PEJ van ErpCD26 / DPPIV in psoriatic skin: Upregulation and topographical changesBr J Dermatol. 2008; 158(6): 1264-72.

RG van Lingen, MKP Poll, MMB Seyger, EMGJ de Jong, PCM van de Kerkhof, PEJ van Erp Distribution of epidermal dipeptidyl-peptidase IV on psoriatic keratinocytes: a com-parison with hyperproliferation and aberrant differentiation markersArch Derm Res. 2008; Online early at http://dx.doi.org/10.1007/s00403-008-0862-1.

RG van Lingen, PCM van de Kerkhof, EMGJ de Jong, MMB Seyger, JBM Boezeman, PEJ van ErpReduced CD26bright expression of peripheral blood CD8+ T-cell subsets in psoriatic patientsExp Dermatol. 2008 Apr; 17 (4): 343-48.

RG van Lingen, PEJ van Erp, MMB Seyger, EMGJ de Jong, RT de Boer-van Huizen, RJB Driessen, PCM van de KerkhofPersistent expression of CD26 / DPPIV after treatment with infliximab in psoriasis de-spite clinical improvementSubmitted.

3.1

3.2

3.3

3.4

This Chapter was based on the following publications:

Expression of CD26 / DPPIV in psoriasis

Chapter 3

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Chapter 3

RG van Lingen, PCM van de Kerkhof, MMB Seyger, EMGJ de Jong, DWA van Rens, MKP Poll, PLJM Zeeuwen, PEJ van ErpCD26 / DPPIV in psoriatic skin: Upregulation and topographical changesBr J Dermatol. 2008; 158(6): 1264-72.

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DERMATOPATHOLOGY DOI 10.1111/j .1365-2133.2008.08515.x

CD26/dipeptidyl-peptidase IV in psoriatic skin:upregulation and topographical changesR.G. van Lingen, P.C.M. van de Kerkhof, M.M.B. Seyger, E.M.G.J. de Jong, D.W.A. van Rens, M.K.P. Poll,P.L.J.M. Zeeuwen and P.E.J. van Erp

Department of Dermatology, Radboud University Medical Centre Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, the Netherlands

CorrespondenceRosanne G. van Lingen.

E-mail: [email protected]

Accepted for publication9 January 2008

Key wordsCD26, dipeptidyl-peptidase IV, DPP-IV, psoriasis

Conflicts of interestNone declared.

Summary

Background Psoriasis is known to affect 2–3% of the population and can be con-sidered an organ-specific autoimmune disease. CD26/dipeptidyl-peptidase IV(DPP-IV) is a membrane-bound protease with diverse properties. In theory, theexpression of CD26/DPP-IV has common grounds with three principal key play-ers of the psoriatic pathogenesis: keratinocytes, T cells and cytokines.Objectives To assess CD26/DPP-IV expression in psoriasis in order to expand onthe search for complementary biomarkers related to inflammation and prolifera-tion in psoriasis.Methods The pattern of expression of CD26/DPP-IV was investigated on themRNA-, protein- and enzyme-functionality level using immunohistochemical,immunofluorescent and enzyme activity labelling techniques.Results An 11-fold significant increase of CD26/DPP-IV on the mRNA level wasdemonstrated in psoriatic epidermal sheets compared with normal skin. Immuno-histochemistry on psoriatic sections showed a distinct patchy honeycomb-likeCD26/DPP-IV staining in the suprapapillary layers. Moreover, a clearly distin-guishable column-like staining pattern throughout the suprabasal compartmentalong the rete ridges was seen, whereas in normal skin these patterns wereabsent. Strikingly, CD26/DPP-IV enzyme activity correlated with this immuno-histochemical reactivity pattern for the CD26/DPP-IV protein. The T-cell boundexpression of CD26/DPP-IV in psoriatic skin was explicitly present, albeit insmall quantities.Conclusions Our data provide clear evidence for a versatile upregulation of CD26/DPP-IV expression in psoriatic (epi)dermis. Although the exact functional contri-bution remains speculative, the topographical distribution of this complex multi-functional protein suggests a suitable role as a complementary biomarker inpsoriasis.

Over recent years, an increasing number of publications have

focused on CD26 or dipeptidyl-peptidase IV (CD26/DPP-IV),

and its various properties have been described. CD26/DPP-IV

is a glycoprotein which belongs to a group of membrane-

bound proteases, which hydrolyse dipeptide sequences that

have proline or, to a lesser extent, alanine at the penultimate

position. Besides its protease activity, CD26/DPP-IV has a

multifunctional role as it can act as a costimulatory protein for

CD45RO, a receptor for adenosine deaminase and an adhesion

molecule for collagen and fibronectin, and it can be involved

in apoptosis.1–6 It is attractive to speculate that CD26/DPP-IV

via these mechanisms may be relevant to pathophysiological

conditions.7 The expression level of CD26/DPP-IV differs

greatly between tissues including T lymphocytes, and endo-

thelial and epithelial cells.8,9 CD26/DPP-IV is known to be

involved in the pathology of a variety of autoimmune diseases

such as multiple sclerosis and rheumatoid arthritis.10–12

Remarkably, data in the literature are sparse with respect to

the expression of CD26/DPP-IV in psoriatic skin.13–15

The pathogenesis of psoriasis comprises three principal key

players. Both keratinocytes and T cells participate actively and

interact (in)directly with each other in psoriatic tissue, orches-

trated by the production of proinflammatory cytokines and

chemokines.16–18 Keratinocyte products influence immune

activation and, vice versa, products of activated immunocytes

alter keratinocyte responses.17 In vivo, CD26/DPP-IV is known

to be expressed on human keratinocytes in variable amounts

in various inflammatory dermatoses.7,14 It was shown that


1264 Journal Compilation � 2008 British Association of Dermatologists • British Journal of Dermatology 2008 158, pp1264–1272

126

DPP-IV inhibitors can suppress keratinocyte proliferation in vitro

and the keratinocyte differentiation can partially be restored

in vivo.7

The expression of CD26/DPP-IV on the surface of the sec-

ond key player, the T cell, has briefly been described in psori-

atic and healthy subjects, mainly in peripheral blood T

cells.19–21 So far, the T-cell bound expression in the skin of

healthy and psoriatic patients has, to our knowledge, remained

untouched.

Cytokines and chemokines represent the third key player in

the psoriatic chronic immune response. They are considered

mediators responsible for activation and recruitment of infil-

trating leucocytes and therefore play a crucial role in the

development and persistence of psoriatic skin lesions. CD26/

DPP-IV is likely to play a pivotal role in processing these mol-

ecules. The extracellular protease domain of CD26/DPP-IV

(both on keratinocytes and T cells) can cleave dipeptides from

the amino terminus of proteins, such as cyto- and chemokines,

which are abundantly present in a chronic immune response

in psoriasis, resulting in alterations in receptor specificity and

subsequently reduced biological activity.22 Taken together, it

is conceivable that in psoriasis, a disease wherein the interplay

between activated T cells, keratinocytes and cytokines is

determinative, CD26/DPP-IV can act on all these three key

players.

The multifunctional characteristics of CD26/DPP-IV

prompted us to investigate its pattern of expression on the

earlier mentioned cell types implicated in the pathogenesis of

psoriasis. The mRNA produced is a good indicator for the

de novo synthesis of protein in the tissue of interest. Therefore

quantitative real-time polymerase chain reaction (qPCR) analy-

sis was performed on epidermal sheets to study CD26/DPP-IV

expression at the mRNA level. Subsequently, frozen sections

of normal and psoriatic skin were collected and examined

using immunohistochemical and immunofluorescent label-

ling techniques, in order to characterize both epidermal and

dermal CD26/DPP-IV expression. In addition, cytochemical

enzyme activity assays were performed in order to see

whether the demonstrated protein is actually enzymatically

active in psoriasis or not. To summarize, our objective was to

assess the expression of CD26/DPP-IV in psoriatic skin and to

expand on the search for complementary (discriminating)

biomarkers related to inflammation and proliferation in

psoriasis.


Patients

For qPCR analysis seven healthy subjects (age range 21–

35 years) and nine patients with psoriasis (age range 49–

70 years) were included. Affected lesional psoriatic skin was

used for the isolation of epidermal sheets.

For the immunohistochemical, enzyme cytochemical and

immunofluorescent staining methods, 11 patients with psoria-

sis (age range 32–59 years) and four healthy volunteers

(age range 18–21 years) were included in the study. Any

topical or systemic treatment was suspended for at least

2 weeks and 4 weeks, respectively, prior to participation.

Written informed consent was obtained before enrolment. All

psoriatic patients had a moderate to severe plaque-type psoria-

sis. The diagnosis of psoriasis was based on clinical and histo-

logical (haematoxylin and eosin staining) criteria.

Clinical scores and biopsies

SUM-scores were performed to assess the clinical status of the

biopsied psoriatic plaques. The SUM-score comprises the sum

of separate scores for erythema (0–4), induration (0–4) and

scaling (0–4). Four-millimetre punch biopsies were taken

from the centre of the psoriatic plaque after anaesthesia with

1% xylocaine/adrenaline.

Real time quantitative polymerase chain reaction

technique for CD26/DPP-IV expression in vivo

Isolation of epidermal sheets

Isolation of epidermal sheets was performed as previously

described.23 Briefly, a 4-mm punch biopsy was incubated for

4 h at 4 �C in PBS (with Ca2+/Mg2+) containing 12 mg mL)1

dispase (Roche Diagnostics, Basel, Switzerland) and 5 lg mL)1

actinomycin-D (Sigma-Aldrich, St Louis, MO, U.S.A.). After

incubation, the dermis was removed with tweezers and the

remaining epidermal sheet was stored in Trizol (for RNA

extraction).

RNA extraction and real-time quantitative polymerase

chain reaction

Epidermal sheets or cultured keratinocytes were used for RNA

extraction, and reverse transcriptase reactions were performed

with 1 lg RNA as described previously.24 The reverse trans-criptase reaction products were used for real-time qPCR ampli-

fication, which was performed with the MyiQ� Single-Color

Real-Time Detection System for quantification with SYBR�

Green Supermix and melting curve analysis (Bio-Rad, Rich-

mond, CA, U.S.A.), as described previously.25 DNA was PCR-

amplified under the following conditions: 4Æ5 min at 95 �Cfollowed by 40 cycles of 15 s at 95 �C and 1 min at 60 �C,with data collection in the last 30 s. For all PCRs, iQ� SYBR

Green Supermix (Bio-Rad) was used in the reaction. All pri-

mer concentrations were 300 nmol L)1 in a total reaction vol-

ume of 25 lL. The amount of mRNA for the CD26/DPP-IV

gene in each sample was normalized to the amount of mRNA

of the human acidic ribosomal protein (hARP; RPLP0, ribo-

somal phosphoprotein P0) reference gene in the same sample.

We used hARP as we have previously found it to be very use-

ful for normalization procedures when using RNA from

biopsy material. The hARP gene is, at least for skin biopsies,

more suitable than commonly used genes such as actin or

glyceraldehyde-3-phosphate dehydrogenase, as hARP correlates



Upregulation of CD26/DPP-IV in the psoriatic lesion, R.G. van Lingen et al. 1265

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127

far better with 28S ribosomal RNA (not shown). Primers for

qPCR were only accepted if their efficiency was 100 ± 10%.

Thus the PCR product concentration was doubled at each cycle

and the threshold cycle (Ct) will be linear with the log of the

initial concentration. For graphic representation of relative

mRNA expression levels we used the DDCt method.26 Primersequences were: CD26/DPP-IV forward 5¢-TCATTCAGTAAAG-AGGCGAAGTATTATC-3¢; CD26/DPP-IV reverse 5¢-CAGTTTTT-TGGAGGGCATCTG-3¢; hARP forward 5¢-CACCATTGAAAT-CCTGAGTGATGT-3¢; and hARP reverse 5¢-TGACCAGCCC-AAAGGAGAAG-3¢. All primers were obtained from Biolegio

(Nijmegen, the Netherlands).


Immunohistochemical staining was performed as previously

described.27,28 The following primary antibodies (mouse anti-

human) were used: anti-CD3 (1 : 100; clone UCTH1; Dako,

Copenhagen, Denmark) and anti-CD26/DPP-IV (1 : 50; clone

BA-5; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.).

Furthermore, from each patient we performed a haematoxylin

and eosin staining to verify that the biopsies under study

fulfilled the psoriasis-specific histological criteria.

Image analysis

With an objective magnification · 20 a representative sectionof the slide was chosen and analysed by two evaluators.

Immunohistochemical staining resulted in a honeycomb-like

and a column-like staining pattern in the psoriatic epidermis.

For characterization of this epidermal CD26/DPP-IV expres-

sion, the following semiquantitative four-point scale was used

to assess both the honeycomb-like and the column-like stain-

ing pattern on the full width of the section (4 mm): ), nostaining; ±, slight staining; +, moderate staining; ++, pro-

nounced staining.

Zenon immunofluorescence labelling technique

The Zenon labelling technique, as described before by Boven-

schen et al.,29 was used to combine the aforementioned

antibodies. Briefly, for double staining, the following fluoro-

chromes were used: Alexa Fluor 488-conjugated IgG1 Fab

fragments (Molecular Probes, Eugene, OR, U.S.A.), a green

fluorochrome, for anti-CD3 and Alexa Fluor 594-conjugated

IgG1 Fab fragments (Molecular Probes), a red fluorochrome,

was used for the anti-CD26/DPP-IV monoclonal antibody.

After incubation of the specific monoclonal antibodies with

the concomitant Alexa Fluor isotype-specific IgG Fab fragment,

the excessive fluorochrome was blocked with IgG-blocking

agent.

Staining procedure

For fluorescent immunohistochemical staining 7-lm sections

were cut on AAS-coated slides. Sections were air-dried, fixed

with acetone, and stored in PBS. After coupling and diluting

the antibodies, sections were incubated at room temperature

for 1 h with 100 lL antibody solution. After washing the

slides with PBS, the sections were fixed in 3Æ8% buffered

formalin (J.T. Baker, Deventer, the Netherlands). Then they

were incubated for 5 min with diamidino-2-phenylindole

(DAPI; 1 000 000 dilution of 1 mg mL)1 stock solution;

Molecular Probes) which is fluorescent upon binding to DNA

and was used to counterstain the cell nuclei. The slides were

mounted in Prolong Gold antifade reagent (Molecular Probes).

During the whole process, the slides were kept in the dark as

much as possible.

Microscopy

The sections and antibodies were examined according to the

method of Bovenschen et al.29 using a 100 W mercury lamp

for immunofluorescence microscopy (Axioskop 2 MOT; Zeiss,

Jena, Germany) with an objective magnification · 40.

Image analysis

In order to analyse the colocalization pattern of the CD26/

DPP-IV on T lymphocytes (CD3+), the photographs of

those single staining patterns representing the same part of

the skin were merged. A representative region of interest

was chosen in each photograph and analysed using Axio-

Vision software (Zeiss), in which the number of lympho-

cytes and specifically the CD26+ T lymphocytes were

counted.29

CD26/DPP-IV enzyme histocytometry

The demonstration of CD26/DPP-IV activity was performed

using the method described by Khalaf et al.,30 with minor

modifications. The obtained 4-mm biopsy specimens were

frozen in Tissue-Tek and sections (10 lm) were cut using acryostat and put on 3-aminopropyl-triethoxy-silane-coated

slides. The sections were air-dried for at least 20 min after

which they were fixed in cold acetone for 10 min.

For the incubation mixture 3 mg of substrate (gly-

pro-MNA; Bachem, Bubendorf, Switzerland) was dissolved in

0Æ2 mL dimethylformamide (DMF) and added to 4Æ6 mL ofPBS (pH 7Æ2). Subsequently 5 mg of fast blue B salt (Sigma-Aldrich) dissolved in 0Æ2 mL DMF was added and filtered. Sec-tions incubated with the DPP-IV inhibitor diprotin A (Bachem,

Bubendorf, Switzerland)31,32 were used as a negative control.

As positive control tissue for CD26/DPP-IV-specific activity,

murine kidney was used.33 This tissue was treated in the same

way as the human samples.

Sections were incubated with the substrate reaction mixture

in a humid chamber (200 lL per slide) at room temperature

for 45 min followed by a short wash with distilled water and

counterstained with Mayer’s haematoxylin. Then the sections

were washed in alkaline tap water and rinsed with distilled

water. Finally they were embedded with glycerol gelatin.



1266 Upregulation of CD26/DPP-IV in the psoriatic lesion, R.G. van Lingen et al.

128

Sections were analysed immediately using transmission light

microscopy.


All analyses were performed using SPSS 12.0.1 software.

Regarding the mRNA expression of CD26/DPP-IV in psoriasis

and in healthy subjects, the Student’s unpaired two-tailed t-test

was performed. Results are presented as means ± SEM. Statisti-

cal significance was set at P < 0Æ05.

Results

Histopathological and clinical severity data

All healthy biopsied volunteers had microscopically and histo-

logically normal skin. The haematoxylin and eosin staining of

the psoriatic biopsies showed psoriasis-specific histological

hallmarks such as hyperplasia of the rete ridges, suprapapillary

thinning or absence of the stratum granulosum.

All participating psoriatic patients had a stable plaque psori-

asis without pinpoint papules surrounding the plaques. The

clinical severity of the psoriatic lesion was assessed using the

SUM-score. Their mean SUM-score at the time of biopsy was

7Æ09 ± 0Æ46 (SEM).

Expression of CD26/DPP-IV in normal and psoriatic skin

in vivo

An 11-fold increase of CD26/DPP-IV on the mRNA level

was found in the examined psoriatic epidermal sheets com-

pared with normal skin (Fig. 1). This strong upregulation

was statistically significant (P = 0Æ002). In normal skin, the

CD26/DPP-IV protein expression on keratinocytes was

primarily shown to be absent (Fig. 2a) except for some focal

positivity of the suprapapillary layers of the epidermis (not

shown). When immunohistochemical staining procedures

were performed on frozen sections of psoriatic lesions,

notable changes in expression were found. A clearly distin-

guishable column-like staining pattern from the basal layers

throughout the suprabasal compartment in rete ridges was

seen in nine of 11 psoriatic sections, whereas in normal skin

this pattern was absent (Table 1, Fig. 2a, b). In addition, a

distinct patchy focal honeycomb-like structure was observed

in the basal layers of the epidermis, in particular just above

the top of the dermal papillae, extending with one to two

keratinocyte layers into the stratum spinosum (Fig. 2c, d).

The semiquantitative analysis of this honeycomb-like staining

in psoriatic sections showed a moderate to pronounced

CD26/DPP-IV staining, compared with a less pronounced

staining in normal skin (Table 1).

The dermal reactivity for CD26/DPP-IV showed a

complex staining pattern not suitable for a clear-cut analysis of

T-cell bound CD26/DPP-IV expression in the dermis (Fig. 2e).

CD26/DPP-IV enzyme histocytochemical activity in the

psoriatic (epi)dermis (Fig. 2f–i) and normal skin (Fig. 2h)

revealed an equal topographical distribution compared with

the immunohistochemical positivity for the CD26/DPP-IV

protein (Fig. 2a–e); the column-like staining and patchy

suprapapillary staining of the basal layers in the psoriatic

epidermis, which were observed by immunohistochemistry,

were also seen using the enzyme histocytometric technique.

Diprotin A, a DPP-IV inhibitor, proved to be a good negative

control in all investigated tissues (Fig. 2j, m). The murine

kidney tissue, which functioned as a positive control tissue,

showed distinct and extensive areas of enzyme activity as

anticipated (Fig. 2k, l).

In order to immunophenotype T lymphocytes, the Zenon

immunofluorescence double-labelling technique in combin-

ation with image analysis was used to identify colocalization

of CD26/DPP-IV on T lymphocytes. Overall, in normal skin

immunofluorescence labelling showed that T lymphocytes

were only sporadically present in both the dermis and epider-

mis. The presence of CD26+ T cells was restricted to a spor-

adic cell in the dermis in only one healthy subject. After

staining the psoriatic sections with the Zenon immunofluores-

cence technique, pronounced differences in T-lymphocyte

expression of CD26/DPP-IV were noted compared with nor-

mal skin (Table 1). In the dermal as well as in the epidermal

compartment, T lymphocytes expressing CD26 on their sur-

face were detected (Fig. 3a–f). The percentage of CD3+ lym-

phocytes which stained positive for CD26, was quantified per

region of interest and ranged between 0% and 17% in the

psoriatic dermis (mean 7Æ9 ± 1Æ72 SEM). In the psoriatic

epidermis, between 0% and 31% of the T lymphocytes

coexpressed CD26/DPP-IV (mean 13Æ9 ± 3Æ38 SEM) (Table 1).The T lymphocytes were mainly situated in groups within the

dermal papillae, surrounding small vessels. Fewer T cells were

localized in the epidermis. To summarize, colocalization of

CD26/DPP-IV on T lymphocytes is explicitly present in psori-

atic plaques, albeit in small quantities in both the dermis and

epidermis.

Fig 1. CD26/dipeptidyl-peptidase IV (DPP-IV) is upregulated in

lesional skin of psoriatic patients. CD26/DPP-IV mRNA levels in

epidermal sheets were significantly higher (P = 0Æ002) in lesional skinfrom psoriatic patients (PS; n = 9) than in normal skin (NS; n = 5).

CD26/DPP-IV mRNA levels were determined by quantitative

polymerase chain reaction. Error bars indicate standard deviation.




Chapter 3


129

(a) (b) (c)

(e)

(g)

(d)

(f)

(j)

(m)(l)

(i)

(k)

(h)




130

Discussion

T-helper (Th) 1 lymphocytes, keratinocytes and cytokines are

believed to play critical roles in the pathogenesis of psoriasis.

Evidence has been provided that CD26/DPP-IV may interact

with all these three key players. We have characterized the

pattern of expression of DPP-IV at the mRNA and protein level

in psoriasis compared with normal skin in order to determine

whether or not its expression can function as a (discriminat-

ing) biomarker in psoriasis. Some restricted, divergent studies

have addressed the expression of the CD26/DPP-IV molecule

in the psoriatic inflammatory condition. Their major findings

comprised a typical focal immunostaining in the epidermis

and an increased overall immunostaining of epidermis in

lesional skin,14 which disappeared after treatment with topical

calcipotriol.15 However, the potential of CD26/DPP-IV to

function as a (discriminating) biomarker related to prolifera-

tion and inflammation in psoriasis was not investigated. These

studies did not comprise homogeneous groups of psoriatic

patients and expression was not validated on the mRNA level

or investigated on T cells.

In the present study we found an evident significant upreg-

ulation (11-fold) of CD26/DPP-IV on the mRNA level in pso-

riatic epidermis compared with normal epidermis. These

findings gave rise to further investigations on the distribution

of CD26/DPP-IV in lesional psoriatic skin and allowed us to

find out in what way this expression is topographically

distributed in the epidermis. We confirmed the observed dif-

ferences at mRNA level after immunohistochemical staining

for CD26/DPP-IV at the protein level. We enriched earlier

findings14 by noting a distinct column-like staining pattern in

the rete ridges of psoriatic epidermis. This is a novel feature

and may indicate that the CD26+ basal keratinocytes are capa-

ble of keeping their CD26-positivity when proliferating and

differentiating upward in the stratum spinosum. Table 1

shows that nine out of 11 psoriatic patients expressed this

column-like staining pattern of keratinocytes. On the one

hand, the robust expression of CD26/DPP-IV in the basal and

Table 1 Keratinocyte expression of CD26/dipeptidyl-peptidase IV (DPP-IV) in normal

and psoriatic skin, and T-cell bound CD26/DPP-IV expression in psoriatic skin

Keratinocyte expression T-cell bound CD26/DPP-IV expression

Honeycomb-like

Column-likepattern

Dermis(% CD3+CD26+ mm)2)

Epidermis(% CD3+CD26+ mm)2)

Patient

1 + + 6 192 + + 7 9

3 ++ ++ 17 314 + ) 3 0

5 + + 10 116 + + ND ND

7 ± + 16 298 + ++ 5 7

9 ± ) 0 410 + + 5 22

11 ++ ++ 10 7Healthy subjects

1 ) ) 0 0

2 + ) 0 03 + ) 1 0

4 ± ) 0 0

), no CD26/DPP-IV staining; ±, slight CD26/DPP-IV staining; +, moderate CD26/DPP-IVstaining; ++, pronounced CD26/DPP-IV staining; ND, not done.

Fig 2. Equal topographical immunohistochemical and enzyme histocytometric staining of psoriatic skin vs. normal skin with CD26/dipeptidyl-

peptidase IV (DPP-IV). (a) Immunohistochemical staining of normal skin with CD26/DPP-IV; (b) in psoriatic skin a clearly distinguishable

column-like staining pattern in the suprabasal compartment in the rete ridges was observed; and (c) a patchy suprapapillary staining of the

psoriatic epidermis extending from the stratum basale up to several keratinocyte layers of the stratum spinosum was observed. (d) In addition, a

distinct focal honeycomb-like structure in the suprabasal layers of the psoriatic epidermis was noted. (e) The dermal reactivity for CD26/DPP-IV

shows a complex staining pattern not suitable for a clear-cut analysis of T-cell bound CD26/DPP-IV expression in the dermis. (f,g,i) Enzyme

histocytometric staining of psoriatic skin vs. normal skin (h) with CD26/DPP-IV (clone BA-5) revealed an equal topographical staining pattern

compared with the immunohistochemical staining pattern of CD26/DPP-IV (a–e). (j) Enzyme histocytometric reactivity was absent in serial

sections using the DPP-IV inhibitor diprotin A. (k, l) Frozen sections of murine kidney were stained with CD26/DPP-IV as a positive control (m)

and with diprotin A.




Chapter 3


131

suprabasal layers of the rete ridges of the epidermis in psoria-

sis suggests that CD26-related processes are relevant in the

maintenance of epidermal hyperproliferation. On the other

hand, the chronic over-reaction of the immune system present

in the psoriatic epidermis causes damage and therefore is

undesirable. Therefore, it is attractive to speculate that the

proteolytic activity of CD26/DPP-IV among other things could

possibly intervene in the unbalanced epidermal immune

activation status and tone down this disequilibrium.

In a previous study, increased CD26/DPP-IV expression was

also shown in spongiotic dermatitis,14 which could exclude

specificity for psoriasis. However, the large difference between

epidermal expression of CD26/DPP-IV in psoriatic and normal

skin suggests that assessment of this marker during treatments

may be useful. In this respect, dose-dependency studies with

individual cytokines with either Th1 or Th2 specificity in both

psoriasis and spongiotic dermatitis could be the subject of

future investigations.

It must be noted that the positive immunohistochemical

staining on sections of psoriatic skin represents a positivity

for that protein and does not necessarily mean that CD26/

DPP-IV enzyme activity is present at that site. For that pur-

pose visualization of enzyme activity in tissue sections was

performed by means of enzyme cytochemical methods. We

found a remarkable correspondence between the CD26/DPP-

IV-positive-stained epidermal areas after immunohistochemi-

cal techniques compared with the enzyme cytochemical

staining procedures, which shows that the expression of

CD26/DPP-IV at protein level is not merely present but also

enzymatically active.

Further specification of the topographical distribution of

CD26/DPP-IV on T cells was possible in our study, devel-

oping a double-staining technique with CD3. To the best of

our knowledge, the colocalization of CD26 on T cells in

the (epi)dermal compartment has never been described

before. We found that dermal T cells and epidermal T cells

both express CD26 on their surface, possibly indicating that

CD26+ T cells are able to migrate to the dermis and epi-

dermis. On the other hand, T cells may possibly be able to

acquire CD26 expression in situ after having migrated to the

skin. Previous investigations showed a decreased number of

CD8 CD26 bright T cells in peripheral blood of patients

with severe psoriasis.21 Our current results show that

between 0% and 31% of the epidermal T lymphocytes

express CD26/DPP-IV on their surface compared with 0–

17% in the dermis. Given the fact that epidermal T cells in

the epidermis are predominantly CD8+,27,28 it would be

interesting to investigate whether these (epi)dermal CD26/

DPP-IV+ T cells are CD8+, to find out whether these cells

express CD26 in a dim or bright way and to define further

the T-cell subsets expressing CD26/DPP-IV. It is attractive to

speculate that epidermal CD8+CD26+ T cells are increased

in the psoriatic skin at the expense of extravasation of these

cells from peripheral blood.

This study reveals and confirms that CD26/DPP-IV can

exert a crucial function and can act on the major patho-

genic key players, as it is expressed both on keratinocytes

and T cells in the psoriatic lesion and has been shown to

have a functional enzyme activity as well. Herewith, poten-

tially important cytokines and chemokines involved in the

psoriatic pathogenesis can be affected or degraded in

(a)

(d)(c)(b)

(e) (f)

Fig 3. Phenotyping of CD26+ T cells in lesional psoriatic

(epi)dermis using the Zenon immunofluorescence labelling technique.

Sections of lesional psoriatic skin were double stained using direct

immunofluorescence labelling for CD3 and CD26/dipeptidyl-peptidase

IV (DPP-IV). (a) Costaining of CD3 (green) and CD26 (red) in

lesional psoriatic skin revealed colocalization of these two proteins

(merge; yellow) in the dermis (inset). (b) Monoclonal anti-

CD26 antibody. (c) Monoclonal anti-CD3 antibody. (d) DAPI

counterstaining of nuclei. (e, f) Colocalization of CD3 (green) and

CD26 (red) was also evident in lesional psoriatic epidermis (merge;

yellow).




132

biological activity. In vitro studies have been reported and

demonstrated an altered receptor specificity, binding or acti-

vation for the chemokines RANTES, IP-10, Mig, SDF-1a and

-b, ITAC, eotaxin and MDC;8 in vivo studies with these indi-

vidual cytokines are of future interest.

Currently, DPP-IV inhibitors are of major interest, due to

their potent immunosuppressive and anti-inflammatory effects

in various disease models. Because of the diverse expression of

CD26/DPP-IV in psoriasis outlined in this study, the skin may

be an important organ for evaluating the effect of these

DPP-IV inhibitors and the potential therapeutic applications in

the future.

To summarize, we have shown that CD26/DPP-IV is

strongly upregulated in psoriatic epidermis and that keratino-

cytes and T cells in psoriatic lesions express CD26/DPP-IV

although in a variable way. Moreover, the CD26/DPP-IV

expression on a protein level in psoriatic skin was demon-

strated to be enzymatically active. This points to a versatile,

complex yet possibly important role of this protein as a com-

plementary biomarker in psoriasis. Focusing on the immuno-

logical and functional expression of CD26/DPP-IV in

keratinocytes in vitro could provide a better insight in CD26/

DPP-IV regulation and seems warranted. Furthermore, the

effects on CD26/DPP-IV expression of known and experimen-

tal antipsoriatic agents are an interesting point of focus as

well. Although the exact functional contribution of these

properties of CD26/DPP-IV needs to be further elucidated, the

topographical distribution of this complex multifunctional

protein suggests a relevant contribution in epidermopoiesis

and T-cell signalling in psoriasis.

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RG van Lingen, MKP Poll, MMB Seyger, EMGJ de Jong, PCM van de Kerkhof, PEJ van Erp Distribution of epidermal dipeptidyl-peptidase IV on psoriatic keratinocytes: a comparison with hyperproliferation and aberrant differentiation markersArch Derm Res. 2008; Online early at http://dx.doi.org/10.1007/s00403-008-0862-1.

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Arch Dermatol ResDOI 10.1007/s00403-008-0862-1

123

ORIGINAL PAPER

Distribution of dipeptidyl-peptidase IV on keratinocytes in the margin zone of a psoriatic lesion: a comparison with hyperproliferation and aberrant diVerentiation markers

R. G. van Lingen · M. K. P. Poll · M. M. B. Seyger · E. M. G. J. de Jong · P. C. M. van de Kerkhof · P. E. J. van Erp

Received: 15 January 2008 / Revised: 3 April 2008 / Accepted: 28 April 2008© The Author(s) 2008

Abstract The inXammation process in psoriatic skin ischaracterized by inXux of leukocytes, hyperproliferationand aberrant diVerentiation of keratinocytes regulated viacytokines. Dipeptidyl-peptidase IV (DPPIV) is known to beupregulated on keratinocytes in the psoriatic lesion. Theobjective was to gain insight into dynamics of DPPIVexpression and enzyme activity together with keratinocyteproliferation and diVerentiation markers during develop-ment of a psoriatic lesion, in order to investigate coherencein mechanisms behind the upregulation of DPPIV in psori-atic skin. The expression of DPPIV, Ki-67 antigen and ker-atin-16 (K16) was studied in the dynamic margin zone ofthe psoriatic lesion, examining skin sections of the clini-cally uninvolved skin, the early lesion and the chroniclesion of psoriatic patients compared to healthy volunteersusing immunohistochemical and enzymehistochemicalstaining methods. DPPIV-expression and enzyme activity,Ki-67 antigen and K16 are signiWcantly upregulated in thecentre and inner margin of the lesion compared to clinicallyuninvolved skin and the healthy volunteers skin. Mutuallybetween the centre and inner margin, this upregulation didnot diVer signiWcantly. The clinical symptomless skinproved to have signiWcantly elevated DPPIV enzyme activ-ity compared to the skin of healthy volunteers. We demon-strate that DPPIV is expressed and enzymatically activewell before the development of an overt psoriatic lesion.The abnormal DPPIV distribution in psoriatic skin does notcoincide with known markers of aberrant growth and diVer-

entiation of keratinocytes, which makes DPPIV (expressionand enzyme activity) a marker standing on its own.

Keywords Psoriasis · Dipeptidyl peptidase IV · Keratin 16 · Ki-67 · CD26

Introduction

Psoriasis is considered to be an organ-speciWc autoimmunedisease, caused by a combination of genetic and environ-mental factors and is triggered and maintained by an acti-vated cellular immune system. Its pathology is mainlycharacterized by cutaneous inXammatory inXux of leuko-cytes and hyperproliferation and aberrant diVerentiation ofkeratinocytes. Within the psoriatic lesion, there is a mutualinteraction between these keratinocytes and mononuclearleukocytes by means of a third involved party, the cyto-kines [5, 11]. The chemical modiWcation of cytokines andhence the modiWcation of their physiological function is ofmajor importance to understand their actual role in thepathophysiology of the psoriatic process.

Dipeptidyl peptidase IV (CD26) is a 110-kDa mem-brane-anchored glycoprotein with a variety of functions [1,4]. In general, it is widely distributed throughout the humanbody and varies in density and form between variousorgans and cell types. Its proteolytic properties enable it todirectly modify substrates such as cytokines in their naturalin vivo form, (e.g., CXCL12, CCL5, CXCL11, CXCL10,GLP-1/2) [1, 4, 7, 14].

In human skin, DPPIV is known to be expressed onkeratinocytes and known to be upregulated in psoriasis [4,13, 19, 22]. However, little data are available concerningthe potential functional implications of such an upregula-tion. Selective blockers of DPPIV activity are currently

R. G. van Lingen (&) · M. K. P. Poll · M. M. B. Seyger · E. M. G. J. de Jong · P. C. M. van de Kerkhof · P. E. J. van ErpDepartment of Dermatology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlandse-mail: [email protected]

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being developed as modulators of immunological responses[15]. Recently, it was shown that such an inhibition couldsuppress keratinocyte proliferation in vitro and partiallyrestore keratinocyte diVerentiation in vivo [19]. Mappingthe DPPIV glycoprotein in psoriasis may therefore add toour current knowledge. Considering the common groundsof the three main pathological components of psoriasis (Tcells, keratinocytes and cytokines) and the various proper-ties of DPPIV, it is attractive to speculate that DPPIV mightbe involved in the modulation of the inXammatory processand the keratinocyte abnormalities in psoriasis.

Ensued from this, our goal was to map the epidermalexpression and enzyme activity of DPPIV in relation toknown markers of proliferation (Ki-67) and abnormal mat-uration (Keratin 16) of psoriatic keratinocytes in the epider-mis [10]. Coherence in dynamics of expression of thesethree markers could result in expanding knowledge on theexisting epidermal disbalance in psoriasis. We studied theexpression and enzyme activity of DPPIV in the dynamicmargin zone of the psoriatic lesion in order to investigatewhether DPPIV distribution diVers with the distribution ofK16 and Ki-67 in clinically uninvolved psoriatic skin, inthe early lesion and subsequently in the chronic psoriaticlesion. Accordingly, coherence in dynamics of DPPIV(expression and enzyme activity) with keratinocyte prolif-eration and diVerentiation markers during the developmentof a psoriatic plaque could reveal more insight into themechanisms behind the known upregulation of DPPIV inpsoriatic skin.


Patients

A total of seven psoriatic patients (mean age § SEM,63 § 3 years) with mild-to-moderate plaque psoriasis [Pso-riasis Area and Severity Index (PASI) ¸2 and ·12] and acontrol group, composed of three healthy volunteers (meanage 52 § 6 years), participated in this study. Patients hadstable plaque-type psoriasis. None of the participants hadused any systemic anti-psoriatic or immune-modulatingtherapy for at least 4 weeks and no topical therapy for atleast 2 weeks prior to the biopsy. The healthy volunteershad no history of dermatological or immunological disease.All participants had given written informed consent beforeenrollment.

Biopsies

From the psoriatic patients three 4-mm punch biopsies wereobtained, respectively, from the centre of the plaque, theinner margin of the plaque comprising the outmost clini-

cally involved area and the clinically uninvolved skin (atleast 2 cm from the lesion). From the healthy volunteersone 4-mm punch biopsy was obtained. All participantsreceived local anesthesia (1% xylocaine/adrenaline) beforethe biopsies were taken. Skin defects were optionallyclosed with one suture. The obtained specimens wereembedded in Tissue-Tek OCT compound (Sakura, Zoeter-woude, Netherlands), snap-frozen in liquid nitrogen andstored at ¡80°C until use.

Histochemical methods

Immunohistochemical procedure

The immunohistochemical staining was performed asdescribed previously [2, 23]. BrieXy, cryostat sections of7 �m were sliced onto (3-amino propyl)triethoxysilane(AAS)-coated slides, air-dried for 10 min and Wxed in coldacetone for 10 min. After blocking for endogenous peroxi-dase for 20 min, sections were incubated with either CD26monoclonal antibody (1:50) (clone BA-5, Santa Cruz Bio-technology, Santa Cruz, FL), Ki-67 (1:25) (clone MIB-1,DAKO, Copenhagen, Denmark) or anti-Keratin-16 (1:20)(clone LL025, Monosan, Uden, Netherlands), diluted in1%BSA/PBS for 1 h. Subsequently, sections underwentsecondary incubation with IgG-labelled polymer, horserad-ish peroxidase antimouse-antibody EnVision+ (DAKO,Copenhagen, Denmark) for 30 min. As a coloring agentaminoethylcarbazole + high-sensitivity substrate chromo-gen (DAKO, Copenhagen, Denmark) was used for 10 minat 37°C. Before and after each incubation, sections werewashed with PBS for 15 min. All incubations were per-formed in a dark, humid environment. Counterstaining wasperformed with Mayer’s hematoxylin. Subsequently, thesections were mounted in glycerol gelatin.

Enzymehistochemical procedure

The demonstration of DPPIV activity was achieved usingthe method, described by Khalaf et al. [9] with some minormodiWcations [22]. The frozen biopsies were sliced into 10-�m sections on AAS-coated slides. These were air-dried for20 min and Wxed in cold acetone for 10 min. For the incu-bation mixture, 3 mg of glycyl-propyl-4-methoxy-2-naph-thylamide (GPMN) (Bachem, Bubendorf, Switzerland) wasdissolved in 0.2 ml dimethylformamide. This substrate wasadded to 4.6 ml of PBS and subsequently mixed with 5 mgof fast blue B salt (FBB) (Sigma–Alderich, St. Louis, MO)dissolved in 0.2 ml dimethylformamide and Wltered. Thesamples were incubated in a humid, dark chamber at roomtemperature for 45 min with 200 �l of the incubation mix-ture added to each section. After incubation the slides werewashed with distilled water, brieXy counterstained with

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being developed as modulators of immunological responses[15]. Recently, it was shown that such an inhibition couldsuppress keratinocyte proliferation in vitro and partiallyrestore keratinocyte diVerentiation in vivo [19]. Mappingthe DPPIV glycoprotein in psoriasis may therefore add toour current knowledge. Considering the common groundsof the three main pathological components of psoriasis (Tcells, keratinocytes and cytokines) and the various proper-ties of DPPIV, it is attractive to speculate that DPPIV mightbe involved in the modulation of the inXammatory processand the keratinocyte abnormalities in psoriasis.

Ensued from this, our goal was to map the epidermalexpression and enzyme activity of DPPIV in relation toknown markers of proliferation (Ki-67) and abnormal mat-uration (Keratin 16) of psoriatic keratinocytes in the epider-mis [10]. Coherence in dynamics of expression of thesethree markers could result in expanding knowledge on theexisting epidermal disbalance in psoriasis. We studied theexpression and enzyme activity of DPPIV in the dynamicmargin zone of the psoriatic lesion in order to investigatewhether DPPIV distribution diVers with the distribution ofK16 and Ki-67 in clinically uninvolved psoriatic skin, inthe early lesion and subsequently in the chronic psoriaticlesion. Accordingly, coherence in dynamics of DPPIV(expression and enzyme activity) with keratinocyte prolif-eration and diVerentiation markers during the developmentof a psoriatic plaque could reveal more insight into themechanisms behind the known upregulation of DPPIV inpsoriatic skin.


Patients

A total of seven psoriatic patients (mean age § SEM,63 § 3 years) with mild-to-moderate plaque psoriasis [Pso-riasis Area and Severity Index (PASI) ¸2 and ·12] and acontrol group, composed of three healthy volunteers (meanage 52 § 6 years), participated in this study. Patients hadstable plaque-type psoriasis. None of the participants hadused any systemic anti-psoriatic or immune-modulatingtherapy for at least 4 weeks and no topical therapy for atleast 2 weeks prior to the biopsy. The healthy volunteershad no history of dermatological or immunological disease.All participants had given written informed consent beforeenrollment.

Biopsies

From the psoriatic patients three 4-mm punch biopsies wereobtained, respectively, from the centre of the plaque, theinner margin of the plaque comprising the outmost clini-

cally involved area and the clinically uninvolved skin (atleast 2 cm from the lesion). From the healthy volunteersone 4-mm punch biopsy was obtained. All participantsreceived local anesthesia (1% xylocaine/adrenaline) beforethe biopsies were taken. Skin defects were optionallyclosed with one suture. The obtained specimens wereembedded in Tissue-Tek OCT compound (Sakura, Zoeter-woude, Netherlands), snap-frozen in liquid nitrogen andstored at ¡80°C until use.

Histochemical methods

Immunohistochemical procedure

The immunohistochemical staining was performed asdescribed previously [2, 23]. BrieXy, cryostat sections of7 �m were sliced onto (3-amino propyl)triethoxysilane(AAS)-coated slides, air-dried for 10 min and Wxed in coldacetone for 10 min. After blocking for endogenous peroxi-dase for 20 min, sections were incubated with either CD26monoclonal antibody (1:50) (clone BA-5, Santa Cruz Bio-technology, Santa Cruz, FL), Ki-67 (1:25) (clone MIB-1,DAKO, Copenhagen, Denmark) or anti-Keratin-16 (1:20)(clone LL025, Monosan, Uden, Netherlands), diluted in1%BSA/PBS for 1 h. Subsequently, sections underwentsecondary incubation with IgG-labelled polymer, horserad-ish peroxidase antimouse-antibody EnVision+ (DAKO,Copenhagen, Denmark) for 30 min. As a coloring agentaminoethylcarbazole + high-sensitivity substrate chromo-gen (DAKO, Copenhagen, Denmark) was used for 10 minat 37°C. Before and after each incubation, sections werewashed with PBS for 15 min. All incubations were per-formed in a dark, humid environment. Counterstaining wasperformed with Mayer’s hematoxylin. Subsequently, thesections were mounted in glycerol gelatin.

Enzymehistochemical procedure

The demonstration of DPPIV activity was achieved usingthe method, described by Khalaf et al. [9] with some minormodiWcations [22]. The frozen biopsies were sliced into 10-�m sections on AAS-coated slides. These were air-dried for20 min and Wxed in cold acetone for 10 min. For the incu-bation mixture, 3 mg of glycyl-propyl-4-methoxy-2-naph-thylamide (GPMN) (Bachem, Bubendorf, Switzerland) wasdissolved in 0.2 ml dimethylformamide. This substrate wasadded to 4.6 ml of PBS and subsequently mixed with 5 mgof fast blue B salt (FBB) (Sigma–Alderich, St. Louis, MO)dissolved in 0.2 ml dimethylformamide and Wltered. Thesamples were incubated in a humid, dark chamber at roomtemperature for 45 min with 200 �l of the incubation mix-ture added to each section. After incubation the slides werewashed with distilled water, brieXy counterstained with

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Mayer’s hematoxylin and immersed for 30 s in alkaline tapwater. After rinsing with distilled water, they were mountedin glycerol gelatin.

Negative and positive controls

In the immunohistochemical procedure, as a negative con-trol the primary antibody was omitted. As a negative con-trol in the enzymehistochemical procedure, inhibition ofDPPIV activity was accomplished by pretreating the slideswith 200 �l of 5 mM Diprotin A (Peptides International,Louisville, KY) for 15 min at 37°C [3]. As positive control,murine kidney tissue was used in the enzymehistochemicalprocedure [18, 21].

Image analysis

QuantiWcation

The immunohistochemical and enzymehistochemicalDPPIV-stained sections were analyzed using transmissionlight microscopy with an objective magniWcation of 200£.Per patient, three of the diVerent biopsy sections (center,inner margin, clinically uninvolved psoriatic skin) wereanalyzed for the full width of the section. For quantiWcationof the epidermal DPPIV expression and enzyme activity,the following semi-quantitative Wve-point scale was used:0 = no staining, 1 = slight staining, 2 = moderate staining,3 = moderately pronounced staining, 4 = pronounced stain-ing.

In order to analyze K16 and Ki-67 positive cells, digitalphotographs were made at 100£ magniWcation. Each pho-tograph was analyzed using IP-lab software (Scanalytics,USA). For quantiWcation of the number of cells positive forKi-67 and the percentage epidermal area positive for K16, adeWned window was set in the epidermis as the region ofinterest (ROI) which was representative for the whole sec-tion. The total ROI and the K16-positive area within theROI were measured. The K16 value was expressed as per-centage K16-positive area of the ROI (%). The number ofKi-67 positive cells was determined by counting the Ki-67positive nuclei in a representative epidermal ROI. A line,following the stratum basale of the ROI, was set out andmeasured. The number of Ki-67 positive nuclei wasexpressed as positive cells per mm length of basementmembrane.


Results are presented as means §SD of the mean. All anal-yses were carried out using Statistica® statistical software,version 7.0. Comparison of the non-parametric immunohis-tochemical and enzymehistochemical DPPIV values for the

plaque centre, inner margin and clinically uninvolved skinwas performed using the Kruskal–Wallis analysis of vari-ance. For analysis of the markers K16 and Ki-67 concern-ing the plaque centre, inner margin and clinicallyuninvolved skin we performed analysis of variance(ANOVA) and if signiWcant, Duncan’s post hoc compari-son was performed. The Student’s t test was carried out tostudy the diVerence in expression of markers between theuninvolved psoriatic skin and the healthy volunteers skin.Correlation was tested with the Spearman’s correlation test.Statistical signiWcance was set at P < 0.05.

Results

Patients

Histologically, the skin of the healthy volunteers was nor-mal (hematoxylin–eosin staining). Seven patients (5 males,2 females) diagnosed with psoriasis participated. In order toassess the severity of the psoriatic lesion used for biopsy,SUM scores (0–12) were determined. SUM score is a psori-atic lesion severity score, comprising erythema (0–4), indu-ration (0–4) and desquamation (0–4) wherein a score of 0represents no psoriasis and 12 the highest possible severityof the plaque. The mean SUM score in this study was5.57 § 0.34.

Epidermal distribution of dipeptidyl-peptidase IV, Ki-67 and Keratin-16 (Fig. 1)

In correspondence with earlier Wndings [22], upregulationof DPPIV on keratinocytes was seen in all of the patients,although to a diVerent extent. In line with these earlier Wnd-ings, the immunohistochemical and the enzymehistochemi-cal staining of DPPIV followed a similar epidermal pattern;focally pronounced patches of staining, mainly displaying ahoneycomb like structure, in psoriatic sections. In those ofthe healthy volunteers this pattern was practically absent. Inbrief, the psoriatic pattern was generally more extensiveand predominantly located para-papillary with an optimumin the epidermal area around the top of the papillae. Usuallythe staining was conWned to the lower two third of the totalepidermis, or more speciWcally to the lower three quartersof the stratum spinosum (Fig. 1). In both healthy and psori-atic skin, no staining in the stratum lucidum was observed.In the psoriatic coupes, additional column-like structurescould regularly be observed.

The DPPIV immunohistochemical expression andenzyme activity in the centre of the plaque, the inner mar-gin and the clinically uninvolved skin showed a comparablepattern with correlations, respectively, of 0.79, 0.85 and0.87 (P<0.02). Considering this comparable pattern, the

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enzyme activity of DPPIV was chosen in this study as a ref-erence for drawing comparisons with the Ki-67 and K16markers as it encompasses more potential to study dynam-ics than the protein expression does. Namely, proteinexpression only conWrms the presence of DPPIV whereasenzyme activity denotes presence and activity of DPPIV.

As far as the proliferation marker Ki-67 is concerned, itpredominantly appeared in the stratum basale in both psori-atic and healthy skin although to a diVerent extent (Fig. 1).Solitary Ki-67 positive cells could be observed sporadicallyin the stratum spinosum of the clinically uninvolved skin ofpsoriatics and the skin of healthy volunteers. Obviously, inthe centre and inner margin of the psoriatic lesion, gener-ally a greater number of Ki-67-positive cells was observedin all patients.

Keratin-16, a diVerentiation marker, only stained posi-tive in the epidermis of the plaque centre and inner marginconWrming the aberrant diVerentiation in these parts asopposed to the clinically uninvolved psoriatic and healthy

skin (Fig. 1). The staining was seen throughout the wholestratum spinosum, with an optimum towards the stratumbasale.

Dynamics in epidermal distribution of dipeptidyl-peptidase IV, Ki-67 and Keratin-16 (Fig. 2)

Throughout the dynamics of the margin zone of the psoriaticlesion, a stronger signiWcant DPPIV enzymatic activity couldbe noticed in both the inner margin and centre of the lesioncompared to the clinically uninvolved psoriatic skin(1.38 § 0.24 and 1.57 § 0.27 vs. 0.62 § 0.15; P < 0.01 andP < 0.05) (Fig. 2). Clinically uninvolved psoriatic skin alsoshowed a signiWcant increase in enzymatic activity comparedto skin of healthy volunteers (P < 0.05). However, whenobserving the expression in the centre and the inner marginmutually, no signiWcant diVerence in expression was detected.

Equal to the DPPIV enzyme activity, Ki-67 antigen wassigniWcantly strongly expressed in both the plaque centre

Fig. 1 Expression of DPPIV protein expression, enzyme activity, Keratin 16 and Ki-67 throughout the margin zone of a psoriatic lesion compared to nor-mal skin. Plaque centre, inner margin, clinically uninvolved skin (UIS): £200 magniWcation. Healthy volunteers skin (HV): £100 magniWcation. Notice-ably, in the skin of healthy vol-unteers the DPPIV enzyme-activity staining creates a brownish background staining

Fig. 2 Mean scores of DPPIV enzyme activity, Ki67 and K16 expression throughout the mar-gin zone. * = signiWcant. (Mean § SEM)

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enzyme activity of DPPIV was chosen in this study as a ref-erence for drawing comparisons with the Ki-67 and K16markers as it encompasses more potential to study dynam-ics than the protein expression does. Namely, proteinexpression only conWrms the presence of DPPIV whereasenzyme activity denotes presence and activity of DPPIV.

As far as the proliferation marker Ki-67 is concerned, itpredominantly appeared in the stratum basale in both psori-atic and healthy skin although to a diVerent extent (Fig. 1).Solitary Ki-67 positive cells could be observed sporadicallyin the stratum spinosum of the clinically uninvolved skin ofpsoriatics and the skin of healthy volunteers. Obviously, inthe centre and inner margin of the psoriatic lesion, gener-ally a greater number of Ki-67-positive cells was observedin all patients.

Keratin-16, a diVerentiation marker, only stained posi-tive in the epidermis of the plaque centre and inner marginconWrming the aberrant diVerentiation in these parts asopposed to the clinically uninvolved psoriatic and healthy

skin (Fig. 1). The staining was seen throughout the wholestratum spinosum, with an optimum towards the stratumbasale.

Dynamics in epidermal distribution of dipeptidyl-peptidase IV, Ki-67 and Keratin-16 (Fig. 2)

Throughout the dynamics of the margin zone of the psoriaticlesion, a stronger signiWcant DPPIV enzymatic activity couldbe noticed in both the inner margin and centre of the lesioncompared to the clinically uninvolved psoriatic skin(1.38 § 0.24 and 1.57 § 0.27 vs. 0.62 § 0.15; P < 0.01 andP < 0.05) (Fig. 2). Clinically uninvolved psoriatic skin alsoshowed a signiWcant increase in enzymatic activity comparedto skin of healthy volunteers (P < 0.05). However, whenobserving the expression in the centre and the inner marginmutually, no signiWcant diVerence in expression was detected.

Equal to the DPPIV enzyme activity, Ki-67 antigen wassigniWcantly strongly expressed in both the plaque centre

Fig. 1 Expression of DPPIV protein expression, enzyme activity, Keratin 16 and Ki-67 throughout the margin zone of a psoriatic lesion compared to nor-mal skin. Plaque centre, inner margin, clinically uninvolved skin (UIS): £200 magniWcation. Healthy volunteers skin (HV): £100 magniWcation. Notice-ably, in the skin of healthy vol-unteers the DPPIV enzyme-activity staining creates a brownish background staining

Fig. 2 Mean scores of DPPIV enzyme activity, Ki67 and K16 expression throughout the mar-gin zone. * = signiWcant. (Mean § SEM)

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and the inner margin compared to the clinically uninvolvedpsoriatic skin (172.65 § 16.06 and 167.60 § 13.84 vs.70.44 § 7.97; both P < 0.01) (Fig. 2). The clinically unin-volved psoriatic skin showed a stronger proliferation ten-dency of keratinocytes than the healthy volunteers skin,albeit borderline signiWcant (P = 0.057). However, whenobserving the Ki-67 expression in the centre and the innermargin mutually again, no signiWcant diVerence in expres-sion was detected.

With respect to the expression of K16 in the variousparts of the margin zone, the plaque centre and the innermargin showed signiWcantly more expression of K16 incomparison to the clinically uninvolved psoriatic skin(67.0 § 3.13 and 65.43 § 2.1 vs. 0 § 0) (Fig. 2). This wasin line with the expected expression pattern because inuninvolved psoriatic skin and healthy volunteers skin thereexists a natural absence of K16 expression. Mutually how-ever, the centre and margin did, once more, not diVer sig-niWcantly in K16 expression.

Correlation of epidermal distribution of dipeptidyl-peptidase IV, Ki-67antigen and K16

In general, there were no signiWcant correlations observedbetween enzymatic cytochemical activity of DPPIV andexpression of aberrant proliferation or diVerentiation ofkeratinocytes throughout the dynamics of the margin zone.

Remarkably, the Ki-67 positive cell count and percent-age of K16 positively stained epidermis showed a strongoverall correlation in the psoriatic patients, taking togetherboth the plaque centre and the inner margin (� = 0.88;P < 0.05). Though, when investigating the correlation ofthese markers per locus separately, no signiWcant correla-tion could be observed (plaque centre � = 0.54; inner mar-gin � = 0.3).

Discussion

Dipeptidyl peptidase IV has diverse properties as an ecto-peptidase, enabling it to play a key role in the chronic pso-riatic immune response which is mainly orchestrated by Tcells, cytokines and keratinocytes in psoriasis. Direct evi-dence that DPPIV inhibitors are relevant to keratinocytebiology has been provided by the observation that suchinhibitors can suppress keratinocyte proliferation in vitroand can partially restore keratinocyte diVerentiation invivo [19]. This would imply that DPPIV may aVect andinteract with the aberrant keratinocyte abnormalities inpsoriasis. In line with previous Wndings [22], the presentstudy conWrms a strong upregulation of DPPIV on kerati-nocytes in psoriatic epidermis but additionally reveals itto be continuously present throughout the dynamics of the

margin zone. Moreover, it aimed to shed light on the func-tional implication of this upregulation combining it withthe expression of markers characteristic for proliferation(Ki-67) and diVerentiation (K16) in psoriatic keratino-cytes as coherence in dynamics of expression of thesemarkers could result in creating an epidermal biomarkerin psoriasis.

Topographically, the distribution of the enzyme activityof DPPIV did not coincide entirely with the areas of hyper-proliferation and aberrant diVerentiation of keratinocytes ina psoriatic section which ultimately makes DPPIV expres-sion (enzyme activity and protein expression) an entity initself in psoriasis. The margin zone was previously provento be a suitable model for studying the dynamics of a psori-atic lesion by representing the chronic and the early processand the uninvolved skin [6, 20, 23]. The inner margin waspresumed to represent an early active lesion and thereforereXecting a diVerent level of expression. DPPIV enzymeactivity, Ki-67 and K16, all three show, throughout themargin zone, signiWcant upregulation in the centre andinner margin of the plaque compared to the clinically unin-volved skin and the healthy volunteers skin. However,mutually the upregulation in expression in the centre andinner margin did not diVer signiWcantly for both the Ki-67,K16 marker as well as for DPPIV enzyme activity. Onecould suggest that in this study within a psoriatic lesionthere exists a homogeneous level of keratinocyte prolifera-tion, diVerentiation and DPPIV enzyme activity as partsrepresenting an early (inner margin) or more chronic (cen-ter) lesion do not seem to make a diVerence in expressionfor the markers under study. Moreover, correlation testsapplied to the margin zone in this investigation showed nocoherence in dynamics of epidermal DPPIV enzyme activ-ity, hyperproliferation and aberrant diVerentiation of kerati-nocytes.

The symptomless skin at 2-cm distance from a psoriaticlesion proved to have signiWcantly elevated DPPIV enzymeactivity as compared to skin of healthy volunteers, whereasKi-67 and K16 were essentially slightly present or absent inthe symptomless skin. Although the symptomless skin mayharbor some “preclinical lesions” or can be subclinicallyinvolved, we can conclude that in an early phase of the pso-riatic process well before an overt lesion has developed, anincreased DPPIV enzyme activity is present clearly show-ing that DPPIV is involved in an early phase of the psoriaticcascade [8, 12].

A number of other molecules exhibiting DPPIV-likeenzyme activity have been introduced recently, and termed“DPP-IV activity and/or structure-homologues” (DASH)[17]. This group comprises several proteases including attr-actin, DPP 8 and 9 [16, 17]. DASH have potential abilitiesto complement and/or functionally substitute DPPIV on thelevel of its enzymatic activity. In this light, a marginal note

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has to be made as the DASH might be involved in DPPIV-like processes (as could be the case in the current investiga-tion). Due to the ubiquitous expression pattern and multi-functional nature of DPPIV speciWcally and the majority ofthe DASH in general, it may be diYcult to deduce theselectivity of DPPIV from the DASH group. However, ourresults did not show discrepancies between the immunohis-tochemical and enzymehistochemical staining, whichimplies that the possible presence of DPPIV homologuesdoes not interfere with the interpretation of the data in thecurrent study.

Our goal was to determine whether the distribution ofDPPIV in the epidermis throughout the dynamics of themargin zone, coheres with markers for proliferation anddiVerentiation of keratinocytes in psoriasis in order toexpand knowledge on the existing epidermal dysbalance inpsoriasis. Considering the localization of DPPIV expres-sion and enzyme activity in the epidermis, being primarilyconcentrated towards the (supra) basal layers, it is possibleto presume an active role for DPPIV in the proliferation ofkeratinocytes in the pathogenesis of psoriasis. On the otherhand, considering the functional enzymatic characteristicsof DPPIV, it may well be feasible that the peptidase activityactually restrains the inXammatory disbalance in the psori-atic skin through down-regulation of cytokines secondarilyaVecting the keratinocyte malfunctioning. Therefore, thepsoriatic skin represents an interesting eVector organ forDPPIV inhibitors which could tackle not only keratinocytesand cytokines but also (DPPIV+) T cells, key players in thepsoriatic pathogenesis all sharing common grounds withDPPIV. Future studies should address the eVect of suchinhibitors in psoriasis.

In conclusion, we have demonstrated diVerential expres-sion and activity of DPPIV in normal and diseased skinthroughout the dynamics of the margin zone. In addition,we have shown that DPPIV (protein expression andenzyme activity) is expressed well before the developmentof the overt psoriatic lesion before the accumulation of Tcells and other inXammatory cells. Moreover, the DPPIVenzyme activity does not increase signiWcantly in the morechronic phase of the psoriatic process. Therefore, DPPIV isa consistent feature of the psoriatic lesion. The abnormalenzyme activity in psoriatic diseased skin does not seem tocorrelate with known markers of aberrant growth anddiVerentiation of keratinocytes, which makes DPPIV amarker standing on its own. Future studies should focus onwhether DPPIV can be regarded a useful biomarker fortherapy evaluation.

Open Access This article is distributed under the terms of theCreative Commons Attribution Noncommercial License whichpermits any noncommercial use, distribution, and reproduction in anymedium, provided the original author(s) and source are credited.

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has to be made as the DASH might be involved in DPPIV-like processes (as could be the case in the current investiga-tion). Due to the ubiquitous expression pattern and multi-functional nature of DPPIV speciWcally and the majority ofthe DASH in general, it may be diYcult to deduce theselectivity of DPPIV from the DASH group. However, ourresults did not show discrepancies between the immunohis-tochemical and enzymehistochemical staining, whichimplies that the possible presence of DPPIV homologuesdoes not interfere with the interpretation of the data in thecurrent study.

Our goal was to determine whether the distribution ofDPPIV in the epidermis throughout the dynamics of themargin zone, coheres with markers for proliferation anddiVerentiation of keratinocytes in psoriasis in order toexpand knowledge on the existing epidermal dysbalance inpsoriasis. Considering the localization of DPPIV expres-sion and enzyme activity in the epidermis, being primarilyconcentrated towards the (supra) basal layers, it is possibleto presume an active role for DPPIV in the proliferation ofkeratinocytes in the pathogenesis of psoriasis. On the otherhand, considering the functional enzymatic characteristicsof DPPIV, it may well be feasible that the peptidase activityactually restrains the inXammatory disbalance in the psori-atic skin through down-regulation of cytokines secondarilyaVecting the keratinocyte malfunctioning. Therefore, thepsoriatic skin represents an interesting eVector organ forDPPIV inhibitors which could tackle not only keratinocytesand cytokines but also (DPPIV+) T cells, key players in thepsoriatic pathogenesis all sharing common grounds withDPPIV. Future studies should address the eVect of suchinhibitors in psoriasis.

In conclusion, we have demonstrated diVerential expres-sion and activity of DPPIV in normal and diseased skinthroughout the dynamics of the margin zone. In addition,we have shown that DPPIV (protein expression andenzyme activity) is expressed well before the developmentof the overt psoriatic lesion before the accumulation of Tcells and other inXammatory cells. Moreover, the DPPIVenzyme activity does not increase signiWcantly in the morechronic phase of the psoriatic process. Therefore, DPPIV isa consistent feature of the psoriatic lesion. The abnormalenzyme activity in psoriatic diseased skin does not seem tocorrelate with known markers of aberrant growth anddiVerentiation of keratinocytes, which makes DPPIV amarker standing on its own. Future studies should focus onwhether DPPIV can be regarded a useful biomarker fortherapy evaluation.

Open Access This article is distributed under the terms of theCreative Commons Attribution Noncommercial License whichpermits any noncommercial use, distribution, and reproduction in anymedium, provided the original author(s) and source are credited.

References

1. Boonacker E, Van Noorden CJ (2003) The multifunctional ormoonlighting protein CD26/DPPIV. Eur J Cell Biol 82:53–73

2. Bovenschen HJ, Seyger MM, van de Kerkhof PC (2005) Plaquepsoriasis versus atopic dermatitis and lichen planus: a comparisonfor lesional T cell subsets, epidermal proliferation and diVerentia-tion. Br J Dermatol 153:72–78

3. Christopherson KW, Cooper S, Broxmeyer HE (2003) Cell sur-face peptidase CD26/DPPIV mediates G-CSF mobilization ofmouse progenitor cells. Blood 101:4680–4686

4. De Meester I, Korom S, Van Damme J, Scharpe S (1999) CD26,let it cut or cut it down. Immunol Today 20:367–375

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6. Gerritsen MJ, Elbers ME, de Jong EM, Van de Kerkhof PC (1997)Recruitment of cycling epidermal cells and expression of Wlaggrin,involucrin and tenascin in the margin of the active psoriaticplaque, in the uninvolved skin of psoriatic patients and in the nor-mal healthy skin. J Dermatol Sci 14:179–188

7. Gorrell MD, Gysbers V, McCaughan GW (2001) CD26: a multi-functional integral membrane and secreted protein of activatedlymphocytes. Scand J Immunol 54:249–264

8. Hammar H, Hellerstrom C (1970) The oxygen consumption of thegerminal epithelium in normal and psoriatic skin. Br J Dermatol83:371–375

9. Khalaf MR, Aqel NM, Hayhoe FG (1987) Histochemistry ofdipeptidyl aminopeptidase (DAP) II and IV in reactive lymphoidtissues and malignant lymphoma. J Clin Pathol 40:480–485

10. Kuijpers AL, Van Pelt JP, Bergers M, Boegheim PJ, Den BakkerJE, Siegenthaler G, Van de Kerkhof PC, Schalkwijk J (1998) TheeVects of oral liarozole on epidermal proliferation and diVerentia-tion in severe plaque psoriasis are comparable with those of acitre-tin. Br J Dermatol 139:380–389

11. Lowes MA, Bowcock AM, Krueger JG (2007) Pathogenesis andtherapy of psoriasis. Nature 445:866–873

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14. Oravecz T, Pall M, Roderiquez G, Gorrell MD, Ditto M, NguyenNY, Boykins R, Unsworth E, Norcross MA (1997) Regulation ofthe receptor speciWcity and function of the chemokine RANTES(regulated on activation, normal T cell expressed and secreted) bydipeptidyl peptidase IV (CD26)-mediated cleavage. J Exp Med186:1865–1872

15. Pratley RE, Salsali A (2007) Inhibition of DPP-4: a new therapeu-tic approach for the treatment of type 2 diabetes. Curr Med ResOpin 23:919–931

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17. Sedo A, Malik R (2001) Dipeptidyl peptidase IV-like molecules:homologous proteins or homologous activities? Biochim BiophysActa 1550:107–116

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dipeptidyl peptidase IV and aminopeptidase N target major patho-genetic steps in acne initiation. J Invest Dermatol 127:1042–1051

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22. Van Lingen RG, Van de Kerkhof PCM, Seyger MMB, De JongEMGJ, van Rens DWA, Poll MKP, Zeeuwen PLJM, Van Erp PEJ(2008) CD26 / DPPIV in psoriatic skin: Upregulation and topo-graphical changes. Br J Dermatol (In Press, online early)

23. Vissers WH, Arndtz CH, Muys L, Van Erp PE, de Jong EM, Vande Kerkhof PC (2004) Memory eVector (CD45RO+) and cytotoxic(CD8+) T cells appear early in the margin zone of spreading pso-riatic lesions in contrast to cells expressing natural killer receptors,which appear late. Br J Dermatol 150:852–859

142

Chapter 3

RG van Lingen, PCM van de Kerkhof, EMGJ de Jong, MMB Seyger, JBM Boezeman, PEJ van ErpReduced CD26bright expression of peripheral blood CD8+ T-cell subsets in psoriatic patientsExp Dermatol. 2008 Apr; 17 (4): 343-48.

3.3


143

Reduced CD26bright expression of peripheral bloodCD8+ T-cell subsets in psoriatic patients

Rosanne G. van Lingen, Peter C. M. van de Kerkhof, Elke M. G. J. de Jong, Marieke M. B. Seyger,

Jan B. M. Boezeman and Piet E. J. van Erp

Department of Dermatology, University Medical Centre St Radboud, Nijmegen, The Netherlands

Correspondence: Rosanne G. van Lingen, Department of Dermatology, University Medical Centre St Radboud, P.O. Box 9101, NL- 6500 HB

Nijmegen, The Netherlands, Tel.: +31 24 3617240, Fax: +31 24 3541184, e-mail: [email protected]

Accepted for publication 18 September 2007

Abstract

Background: T cells have been shown to be highly relevant in

psoriasis. CD26 is a novel T-cell activation marker involved in

various T-cell functions, e.g. (i) co-stimulation, (ii) migration and

(iii) T-cell memory response. In particular, CD26bright peripheral

blood T cells have been shown to be altered in several

autoimmune diseases.

Objective: To characterize CD26-expression of T-cell subsets in

psoriatic patients compared to healthy subjects.

Methods: Peripheral blood was obtained from 15 untreated

patients with severe psoriasis and from nine healthy subjects. The

presence of specific CD26-related T-cell subsets was assessed by

flow cytometry.

Results: The CD26bright expression of CD8+ lymphocytes

revealed a statistically significant (P<0.05) decrease in psoriatic

patients. The majority of CD4+CD26bright cells are CD45RO+,

whereas the minority of CD8+CD26bright cells are CD45RO+ in

both groups.

Conclusions: The present study demonstrates that the

CD8CD26bright T-cell population is markedly decreased in

peripheral blood of psoriatic patients. Moreover, CD26 expression

did not show a restriction to memory T cells. As CD26 is of

relevance for T-cell functions, future investigations should focus

on elucidating these functions in psoriasis. It is attractive to

speculate that the reduction in the CD8CD26bright subpopulation

may represent a biomarker for recompartimentalization of

activated T cells and a reduced CD8CD26bright count may

correlate with increased responsiveness to T-cell targeted

treatments.

Key words: CD26 – peripheral blood – psoriasis – T-cells

Please cite this paper as: Reduced CD26bright expression of peripheral blood CD8+ T-cell subsets in psoriatic patients. Experimental Dermatology 2008; 17:

343–348.

Introduction

Psoriasis vulgaris can be considered one of the most com-

mon disfiguring skin diseases, in which a multifactorial

basis is the input for divergent therapeutic modalities since

decades. The aetiology regarding the initial trigger for

developing this skin disease and its matching characteristic

features, remains speculative. Unravelling the psoriatic

pathogenesis, one of the adhered hypothesis states that pso-

riasis is thought to be a T-cell-mediated skin disease in

which activated T cells have been shown of major signifi-

cance. These cells are known to be present in skin lesions

and in peripheral blood of psoriatic patients (1,2). A spec-

trum of cytokines and chemokines are produced by these T

cells which ultimately results in hyperproliferation of kerat-

inocytes (3). In particular, Th1 cytokines, such as tumor

necrosis factor-a (TNF-a), Interleukin (IL) 2 and -12 and

interferon-c have been shown to dominate over Th2

cytokines, such as IL- 4 and IL-10 (4). Activation of Th1-

producing T cells by antigen presenting cells (APCs), with

IL-12 ⁄ Il-23 as important mediators, have been suggested tobe of great importance in the pathogenesis of psoriasis (5).

More recently, biologic therapies, targeted specifically on

T-cell epitopes have been shown to be effective in psoriasis,

including alefacept (CD2-LFA3 co-stimulatory pathway)

(6), efalizumab (LFA1-ICAM1 co-stimulatory pathway)

(7,8) and anti-IL-12 ⁄ -23 (9). Psoriasis can therefore be seenas a disease in which the interplay between activated T

cells, keratinocytes and cytokines is determinative.

CD26 is a surface glycoprotein of T cells, belonging to

the class of membrane-associated peptidases and is identical

to dipeptidylpeptidase IV (DPPIV). Evidence is accumulat-

ing that CD26 is a key player in three different T-cell func-

tions: (i) co-stimulation, (ii) migration, and (iii) memory

DOI:10.1111/j.1600-0625.2007.00650.x

www.blackwellpublishing.com/EXDOriginal Article

ª 2007 The Authors

Journal compilation ª 2007 Blackwell Munksgaard, Experimental Dermatology, 17, 343–348 343

144

response (10–15). To induce T-cell proliferation and lym-

phokine secretion T cells require a second co-stimulatory

signal, provided by a number of so-called accessory mole-

cules expressed on the T-cell surface interacting with mole-

cules on APCs (16). It has been suggested that CD26, as

such an accessory molecule, in concerted action with

CD45RO (memory T-cell marker) on the T-cell surface,

can interact with an undefined counter-receptor on APCs

resulting in T-cell activation (17). In addition, the enzyme

activity of CD26 ⁄DPPIV is capable of cleaving dipeptidesat the N-terminus of polypeptides with proline, or alanine

in a lesser extent, at the penultimate position. Inflamma-

tory cyto- and chemokines can be processed by CD26, such

as CCL5(RANTES) and IL-2 (18). Interestingly, CD26 is

described to be expressed on keratinocytes as well (19). To

summarize, ensued from the above-mentioned all three

major participants in the psoriatic interplay (T cells, cyto-

kines, and keratinocytes) could be targeted performing an

expression-analysis of CD26.

The aim of the present study was to focus on the T-cell-

related expression of CD26 in peripheral blood of psoriatic

patients. In vitro studies have shown that CD26 is constitu-

tively expressed by 56% of CD4+ T cells and by 35% of

CD8+ T cells (20). In resting peripheral blood mononu-

clear cells a small subpopulation expresses CD26 at high

density, which subpopulation is designated as ‘CD26bright

T cells’. Several authors have claimed that the CD26bright

T cells belong to a CD45RO+ subpopulation (21,22).

Further studies are required to find out whether this

co-expression is the reality of the in vivo situation. Remark-

ably, in vivo, peripheral blood CD26bright T cells have

been shown to be increased in patients during active phases

of other autoimmune diseases, such as rheumatoid arthri-

tis, Graves disease and multiple sclerosis and decreased in

immunocompromised patients (HIV) (23–28). In psoriasis,

this CD26bright population has not been studied yet. The

identification of these T cells in normal and psoriatic con-

ditions may contribute to a better understanding of the

underlying pathogenesis of psoriasis. Therefore, we have

characterized peripheral blood lymphocytes of patients with

untreated severe widespread psoriasis and healthy volun-

teers by multiparameter flow cytometric analysis.

Methods

Study populationFifteen patients (six females, nine males between 31 and

59 years of age) with severe psoriasis, defined as with a

Psoriasis and Severity Index (PASI) of 12 or greater, and

nine healthy subjects (three females and six males between

19 and 50 years of age) without any history or signs of skin

diseases participated in this study after giving informed

consent. All patients had a washout of systemic treatments

or phototherapy for at least 4 weeks and had not used top-

ical treatment for the last 2 weeks at time of venapuncture.

Furthermore, they did not have comorbidities and did not

use interfering medication.

Isolation of peripheral blood lymphocytesPeripheral blood specimens were collected in 3 ml vacu-

tainer tubes (BD Vacutainer Systems, Plymouth, UK) con-

taining ethylenediaminetetraacetate (EDTA). Aliquots of

100 ll were placed directly into staining tubes containingthe appropiate volumes of fluorochrome-conjugated mono-

clonal antibodies. The tubes were mixed by gentle vor-

texing and were incubated at room temperature (20�C) for30 min. in the dark. Two millilitres of commercially avail-

able lysing solution (FACS Lysing Solution; BD Biosciences

Pharmingen, San Diego, CA, USA) diluted 1 in 10 into

water, was added to each tube. The tubes were vortexed

and incubated for exactly 10 min at room temperature in

the dark, after which they were centrifuged for 10 min at

200·g. Subsequently, the supernatant was discarded andthe pellet was washed and centrifuged in phosphate buffer

saline (Braun, Melsungen AG, Germany) for 10 min at

200·g. After the supernatant was discarded again, the

resulting cell pellets were resuspended and fixed in 1%

paraformaldehyde (Cytofix 1:4 diluted, BD Fixation Buffer,

BD Biosciences Pharmingen, San Diego, CA, USA).

Monoclonal antibodiesFluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-,

PE-Cy5 and PE-Cy7-conjugated monoclonal antibodies

were used in various designated combinations. The follow-

ing combinations of FITC-PE-Pc5 and Pc7-conjugated

reagents were arranged to assess the CD26 expression in

peripheral blood: anti-CD3+ (T cells) and anti-CD4+

(T-helper cells), CD8+ (cytotoxic T cells) and CD26

(quadruple staining). Anti-CD45RO+ (memory T cells),

anti-CD25+ (early-activated T cells), and anti-CD26+ were

coupled with either anti-CD4+ or anti-CD8+ (quadruple

staining). Table 1 lists the antibodies used.

Flow cytometryAll assays were run on a FC500 flow cytometer (Beckman

Coulter, Cytomics FC500, Miami, FL, USA) equipped with

an argon ion laser emitting at 488 nm. The flow cytometer

was calibrated routinely, as recommended by the manufac-

turer. List mode data were acquired (CXP Software Version

2.1, 2005, Miami, FL, USA). All samples were processed in

duplo within 24 h of venapuncture.

List mode data analysisList mode files were analysed using WinList (Verity Soft-

ware House Inc, Winlist Version 5.0, Topsham, ME, USA)

software. A regional gate was set on CD3+ (FITC) versus

van Lingen et al.

ª 2007 The Authors

344 Journal compilation ª 2007 Blackwell Munksgaard, Experimental Dermatology, 17, 343–348

Chapter 3

forward scatter to gate for T lymphocytes in the sample

being studied. At least 20 000 gated events were acquired

for each antibody combination.

Statistical analysisThe statistical significance between the two groups involved

the Student’s unpaired two-tailed t-test. P-values below

0.05 were considered statistically significant. Data analysis

was performed using SPSS 12.0.1 software.

Results

Participating patients had longstanding psoriasis with a

mean PASI of 16.6 at time of venapuncture. Overall, there

was no significant increase with respect to the extent of

lesions during the washout period (mean delta PASI

1.3 ± 1.1 (SEM)).

Distribution of CD26-subsets in T cells in healthycontrols and untreated psoriatic patientsLymphocyte subsets were measured as a percentage of

total gated T cells (CD3+). The CD26 expression in the

CD3+population showed a negative-, a dim- and a

bright positive fraction. These three fractions were

quantitatively similar in psoriatic patients versus healthy

controls (Fig. 1a,b), without statistically significant

differences.

The CD3+ population in the psoriatic patients consisted

of similar proportions of CD4+ and CD8+ T cells com-

pared to healthy controls (Fig. 2). With respect to the

CD4+ population, the intensity of the CD26 expression

(negative ⁄dim ⁄bright) showed no significant differences

between psoriatic patients and the healthy controls. How-

ever, the CD26bright expression on CD8+ lymphocytes

revealed a marked and statistically significant (P = 0.014)

decrease in psoriatic patients compared to the healthy

volunteers (Fig. 1c,d).

Association of CD45RO+ and CD26brightexpression on T cells in healthy controlsand untreated psoriatic patientsTable 2A summarizes quantitatively the co-expression of

CD45RO and CD26 on either the CD4+ or CD8+ subsets

in psoriatics and healthy controls. These data show that

CD26bright cells are not specifically restricted to the

CD45RO+ population. In the CD4+ T cells, 72.1% of the

CD26 bright subset are CD45RO+ in psoriatics, corre-

sponding to 67.6% in healthy volunteers.

Of the CD8+ cells, however, only 24.4% of the CD26po-

sitive subset is CD45RO+ in psoriatics, compared to 10.1%

in healthy subjects. Thus, whereas the majority of

Table 1. Monoclonal antibodies (MAb) used

CD MAb ⁄ clone Label Source

CD3 UCTH1 FITC Beckman Coulter

CD4 SFCI12T4D11(T4) PC7 Beckman Coulter

CD8 SFCI12Thy2D3(T8) PC7 Beckman Coulter

CD8 RPA-T8 PC5 Becton Dickinson

CD25 M-A251 FITC Becton Dickinson

CD26 L272 PE Becton Dickinson

CD45RO UCHL1 PC5 Becton Dickinson

FITC, fluorescein isothiocyanate; PC7, phycoerythrin-Cy7; PC5,

phycoerythrin-Cy5; PE, phycoerythrin.

(a) (c)

(d)(b)

Healthy subject

CD26 fractions of CD3+ cells (%)

100 101 102

CD26-PE CD26-PE CD26-PE CD26-PE

CD26-PECD26-PECD26-PECD26-PE

103 100 101 102 103 104 100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103 104104 100 101 102 103 100 101 102 103 110

01

01

10

21

03

10

4

10

01

01

10

21

03

10

4

10

01

01

10

21

03

10

4

10

01

01

10

2C

D3

-FIT

C

CD

3-F

ITC

CD

8-P

C5

CD

8-P

C5

10

31

04

05

01

00

Nu

mb

er

Nu

mb

er

Nu

mb

er

Nu

mb

er1

50

040

60

80

20

100

040

60

80

20

100

040

60

80

20

100

120

120

140160

CD8+CD26 bright

Psoriasis

Healthy subjects PsoriasisHealthy subjects Psoriasis

22.922.28

65.49

12.23 8.58

68.97 CD26 brightCD26 dimCD26-

0%0

10%

10% c

ells

20%30%

20

30

40%50%60%70%80%90%

100%

Healthy subject Psoriasis

Figure 1. Expression of CD26 in peripheral

blood of psoriatic patients and healthy

subjects. (a): Lymphocytes were gated based

on morphology by side- and forward scatter

properties as discriminators. Example of

statistically equal CD26-, CD26dim and

CD26bright expression on T cells (CD3+) in

both groups. (b): Representation of

(statistically equal) mean percentages of CD26

fractions on T cells. (c): Representative

example of CD8+CD26 expression in

peripheral blood of psoriatic patients and

healthy subjects. (d) Dispersal of individual

CD8CD26bright expression in both groups.

The CD8+CD26bright population is markedly

reduced in the psoriatic patient group.

CD26 T-cell expression in psoriatic patients

ª 2007 The Authors



145

response (10–15). To induce T-cell proliferation and lym-

phokine secretion T cells require a second co-stimulatory

signal, provided by a number of so-called accessory mole-

cules expressed on the T-cell surface interacting with mole-

cules on APCs (16). It has been suggested that CD26, as

such an accessory molecule, in concerted action with

CD45RO (memory T-cell marker) on the T-cell surface,

can interact with an undefined counter-receptor on APCs

resulting in T-cell activation (17). In addition, the enzyme

activity of CD26 ⁄DPPIV is capable of cleaving dipeptidesat the N-terminus of polypeptides with proline, or alanine

in a lesser extent, at the penultimate position. Inflamma-

tory cyto- and chemokines can be processed by CD26, such

as CCL5(RANTES) and IL-2 (18). Interestingly, CD26 is

described to be expressed on keratinocytes as well (19). To

summarize, ensued from the above-mentioned all three

major participants in the psoriatic interplay (T cells, cyto-

kines, and keratinocytes) could be targeted performing an

expression-analysis of CD26.

The aim of the present study was to focus on the T-cell-

related expression of CD26 in peripheral blood of psoriatic

patients. In vitro studies have shown that CD26 is constitu-

tively expressed by 56% of CD4+ T cells and by 35% of

CD8+ T cells (20). In resting peripheral blood mononu-

clear cells a small subpopulation expresses CD26 at high

density, which subpopulation is designated as ‘CD26bright

T cells’. Several authors have claimed that the CD26bright

T cells belong to a CD45RO+ subpopulation (21,22).

Further studies are required to find out whether this

co-expression is the reality of the in vivo situation. Remark-

ably, in vivo, peripheral blood CD26bright T cells have

been shown to be increased in patients during active phases

of other autoimmune diseases, such as rheumatoid arthri-

tis, Graves disease and multiple sclerosis and decreased in

immunocompromised patients (HIV) (23–28). In psoriasis,

this CD26bright population has not been studied yet. The

identification of these T cells in normal and psoriatic con-

ditions may contribute to a better understanding of the

underlying pathogenesis of psoriasis. Therefore, we have

characterized peripheral blood lymphocytes of patients with

untreated severe widespread psoriasis and healthy volun-

teers by multiparameter flow cytometric analysis.

Methods

Study populationFifteen patients (six females, nine males between 31 and

59 years of age) with severe psoriasis, defined as with a

Psoriasis and Severity Index (PASI) of 12 or greater, and

nine healthy subjects (three females and six males between

19 and 50 years of age) without any history or signs of skin

diseases participated in this study after giving informed

consent. All patients had a washout of systemic treatments

or phototherapy for at least 4 weeks and had not used top-

ical treatment for the last 2 weeks at time of venapuncture.

Furthermore, they did not have comorbidities and did not

use interfering medication.

Isolation of peripheral blood lymphocytesPeripheral blood specimens were collected in 3 ml vacu-

tainer tubes (BD Vacutainer Systems, Plymouth, UK) con-

taining ethylenediaminetetraacetate (EDTA). Aliquots of

100 ll were placed directly into staining tubes containingthe appropiate volumes of fluorochrome-conjugated mono-

clonal antibodies. The tubes were mixed by gentle vor-

texing and were incubated at room temperature (20�C) for30 min. in the dark. Two millilitres of commercially avail-

able lysing solution (FACS Lysing Solution; BD Biosciences

Pharmingen, San Diego, CA, USA) diluted 1 in 10 into

water, was added to each tube. The tubes were vortexed

and incubated for exactly 10 min at room temperature in

the dark, after which they were centrifuged for 10 min at

200·g. Subsequently, the supernatant was discarded andthe pellet was washed and centrifuged in phosphate buffer

saline (Braun, Melsungen AG, Germany) for 10 min at

200·g. After the supernatant was discarded again, the

resulting cell pellets were resuspended and fixed in 1%

paraformaldehyde (Cytofix 1:4 diluted, BD Fixation Buffer,

BD Biosciences Pharmingen, San Diego, CA, USA).

Monoclonal antibodiesFluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-,

PE-Cy5 and PE-Cy7-conjugated monoclonal antibodies

were used in various designated combinations. The follow-

ing combinations of FITC-PE-Pc5 and Pc7-conjugated

reagents were arranged to assess the CD26 expression in

peripheral blood: anti-CD3+ (T cells) and anti-CD4+

(T-helper cells), CD8+ (cytotoxic T cells) and CD26

(quadruple staining). Anti-CD45RO+ (memory T cells),

anti-CD25+ (early-activated T cells), and anti-CD26+ were

coupled with either anti-CD4+ or anti-CD8+ (quadruple

staining). Table 1 lists the antibodies used.

Flow cytometryAll assays were run on a FC500 flow cytometer (Beckman

Coulter, Cytomics FC500, Miami, FL, USA) equipped with

an argon ion laser emitting at 488 nm. The flow cytometer

was calibrated routinely, as recommended by the manufac-

turer. List mode data were acquired (CXP Software Version

2.1, 2005, Miami, FL, USA). All samples were processed in

duplo within 24 h of venapuncture.

List mode data analysisList mode files were analysed using WinList (Verity Soft-

ware House Inc, Winlist Version 5.0, Topsham, ME, USA)

software. A regional gate was set on CD3+ (FITC) versus

van Lingen et al.

ª 2007 The Authors


forward scatter to gate for T lymphocytes in the sample

being studied. At least 20 000 gated events were acquired

for each antibody combination.

Statistical analysisThe statistical significance between the two groups involved

the Student’s unpaired two-tailed t-test. P-values below

0.05 were considered statistically significant. Data analysis

was performed using SPSS 12.0.1 software.

Results

Participating patients had longstanding psoriasis with a

mean PASI of 16.6 at time of venapuncture. Overall, there

was no significant increase with respect to the extent of

lesions during the washout period (mean delta PASI

1.3 ± 1.1 (SEM)).

Distribution of CD26-subsets in T cells in healthycontrols and untreated psoriatic patientsLymphocyte subsets were measured as a percentage of

total gated T cells (CD3+). The CD26 expression in the

CD3+population showed a negative-, a dim- and a

bright positive fraction. These three fractions were

quantitatively similar in psoriatic patients versus healthy

controls (Fig. 1a,b), without statistically significant

differences.

The CD3+ population in the psoriatic patients consisted

of similar proportions of CD4+ and CD8+ T cells com-

pared to healthy controls (Fig. 2). With respect to the

CD4+ population, the intensity of the CD26 expression

(negative ⁄dim ⁄bright) showed no significant differences

between psoriatic patients and the healthy controls. How-

ever, the CD26bright expression on CD8+ lymphocytes

revealed a marked and statistically significant (P = 0.014)

decrease in psoriatic patients compared to the healthy

volunteers (Fig. 1c,d).

Association of CD45RO+ and CD26brightexpression on T cells in healthy controlsand untreated psoriatic patientsTable 2A summarizes quantitatively the co-expression of

CD45RO and CD26 on either the CD4+ or CD8+ subsets

in psoriatics and healthy controls. These data show that

CD26bright cells are not specifically restricted to the

CD45RO+ population. In the CD4+ T cells, 72.1% of the

CD26 bright subset are CD45RO+ in psoriatics, corre-

sponding to 67.6% in healthy volunteers.

Of the CD8+ cells, however, only 24.4% of the CD26po-

sitive subset is CD45RO+ in psoriatics, compared to 10.1%

in healthy subjects. Thus, whereas the majority of

Table 1. Monoclonal antibodies (MAb) used

CD MAb ⁄ clone Label Source

CD3 UCTH1 FITC Beckman Coulter

CD4 SFCI12T4D11(T4) PC7 Beckman Coulter

CD8 SFCI12Thy2D3(T8) PC7 Beckman Coulter

CD8 RPA-T8 PC5 Becton Dickinson

CD25 M-A251 FITC Becton Dickinson

CD26 L272 PE Becton Dickinson

CD45RO UCHL1 PC5 Becton Dickinson

FITC, fluorescein isothiocyanate; PC7, phycoerythrin-Cy7; PC5,

phycoerythrin-Cy5; PE, phycoerythrin.

(a) (c)

(d)(b)

Healthy subject

CD26 fractions of CD3+ cells (%)

100 101 102

CD26-PE CD26-PE CD26-PE CD26-PE

CD26-PECD26-PECD26-PECD26-PE

103 100 101 102 103 104 100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103 104104 100 101 102 103 100 101 102 103 110

01

01

10

21

03

10

4

10

01

01

10

21

03

10

4

10

01

01

10

21

03

10

4

10

01

01

10

2C

D3

-FIT

C

CD

3-F

ITC

CD

8-P

C5

CD

8-P

C5

10

31

04

05

01

00

Nu

mb

er

Nu

mb

er

Nu

mb

er

Nu

mb

er1

50

040

60

80

20

100

040

60

80

20

100

040

60

80

20

100

120

120

140160

CD8+CD26 bright

Psoriasis

Healthy subjects PsoriasisHealthy subjects Psoriasis

22.922.28

65.49

12.23 8.58

68.97 CD26 brightCD26 dimCD26-

0%0

10%

10% c

ells

20%30%

20

30

40%50%60%70%80%90%

100%

Healthy subject Psoriasis

Figure 1. Expression of CD26 in peripheral

blood of psoriatic patients and healthy

subjects. (a): Lymphocytes were gated based

on morphology by side- and forward scatter

properties as discriminators. Example of

statistically equal CD26-, CD26dim and

CD26bright expression on T cells (CD3+) in

both groups. (b): Representation of

(statistically equal) mean percentages of CD26

fractions on T cells. (c): Representative

example of CD8+CD26 expression in

peripheral blood of psoriatic patients and

healthy subjects. (d) Dispersal of individual

CD8CD26bright expression in both groups.

The CD8+CD26bright population is markedly

reduced in the psoriatic patient group.


ª 2007 The Authors


146

CD4CD26bright cells are CD45RO+, the minority of

CD8CD26bright cells are CD45RO+ in both groups (Fig 3,

Table 2A).

CD26 expression on CD4+CD25+ T cells inhealthy controls and untreated psoriatic patientsIn the patients with psoriasis the activation and Treg

marker CD25 was significantly elevated (P = 0.006) on

CD4+ T cells compared to healthy subjects althought these

populations were small (6.3 ± 0.6 vs 4.1 ± 0.4). Remark-

ably, this CD4+CD25+ expression was not specifically

associated with CD26 dim ⁄bright expression (Table 2B).

Discussion

T cells are thought to play a crucial pathogenic role in the

development and maintenance of psoriasis. In this study,

we investigated the T-cell expression of CD26 in untreated

severe psoriatic patients in comparison with healthy sub-

jects, to strive to identify a character profile of CD26

expression in relation to psoriasis-associated T-cell subsets.

The present study demonstrates a substantial decrease in

the CD8+CD26+ T-cell subset in psoriasis which is in

accordance with a previous study (29). Our results extend

this observation, however, by demonstrating that it con-

cerns the ‘CD8+CD26bright T-cell subset’ which is reduced

in psoriasis. Contradictory, the CD4+CD26bright popula-

tion in psoriasis showed no significant difference to that of

the healthy subjects. Yet, in other auto-immune processes

increased levels of CD4+CD26bright T cells were actually

prominent findings. As CD26 is of relevance to transendo-

thelial migration in vitro (11,30), it is attractive to speculate

that such a decrease of CD8+CD26bright T cells is the

result of efficient extravasation and migration into the skin

of this CD26bright subset in patients with widespread

psoriasis. Strikingly, CD8+ T cells are described to be

abundantly present in lesional psoriatic skin (31–36).

Hypothetically, the CX3CL1-CX3CR1 system is suggested

to play a role in the extravasation of leukocytes towards

the psoriatic skin (37) and could be a keyplayer in this

migratory movement of the CD26bright subset. On the

other hand, deficient upregulation of CD26 expression in

CD8+ T cells can be causally related to the decrease as

well. Future investigations could give insight into such a

mechanism.

It is intriguing that psoriasis differs from those autoim-

mune diseases characterized by increased CD26+ T-cell

Table 2. (A): Distribution of CD45RO in the CD26 neg ⁄ dim ⁄ bright subpopulations of CD4+ and CD8+ T cells in psoriatic patients compared to

healthy controls (triple staining). Data reported as mean percentages (ranges). (B): Association of CD26 (negative vs dim ⁄ bright) expression with the

activation and Treg marker CD25 on CD4+ T cells. Overview of Mean percentages (ranges)

CD26 Expression

Psoriasis Healthy controls

Negative Dim Bright Negative Dim Bright

A

CD4+

% CD45RO+ 54.9 (10.4–78.7) 27.6 (8.2–55.3) 72.1 (39.2 –99.7) 33.8 (18–59.2) 19.5 (10.8–30.4) 67.6 (43.4–80.3)

% CD45RO) 45.1 (21.3–89.6) 72.4 (44.7–91.8) 27.9 (0.3–60.8) 66.2 (40.8–82) 80.5 (69.6–89.2) 32.4 (19.7–56.6)

CD8+

% CD45RO+ 16.5 (2.8–39.1) 15.6 (2.8–40.4) 24.4 (3.2–80.9) 9.1 (0.6–15.1) 4.2 (0.7–10) 10.1 (0.005–27.8)

% CD45RO) 83.5 (60.9–97.2) 84.4 (59.6–97.3) 75.6 (19.1–96.8) 90.9 (84.9–99.4) 95.8 (90–99.3) 89.9 (72.3–99.9)

B

Negative Dim ⁄ bright Negative Dim ⁄ bright

CD4+

% CD25+ 47 32.9–65.9) 56.1 (34.1–78.2) 46.2 (33–61) 53.8 (39–67)

(a)

(b) (c)

100%90%80%70%60%50%40%30%20%10%

0%Healthy subjects

CD3CD4

T-cell population (CD3+)

CD3CD8

Psoriasis

4.94

64.58

CD8+

CD4+

CD4-CD8-

29.9830.9

62.59

6.16

Healthy subjects40

50

40

30

20

10

50

60

70

% c

ells

% c

ells

80

90

Psoriasis Healthy subjects Psoriasis

Figure 2. CD4 and CD8 fractions of the overall T-cell population

found in the peripheral blood of psoriatic patients and healthy subjects.

(a): Mean percentages (statistically equal) (b) and (c): Dispersal of

individual CD4 and CD8 expression in both groups. These data show

comparable expression.

van Lingen et al.

ª 2007 The Authors


Chapter 3


147

CD4CD26bright cells are CD45RO+, the minority of

CD8CD26bright cells are CD45RO+ in both groups (Fig 3,

Table 2A).

CD26 expression on CD4+CD25+ T cells inhealthy controls and untreated psoriatic patientsIn the patients with psoriasis the activation and Treg

marker CD25 was significantly elevated (P = 0.006) on

CD4+ T cells compared to healthy subjects althought these

populations were small (6.3 ± 0.6 vs 4.1 ± 0.4). Remark-

ably, this CD4+CD25+ expression was not specifically

associated with CD26 dim ⁄bright expression (Table 2B).

Discussion

T cells are thought to play a crucial pathogenic role in the

development and maintenance of psoriasis. In this study,

we investigated the T-cell expression of CD26 in untreated

severe psoriatic patients in comparison with healthy sub-

jects, to strive to identify a character profile of CD26

expression in relation to psoriasis-associated T-cell subsets.

The present study demonstrates a substantial decrease in

the CD8+CD26+ T-cell subset in psoriasis which is in

accordance with a previous study (29). Our results extend

this observation, however, by demonstrating that it con-

cerns the ‘CD8+CD26bright T-cell subset’ which is reduced

in psoriasis. Contradictory, the CD4+CD26bright popula-

tion in psoriasis showed no significant difference to that of

the healthy subjects. Yet, in other auto-immune processes

increased levels of CD4+CD26bright T cells were actually

prominent findings. As CD26 is of relevance to transendo-

thelial migration in vitro (11,30), it is attractive to speculate

that such a decrease of CD8+CD26bright T cells is the

result of efficient extravasation and migration into the skin

of this CD26bright subset in patients with widespread

psoriasis. Strikingly, CD8+ T cells are described to be

abundantly present in lesional psoriatic skin (31–36).

Hypothetically, the CX3CL1-CX3CR1 system is suggested

to play a role in the extravasation of leukocytes towards

the psoriatic skin (37) and could be a keyplayer in this

migratory movement of the CD26bright subset. On the

other hand, deficient upregulation of CD26 expression in

CD8+ T cells can be causally related to the decrease as

well. Future investigations could give insight into such a

mechanism.

It is intriguing that psoriasis differs from those autoim-

mune diseases characterized by increased CD26+ T-cell

Table 2. (A): Distribution of CD45RO in the CD26 neg ⁄ dim ⁄ bright subpopulations of CD4+ and CD8+ T cells in psoriatic patients compared to

healthy controls (triple staining). Data reported as mean percentages (ranges). (B): Association of CD26 (negative vs dim ⁄ bright) expression with the

activation and Treg marker CD25 on CD4+ T cells. Overview of Mean percentages (ranges)

CD26 Expression

Psoriasis Healthy controls

Negative Dim Bright Negative Dim Bright

A

CD4+

% CD45RO+ 54.9 (10.4–78.7) 27.6 (8.2–55.3) 72.1 (39.2 –99.7) 33.8 (18–59.2) 19.5 (10.8–30.4) 67.6 (43.4–80.3)

% CD45RO) 45.1 (21.3–89.6) 72.4 (44.7–91.8) 27.9 (0.3–60.8) 66.2 (40.8–82) 80.5 (69.6–89.2) 32.4 (19.7–56.6)

CD8+

% CD45RO+ 16.5 (2.8–39.1) 15.6 (2.8–40.4) 24.4 (3.2–80.9) 9.1 (0.6–15.1) 4.2 (0.7–10) 10.1 (0.005–27.8)

% CD45RO) 83.5 (60.9–97.2) 84.4 (59.6–97.3) 75.6 (19.1–96.8) 90.9 (84.9–99.4) 95.8 (90–99.3) 89.9 (72.3–99.9)

B

Negative Dim ⁄ bright Negative Dim ⁄ bright

CD4+

% CD25+ 47 32.9–65.9) 56.1 (34.1–78.2) 46.2 (33–61) 53.8 (39–67)

(a)

(b) (c)

100%90%80%70%60%50%40%30%20%10%

0%Healthy subjects

CD3CD4

T-cell population (CD3+)

CD3CD8

Psoriasis

4.94

64.58

CD8+

CD4+

CD4-CD8-

29.9830.9

62.59

6.16

Healthy subjects40

50

40

30

20

10

50

60

70

% c

ells

% c

ells

80

90

Psoriasis Healthy subjects Psoriasis

Figure 2. CD4 and CD8 fractions of the overall T-cell population

found in the peripheral blood of psoriatic patients and healthy subjects.

(a): Mean percentages (statistically equal) (b) and (c): Dispersal of

individual CD4 and CD8 expression in both groups. These data show

comparable expression.

van Lingen et al.

ª 2007 The Authors


numbers (25–27,38,39). Indeed, psoriasis may not be

regarded as a classical autoimmune disease. In fact, evi-

dence is accumulating that psoriasis is characterized by an

abnormality in innate immunity (40–42). In contrast to

previous observations in HIV-infected patients and

MS-patients (17,22,28), CD26bright expression was not

specifically restricted to CD45RO+ T cells. Our results

reveal that both in healthy subjects as psoriatic patients the

vast majority of CD8+CD26bright cells were CD45RO-

(89.9% and 75.6%). Contradictory, the majority of

CD4+CD26bright cells were CD45RO+ in healthy subjects

and psoriatic patients (67.6% and 72.1%). These findings

insinuate that such a distinct restriction of CD26bright T

cells to the CD45RO+subpopulation in psoriasis is not

requisite for the defined co-stimulatory properties that

CD26bright expression is described to induce in concerted

action with CD45RO.

In conclusion, we demonstrated for the first time that

the CD8+CD26bright T cells are reduced in psoriasis. It is

remotely possible that such a decrease in an individual

patient represents influx of activated T cells into the

dermal compartment and that such may represent a posi-

tive sign that some T-cell targeted treatments are indicated

in such a patient. Introcaso et al. suggested, that as some

patients respond to therapy, their CD26cell populations go

through stages of change or alternatively that there is

something different about the pathophysiology of patients

who have CD26- versus CD26dim or CD26bright subpopu-

lations (51). At this point, it is unclear what distinguishes

the three subpopulations from a functional point of view

in psoriasis. The reduction in the CD8+CD26bright sub-

population, however, remains a challenging observation

because it differs from findings in other (classical) auto-

immune diseases. Further studies are indicated to find

out whether patients with a profound reduction of

CD8+CD26bright cells in peripheral blood may be excellent

responders to some T-cell targeted treatments.

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FS

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FS

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FS

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ª 2007 The Authors


Chapter 3


149

150

RG van Lingen, PEJ van Erp, MMB Seyger, EMGJ de Jong, RT de Boer-van Huizen, RJB Driessen, PCM van de KerkhofPersistent expression of CD26 / DPPIV after treatment with infliximab in pso-riasis despite clinical improvementSubmitted.

Chapter 3

3.4


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General Discussion

Chapter 4

General Discussion

161

4.1 Introduction

The centre of interest of the present thesis was to study the dynamics of on the one hand well-established psoriasis-associated markers of the T-cell and on the other hand of a potential novel marker (CD26 / DPPIV) relevant to the pathogenesis of psoriasis. In particular we studied these processes during treatment with established and innovative anti-psoriatic therapies. In order to expand knowledge on the complex puzzle of the pathogenesis of psoriasis, corresponding aims and questions were formulated in the gen-eral introduction of this thesis (1.6). Here, we will discuss and position the current observations made in chapters 2 and 3 of this thesis in the light of relevant findings in literature. In the end we will propose some directionsfor future investigations.

4.2 AIM I Assessing clinical observations and interference with T-cell subsets of pathogenesis-based therapeutic interventions

4.2.1 AIM IATo define the effect of immune-targeted therapies on well-established pso-riasis relevant T-cell subsets in the cutaneous and peripheral blood com-partment, and to address alleged compartmentalization between them in the context of clinical efficacy.

An immune-mediated disease requires immune-targeted therapies. The T-cell as central keyplayer (amongst others) is not to be undervalued in the pathogenesis of psoriasis. Evaluating the effect of immune-targeted thera-pies on the T-cell is important in our search for prognostic markers and po-tential markers to evaluate therapeutic responses. In line, numerous studies have addressed the effect of such therapies in the cutaneous and peripheral blood compartment separately.1-4 Migration of T-lymphocytes from the cir-culation to lesional skin and vice versa is a well-known phenomenon (see paragraph 1.3.2), even the migration of immune cells to the site of inflam-mation in psoriasis is described to be enhanced.5

In chapter 2.1 & 2.2 we aimed to address shifting of T-cells between the le-sional skin and circulation under influence of immune-targeted therapies.In particular, several well-established subsets, as coworkers have studied these in specific and subsets were found to be of pathogenic relevance inpsoriasis e.g. CD3, CD4, CD8, CD45RO, NKT-cells. When performing observa-tions and analyses concerning a single compartment and thus with a staticcharacter, no conclusions can be drawn on the interaction between the compartments. Correlating disease activity or response to therapy with migration of T-cells between compartments is of interest in expanding

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knowledge on the dynamic role of the T-cell in psoriasis. We observed that after a 12-week period of treatment with etanercept, adalimumab and methotrexate some significant reductions in lesional T-cell subsets werefound (see chapter 2.1 and 2.2) in contrast to no significant changes in theT-cell subsets in the peripheral blood. The clinical response to these treat-ments, reflected in a 48-85% reduction of PASI score from baseline, can beregarded as a good response. Changes in quantities of T-cell subsets in re-sponse to therapy is hypothesized to cohere with an altered state of im-mune system activity and in line with a certain extent of clinical efficacy.Given the low amount of significant alterations in T-cell subsets in the cu-taneous and peripheral blood compartment after these therapies, no cor-responding T-cell subsets could be detected which in some degree would reflect a substantial rearrangement of these subsets between the two com-partments. (Re)compartmentalization of T-cell subsets in response to clini-cal effective therapy could not be justified for the immune-targeted thera-pies etanercept, adalimumab and methotrexate. These three treatments indirectly target the T-cell as they primarily target other key aspects of the psoriatic pathogenesis. Efalizumab, however, showed to act entirely different compared to theaforementioned therapies. Important to note herein is that efalizumab has a mode of action primarily targeting the T-cell as it inhibits T-lymphocyte activation and trafficking of T-cells. The observations in this thesis demon-strated that a 12-week period treatment with efalizumab caused significantreductions in multiple lesional T-cell subsets in dermis and epidermis and partly corresponding shifts of these subsets in the peripheral blood. In par-ticular a remarkable lymphocytosis was observed after therapy. This nota-ble (re)compartmentalization of T-cells subsets in response to therapy was not reflected in an acceptable clinical efficacy as the PASI was not signifi-cantly reduced in these patients. In addition, the (re)compartmentalization of T-cells could not be used as predictive marker for responsiveness to efali-zumab therapy, as both clinical responders and non-responders had a com-parable extent of shifting of T-cells.Therefore, shifts in T-cell subsets in response to immune-targeted thera-pies, in general, do not represent a prognostic marker for the degree of clinical efficacy and consequently cannot be used as a predictive marker.It is the mode of action of immune-targeted therapies, including biologi-cals, which seems to play a prominent role in the actual shifting of T-cell subsets between the cutaneous and systemic compartment as a marked difference was detected between primarily and secondarily T-cell target-ing treatments. The fact that one patient responds well to a certain therapy and the other does not, still puzzles the researchers in the field of dermato-logy. Consequently, the longing for predictive markers continues to exist to date.

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Lymphocytes are known to acquire a certain predilection, based on the en-vironment in which they first encounter foreign antigen, to home or to re-circulate through that same environment.6 In skin, cutaneous lymphocyte-assocated antigen (CLA) is a homing receptor which determines the specific migration of T-cells to the site of (psoriatic) inflammation.7 When considering (re)compartmentalization of T-cell subsets, the assessment of the CLA-expression as a skin-homing marker could be of additional value in elucidating the migratory capacity of T-cells. However, literature comprises conflicting data on the exact amount of CLA+ T-cells that infiltrate the pso-riatic skin varying between approximately 50 to 80 %.8-11 Moreover as in un-involved skin, distant from the active plaque edge, a significant infiltrationof CLA+ cells was reported before any signs of epidermal hyperprolifera-tion were present8 and in an increased extent compared to lesional psori-atic skin, in our opinion no reliable impression of the extent of homing of T-cells can be acquired in order to assess (re)compartmentalization of CLA+ T-cells . In peripheral blood, previous studies (also at our own department) showed that CLA+ T-cells were present only in relatively small amounts3;11-13 difficult to analyze in smaller sample sizes of patients. As a result, we rea-soned to exclude the CLA-marker for evaluating shifting of T-cells between the compartments of the skin and peripheral blood in this thesis.As mentioned, the immune system is organized into distinct functional compartments, allowing the extravasation and continuous recirculation of specific populations of lymphocytes into anatomically distinctive sites.Lymphocytes continually patrol the body for foreign antigen by recircu-lating from blood, through skin, into lymph and back to blood regulated through receptor/ligand pairs.6;14 Besides the peripheral blood and cutane-ous compartment, a third compartment may comprise amounts of T-lym-phocytes; the lymphatic system including afferent and efferent lymph.15 Although it is more difficult to gain access to a lymph node, since one hasto perform a more invasive procedure compared to venapunction or skin biopsy, it would be interesting to analyze and quantify the amount of T-cell subsets in psoriasis which reside in the lymphatic system in order to create a comprehensive view on the occurrence of (re)compartmentalization of T-cells in response to therapy.

4.2.2 AIM IBTo position vascular laser treatment compared to several established treat-ments in the therapeutic array for psoriasis.

The superficial microvasculature as a key aspect of the psoriatic patho-genesis represents an interesting underestimated/neglected target in the array of antipsoriatic therapies. The presence of dilated, elongated and

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increased prominent superficial capillaries in the dermis can facilitate anincreased influx of T-cells and other immune mediators into the skin main-taining the chronic inflammatory reaction and consequently clinical lesionsin psoriasis.16 The exact mechanisms behind the maintenance of this reac-tion are unclear however. Previous studies have indicated that intervention through vascular targeted treatments, such as with vascular lasers, induces moderate to good clinical efficacy in chronic plaque type psoriasis with nor-malization of the epidermal hyperproliferation and of the T-cell influx.16-21 In addition, the remission time of the pulsed dye laser in psoriasis is reported to be long-lasting up to 6 months after cessation compared to treatment with topical therapies.17 Nevertheless, applying the PDL is a time-consum-ing event and possible side-effects include pain, formation of crusts andtransient hyperpigmentation. In order to underline or expand the clinical effect of the PDL, direct comparisons with commonly used therapeuticsextends knowledge on the use and position of PDL in the array of treat-ment modalities for psoriasis. Especially when two therapies are combined each with a different mode of action targeting a different key aspect in thepathogenesis of psoriasis, a synergistic additional clinical effect can poten-tially be achieved.Chapter 2.3 comprises a study combining PDL- and UVB-therapy thereby evaluating the potential synergistic clinical effect in psoriasis as the first af-fects the dermal superficial vessels and the latter affects the keratinocyteproliferation and induces immunosuppression.22-24 Through combination of these divergent modes of action of PDL and UVB, different but crucialpathways in the immunopathogenesis of psoriasis tend to be targeted. Al-though a comprehensive immunohistochemical study is not available, it was found that all tested treatment modalities appeared to be efficacious inthe treatment of localized plaque type psoriasis as no statistically significantdifferences were found between the clinical efficacy of PDL treatment andUVB treatment. A combination of both treatments did not result in syner-gism with respect to clinical effect. No major insuperable side effects weredetected during or after PDL treatment which makes it a safe therapeutic option. Therefore, suitable psoriatic candidates proposed for vascular laser therapy include patients with a limited amount of persistent plaques not responding to the available medications, who are unfit for several therapiesdue to side-effects or comorbidity, who are at risk for skin cancer causedby actinic damage or immunosuppression and patients having plaques on non-covered body-sites. Chapter 2.4 on the other hand evaluates the position of the Nd:YAG laser in the therapeutic array for psoriasis, incorporating immunohistochemical studies to shed light on the mode of action of this laser. Although the study design had a pilot character, targeting the deeper abnormal vasculature in the psoriatic dermis seems justified in order to achieve a longer lasting

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effect of clearance of psoriatic plaques compared to the more superficial-ly targeted PDL. Yet, the clinical and immunohistochemical results of thisNd:YAG study did not lend support for this hypothesis. However, consist-ent with earlier findings17;25-27, the active comparator (CBD ointment) used in this study, proved to reduce the clinical severity of the treated plaques significantly, together with the epidermal thickness.Taken together, as a result, targeting more superficial located vasculaturein psoriasis (PDL) is likely to be of stronger importance in achieving clinical effect than the deeper abnormal vasculature (Nd:YAG).The need for safe and cost-effective treatments continues to exists as eachpatient deserves tailored psoriasis management. Psoriasis is not a single disease entity and multiple patterns of expression are observed ranging in severity, extent and aspect of the lesions. The option for a specific treat-ment is dependent on psoriasis-related factors (type of psoriasis, severity, extent, disease duration and localization), but also on treatment-associated factors (efficacy, side-effects, contra-indications, availability, duration of remission, preceding treatments) and patient-associated factors (age, sex, physical and mental health, patients preference) in order to reach induction of remission. Mostly, at our department, a ‘stepwise approach’ or therapeutic ladder is adopted starting with the medications with the least potential toxicity, to

Figure 13 Proposed positioning of the Pulsed Dye Laser (PDL) on the anti-psoriatic treatment ladder.

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define optimal treatment for the individual psoriatic patient. With respectto the vascular laser in the treatment of psoriasis, a position in this approach or on this ladder has not yet been defined. Based on our own data in thisthesis and the efficacy and safety data in literature on the PDL17;19;21;28, we propose PDL next to phototherapy in the treatment ladder after the topical therapies and before the systemic and biological treatments (figure 13). However, one should keep in mind the characteristics (for treatment with the PDL) of the proposed suitable patient population for this treatment. Whereas widespread psoriasis is a proper indication for UVB phototherapy, localized (recalcitrant) plaque psoriasis is a proper one for PDL laser treat-ment.With regard to the Nd:YAG laser well-designed (comparative) studies are required to further delineate the positioning of this laser in the treatment array of psoriasis.

4.3 AIM II Defining the expression of CD26 / DPPIV and char-acterizing it as a potential novel marker in the immunopatho-genesis of psoriasis

Studies on the pathogenesis, cause and treatment of psoriasis are continu-ally arising and considerable progress has been made the last ten years with respect to the immunopathogenesis of psoriasis. Whereas the antigen re-mains undefined, the underlying immunopathogenesis incorporating therole and function of certain cells equally challenges and puzzles research-ers to date.Psoriasis shares many features with other inflammatory autoimmune diseas-es such as multiple sclerosis, rheumatoid arthritis, autoimmune diabetes and inflammatory bowel disease. Several break-throughs concerning discovery of markers or new treatment options in the latter, sooner or later appeared to be also of importance in psoriasis. The last two decades, an increasing number of articles have focused on the expression of CD26 / DPPIV sharing common grounds with or directly focusing on the immunopathogenesis of above-mentioned autoimmune diseases and even CD26 / DPPIV inhibitors have been developed.29-37 A number of important functions, including the regulation of immune reactions, have been attributed to the multifunction-al CD26 / DPPIV. Altered levels of expression have been found in several au-toimmune diseases (see table 3, paragraph 1.4.5). In psoriasis, however, less is known about the level of expression of CD26 / DPPIV. Given that, on the one hand, the regulation of inflammatory processes in psoriasis have notbeen completely unraveled and, on the other hand, CD26 / DPPIV inhibitors are currently being explored as immune modulators in chronic inflamma-tion, mapping the CD26 / DPPIV enzyme in psoriasis was our defined aim.

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In chapter 3, we characterized the pattern of expression of CD26 / DPPIV in psoriasis in four divergent studies. In general, the point of focus consisted of the psoriatic interplay between cytokines, T-cells and keratinocytes with-in the field of adaptive immunity. Considering the attributed moonlightingproperties of CD26 / DPPIV, two mainstays can be described herein: (1) itsability to regulate the effect of biological factors through its enzymaticactivity (see paragraph 1.4.4) and (2) its essential role in human T-cellphysiology (see paragraph 1.4.2 & 1.4.3). In the first paragraph of the third chapter (3.1), we observed a versatile up-regulation in psoriatic (epi)dermis providing evidence that CD26 / DPPIV is expressed on keratinocytes cohering with corresponding enzymatic activ-ity, and that T-cells express CD26 to an increased extent on their surface compared to healthy volunteers. On the whole, considering the common grounds of the pattern of expression of CD26 / DPPIV with main pathologi-cal components of the psoriatic pathogenesis (T-cells, keratinocytes and cytokines) and the consequent topographical distribution of CD26 / DPPIV, further defining the role and function of CD26 / DPPIV seems valuable inthe attempt to shed light on the psoriatic puzzle. As we found this distinct upregulation and as CD26 / DPPIV is able to trun-cate several chemokines and can influence immune responses via modula-tion of cell adhesion and T-cell activation, inhibition of CD26 / DPPIV could possibly represent a promising therapeutic approach. Although one should consider whether simultaneous inhibition of both above-defined main-stays is desirable, as on the one hand the enzyme activity of CD26 / DPPIV can induce the processing of chemokines whereby reducing chemotaxis38 which is favourable and thus not to be inhibited in the psoriatic inflam-mation process. On the other hand, inhibiting the supposedly regulatory role of CD26 / DPPIV in T-cell activation39 brings about a suppressive effecton T-cell functions in the psoriatic inflammation process which may have atherapeutic potential. Thus, a differential inhibition of these two main prop-erties of CD26 / DPPIV should be considered when investigating the thera-peutic potential of CD26 / DPPIV inhibitors in psoriasis. In order to serve as a diagnostic marker, however, CD26 / DPPIV has to show an expression which is distinct from other inflammatory skin disorders e.g.lichen planus, atopic dermatitis. Actually, an increased expression of CD26 / DPPIV in the skin of these diseases has been reported40, but only small patient groups were incorporated not further defined by the extent or typeof disease and thus not comparable with the homogeneous patient group in this thesis (chronic plaque type psoriasis). Furthermore, as the enzyme activity of CD26 / DPPIV in the skin of healthy volunteers was found to be absent, one could hypothesize that the actual activity might be inflamma-tion-related and not specifically psoriasis-related. Although reports have in-dicated a relation to hyperproliferative and inflammatory skin diseases40;41,

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no comprehensive data are available concerning the actual disease-specifi-city of the upregulation of CD26 / DPPIV in psoriasis.In addition, in order to serve as a prognostic marker, the dynamics of CD26 / DPPIV during the disease process in psoriasis has to be investigated. Such dynamic studies were integrated in the second paragraph (3.2). We observed that upregulation of DPPIV expression and enzyme activity in uninvolved skin of psoriatic patients preceded epidermal hyperprolifera-tion and the presence of an overt psoriatic lesion. Therefore, the increasedDPPIV enzyme activity is involved in an early phase of the lesional psoriatic process, possibly inflicting a role in the immunological cascade during trig-gering or initiation of new lesions. Analogue expression with markers for aberrant growth and differentiation of keratinocytes during the develop-ment of a psoriatic lesion was not observed, as a result of which coherence in mechanisms behind the upregulation of CD26 / DPPIV in psoriatic skin were improbable taking into account the small sample size in this study and the semi-quantitative scoring method for CD26 / DPPIV. The third study investigated the (dynamics in) expression of CD26 on pe-ripheral blood T-cells of psoriatic patients (paragraph 3.3). The CD26 expres-sion in the T-cell population showed an negative-, a dim- and a bright posi-tive fraction. Whether the intensity of expression is relevant functionally, remains unclear. However, the CD8CD26bright T-cell population showed to be markedly decreased compared to healthy volunteers. This reduced pop-ulation of T-cells in peripheral blood could be speculated to correlate with an increased population in the cutaneous compartment, as CD26bright expression has been reported to be of relevance to transendothelial migra-tion in vitro.42;43 Moreover, as CD8+ T-cells are described to be abundantly present in lesional psoriatic skin44-47, characterizing and specifying this epi-dermal CD8+ population with respect to expression of CD26bright would further elucidate such a compartmentalization mechanism. Furthermore, the specific migratory capacity of CD26bright T-cells is reported to be pri-marily exhibited by the CD26bright CD4+CD45RO+ T-cells.48 Interestingly, the rather small CD26bright population has been shown to belong to the CD45RO+ (memory) T-cell population and CD26 in general showed to be a costimulatory protein for CD45RO in vitro and in vivo.48-51 Our results did not show such a distinct restriction of CD26 to the CD45RO+ T-cell population in peripheral blood, contrasting previous observations in HIV-infected and MS-patients33;48;51 and emphasizing the need for additional investigations on this issue. The dynamics in regulation of CD26 / DPPIV under treatment, was investi-gated in paragraph 3.4 as infliximab was introduced to further assess thepotential of CD26 / DPPIV to correlate with disease activity. The expression of CD26 / DPPIV on keratinocytes on the one hand, and T-cells in the pe-ripheral blood of psoriatic patients on the other hand, showed to be inde-

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pendent of the major (and rapid) clinical effect of treatment with inflixi-mab. Herewith, the expression of CD26 / DPPIV, though sharing common grounds with key players of the psoriatic pathogenesis, showed to be a persistent feature of ‘post-treatment’ skin after anti-TNFα therapy, possibly encompassing a triggering role in the initiation or maintenance of the lo-cal immune system in psoriasis. Moreover, because we previously reported that in uninvolved skin an upregulation of CD26 / DPPIV enzyme activity is present (paragraph 3.2). The previously observed decreased CD26bright subpopulation of CD8+T-cells in peripheral blood (paragraph 3.3) showed to remain consistently reduced after treatment with infliximab. However,one should bear in mind that this study only reflects data on the expressionof CD26 / DPPIV in response to anti-TNFα therapy. Consequently, the ques-tion whether CD26 / DPPIV could possibly function as a marker correlating with disease activity with respect to anti-psoriatic treatment, needs further assessment as the array of available treatment modalities for psoriasis com-prises several other effective modes of action.

4.4 Concluding comments

Cytokines and chemokines involved in (psoriatic) inflammation form acomplex web between cells and support the initiation and perpetuation of psoriasis. The chemical modification of cytokines and hence the modifica-tion of their physiological function (as CD26 / DPPIV can exert) is of major importance to understand their actual role in the pathophysiology of the psoriatic process. In addition, T-cells are considered to be of fundamental importance for the initiation and maintenance of a variety of immune dis-orders, including psoriasis. Moreover, as the robuste keratinocyte changes in the epidermis are elemental in the psoriatic pathogenesis, a set of three principal keyplayers is appointed within the context of innate and adaptive immunity. In this thesis, evidence has been provided that (re)compartmentalization of T-cell subsets in response to immune targeted anti-psoriatic therapy tends to depend on the primary mode of action of the treatment in ques-tion. (Re)Compartmentalization of T-cell subsets in response to immune-targeted therapies did not prove to be a valuable marker for the degree of clinical efficacy. In order to assess the circulating T-cell subsets betweenskin and peripheral blood comprehensively in response to therapy, ideally T-cell counts in the lymphatic compartment should be included in future studies.In this thesis, on the one hand pulsed dye laser treatment for psoriasis was put forward as a safe and effective treatment option for a selective groupof psoriatic patients primarily characterized by having a limited amount of

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persistent plaques. Combination with UVB TL-01 therapy did not result in a clinical synergistic effect as would the divergent mode of actions suggest.On the other hand, the Nd:YAG vascular laser did not prove effective in thetreatment for psoriasis. Targeting the more superficial located vasculaturein psoriasis is likely to be of stronger importance in achieving clinical effectthan the deeper abnormal vasculature. With respect to its positioning on the anti-psoriatic treatment ladder, we proposed the PDL on the level of phototherapy in between the topical and systemic therapies of psoriasis.In this thesis, CD26 / DPPIV was found to be upregulated on keratinocytes and T-cells in lesional psoriatic skin. This upregulated protein expression on keratinocytes proved to be enzymatically active and similarly topographi-cally distributed, present in uninvolved skin well before an overt lesion de-veloped and independent of the expression of hyperproliferation and dif-ferentiation markers of keratinocytes. In peripheral blood, in particular the CD8CD26bright population was found to decreased as opposed to healthy volunteers. In general, CD26 / DPPIV seems to represent a potential novel marker with respect to psoriatic key players but it remains to be determined whether or not its properties are directly involved in the immunologic pso-riatic context and in line its specificity needs further elucidation.All of these issues are complex and multifactorial in nature. As theories re-garding the pathogenesis of psoriasis have changed over decades, the fu-ture tells us to be open to new hypotheses as researchers are continuously investigating psoriatic aspects in order to contribute to the ongoing discus-sion and understanding of this complex disease.

4.5 Future outlooks

Research generates additional research. It is an intriguing question whether different patterns of skin involvement in psoriasis imply distinct pathogenicmechanisms and consequently whether the expression of distinct patho-genic principles may explain the interindividual differences between theresponses to treatment. In order to further evaluate and define the posi-tion of the vascular laser and the expression of CD26 / DPPIV in psoriasis, well-designed larger studies assessing patterns of skin involvement and functional properties respectively are required to further expand on the findings of this thesis.As the PDL has proven to generate a good clinical effect for a selectedgroup of patients, immunohistochemical assessments after PDL-therapy evaluating the response in terms of pathogenic aspects could further in-spire the search for optimal time-intervals between successive treatments as no data exist on ideal time-intervals, or substantiate and expand insights into the mode of action. Regarding the Nd:YAG laser, the study in this thesis

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presents preliminary findings which promotes further research elucidatingthe possible position of the Nd:YAG in the array of treatment modalities for psoriasis. In particular, the settings should be optimized and validated in a larger population in order to judge on the anti-psoriatic therapeutic potential.In studies on the pathogenesis of psoriasis, the search for diagnostic and prognostic markers represents an important approach as the responsible antigen still has to be identified. In this thesis, mapping the expression ofCD26 / DPPIV and defining its position in the pathogenesis of psoriasis is anattempt to contribute to this search. The initial trigger for the investigations done, was the pattern of expression of CD26 / DPPPIV observed in several autoimmune diseases. As our findings were not always compatible andcomparable to those in other autoimmune diseases, further research could incorporate analyses on peripheral blood of patients with RA, MS, psoriasis and IBD to make a direct comparison and judge on whether the expression of CD26 / DPPIV in these autoimmune diseases is of similar nature.The results of this thesis give emphasis to the observed upregulation of CD26 / DPPIV in psoriasis. Due to its moonlighting properties, we feel we have only touched upon the tip of the iceberg considering the complex nature, proper-ties, functions and relevance of the expression of CD26 / DPPIV in psoriasis. In line, the exact picture of all biological functions of CD26 / DPPIV is difficult tocompose as CD26 / DPPIV exerts its different functions in different cell typesand thus in different cellular contexts. Further research should involve other inflammatory dermatoses in order to shed light on the specificity of the up-regulation of CD26 / DPPIV in psoriasis. In addition, it would be fascinating to work out a method to typify CD26dim or CD26bright T-cells in psoriatic skin to investigate whether the reduced amount of CD8CD26bright T-cells in peripheral blood is caused by an increased expression of CD8CD26bright T-cells in lesional skin. Besides, performing experiments addressing the role of CD26 / DPPIV for cellular functions, e. g. T cell activation, proliferation and cytokine production in vitro would add functional information to our ob-servations. In the end, CD26 / DPPIV expression and -activity measurement may be useful for the assessment of the severity of various inflammatorydiseases, the response to therapy or the prognosis.In this thesis, we have studied the effect of the anti-TNF treatment infliximabon the expression of CD26 / DPPIV in lesional skin and peripheral blood. The effect of other anti-psoriatic therapies such as topical therapies or UVB-treatment, is still to be investigated to create a comprehensive view on dy-namics in CD26 / DPPIV expression and to further investigate its potential to function as a diagnostic and prognostic marker in response to therapy.As DASH-proteins, exhibiting DPPIV-like enzyme activity, have been intro-duced in inflammatory conditions with potential abilities to complementand/or associate to DPPIV on the level of its enzymatic activity, future studies

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should assess the DASH-proteins in psoriasis in order to define the exactplace of CD26 / DPPIV within the array of DASH-proteins. To conclude, the everlasting question in psoriasis remains as (immuno)dermatologists and researchers in the field still wonder whetherthe aberrant keratinocyte differentiation is a primary defect or that it is theconsequence of an influx of pathogenic immunocytes, or maybe both. Per-haps an even more intriguing question is the nature of the factor(s) that trigger psoriasis because that answer is still wide open. A better apprecia-tion and approach is needed to understand not only the pathogenesis but also the role of CD26 / DPPIV in psoriasis. Although a new Th17 T-cell para-digm with potential relevance is precautiously gaining ground in psoriasis, it will probably still take years and certainly further research efforts until allthe pieces of the psoriatic puzzle fall into their places and reveal a compre-hensive view on this disease.

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Bovenschen HJ, Van de Kerkhof PC, Gerritsen WJ et al. The role of lesional T cells in recal-citrant psoriasis during infliximab therapy. Eur.J.Dermatol. 2005; 15: 454-8.Langewouters AM, Van Erp PE, De Jong EM et al. The added therapeutic efficacy andsafety of alefacept in combination with other (systemic) anti-psoriatics in refractory psoriasis. J.Dermatolog.Treat. 2006; 17: 362-9.Langewouters AM, Bovenschen JH, De Jong EM et al. The effect of topical corticosteroidsin combination with alefacept on circulating T-cell subsets in psoriasis. J.Dermatolog.Treat. 2007; 18: 279-85.Bedini C, Nasorri F, Girolomoni G et al. Antitumour necrosis factor-alpha chimeric an-tibody (infliximab) inhibits activation of skin-homing CD4+ and CD8+ T lymphocytesand impairs dendritic cell function. Br.J.Dermatol. 2007; 157: 249-58.Sabat R, Philipp S, Hoflich C et al. Immunopathogenesis of psoriasis. Exp.Dermatol.2007; 16: 779-98.Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: themultistep paradigm. Cell 1994; 76: 301-14.Berg EL, Yoshino T, Rott LS et al. The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1. J.Exp.Med. 1991; 174: 1461-6.Davison SC, Ballsdon A, Allen MH et al. Early migration of cutaneous lymphocyte-associ-ated antigen (CLA) positive T cells into evolving psoriatic plaques. Exp.Dermatol. 2001; 10: 280-5.Pont-Giralt M, Gimenez-Arnau AM, Pujol RM et al. Circulating CLA(+) T cells from acute and chronic psoriasis patients manifest a different activation state and correlation withdisease severity and extension. J.Invest Dermatol. 2006; 126: 227-8.Pitzalis C, Cauli A, Pipitone N et al. Cutaneous lymphocyte antigen-positive T lym-phocytes preferentially migrate to the skin but not to the joint in psoriatic arthritis. Arthritis Rheum. 1996; 39: 137-45.Santamaria-Babi LF. CLA(+) T cells in cutaneous diseases. Eur.J.Dermatol. 2004; 14: 13-8.Hollo P, Marschalko M, Temesvari E et al. Follow-up analysis of circulating mononuclear cell CLA expression in patients with psoriasis. J.Dermatol.Sci. 2005; 39: 131-3.Sigmundsdottir H, Gudjonsson JE, Jonsdottir I et al. The frequency of CLA+ CD8+ T cells in the blood of psoriasis patients correlates closely with the severity of their disease. Clin.Exp.Immunol. 2001; 126: 365-9.Picker LJ, Treer JR, Ferguson-Darnell B et al. Control of lymphocyte recirculation in man. II. Differential regulation of the cutaneous lymphocyte-associated antigen, a tissue-se-lective homing receptor for skin-homing T cells. J.Immunol. 1993; 150: 1122-36.Mackay CR, Kimpton WG, Brandon MR et al. Lymphocyte subsets show marked differ-ences in their distribution between blood and the afferent and efferent lymph of pe-ripheral lymph nodes. J.Exp.Med. 1988; 167: 1755-65.Hacker SM, Rasmussen JE. The effect of flash lamp-pulsed dye laser on psoriasis. Arch.Dermatol. 1992; 128: 853-5.Erceg A, Bovenschen HJ, Van de Kerkhof PC et al. Efficacy of the pulsed dye laser in thetreatment of localized recalcitrant plaque psoriasis: a comparative study. Br.J.Dermatol. 2006; 155: 110-4.Hern S, Allen MH, Sousa AR et al. Immunohistochemical evaluation of psoriatic plaques following selective photothermolysis of the superficial capillaries. Br.J.Dermatol. 2001;145: 45-53.Hern S, Stanton AW, Mellor RH et al. In vivo quantification of the structural abnormali-ties in psoriatic microvessels before and after pulsed dye laser treatment. Br.J.Dermatol. 2005; 152: 505-11.

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Ros AM, Garden JM, Bakus AD et al. Psoriasis response to the pulsed dye laser. Lasers Surg.Med. 1996; 19: 331-5.Zelickson BD, Mehregan DA, Wendelschfer-Crabb G et al. Clinical and histologic evalu-ation of psoriatic plaques treated with a flashlamp pulsed dye laser. J.Am.Acad.Derma-tol. 1996; 35: 64-8.Aubin F. Mechanisms involved in ultraviolet light-induced immunosuppression. Eur.J.Dermatol. 2003; 13: 515-23.Lebwohl M. Psoriasis. Lancet 2003; 361: 1197-204.Ozawa M, Ferenczi K, Kikuchi T et al. 312-nanometer ultraviolet B light (narrow-band UVB) induces apoptosis of T cells within psoriatic lesions. J.Exp.Med. 1999; 189: 711-8.Kragballe K, Austad J, Barnes L et al. A 52-week randomized safety study of a calcipo-triol/betamethasone dipropionate two-compound product (Dovobet/Daivobet/Ta-clonex) in the treatment of psoriasis vulgaris. Br.J.Dermatol. 2006; 154: 1155-60.Saraceno R, Andreassi L, Ayala F et al. Efficacy, safety and quality of life of calcipot-riol/betamethasone dipropionate (Dovobet) versus calcipotriol (Daivonex) in the treat-ment of psoriasis vulgaris: a randomized, multicentre, clinical trial. J.Dermatolog.Treat. 2007; 18: 361-5.Van de Kerkhof PC, Wasel N, Kragballe K et al. A two-compound product containing cal-cipotriol and betamethasone dipropionate provides rapid, effective treatment of pso-riasis vulgaris regardless of baseline disease severity. Dermatology 2005; 210: 294-9.Katugampola GA, Rees AM, Lanigan SW. Laser treatment of psoriasis. Br.J.Dermatol. 1995; 133: 909-13.Bank U, Heimburg A, Helmuth M et al. Triggering endogenous immunosuppressive mechanisms by combined targeting of Dipeptidyl peptidase IV (DPIV/CD26) and Ami-nopeptidase N (APN/ CD13)--a novel approach for the treatment of inflammatory bow-el disease. Int.Immunopharmacol. 2006; 6: 1925-34.Ellingsen T, Hornung N, Moller BK et al. In active chronic rheumatoid arthritis, dipepti-dyl peptidase IV density is increased on monocytes and CD4(+) T lymphocytes. Scand.J.Immunol. 2007; 66: 451-7.Gerli R, Muscat C, Bertotto A et al. CD26 surface molecule involvement in T cell activa-tion and lymphokine synthesis in rheumatoid and other inflammatory synovitis. Clin.Immunol.Immunopathol. 1996; 80: 31-7.Hildebrandt M, Rose M, Ruter J et al. Dipeptidyl peptidase IV (DP IV, CD26) in patients with inflammatory bowel disease. Scand.J.Gastroenterol. 2001; 36: 1067-72.Krakauer M, Sorensen PS, Sellebjerg F. CD4(+) memory T cells with high CD26 surface expression are enriched for Th1 markers and correlate with clinical severity of multiple sclerosis. J.Neuroimmunol. 2006; 181: 157-64.McIntosh CH. Dipeptidyl peptidase IV inhibitors and diabetes therapy. Front Biosci. 2008; 13: 1753-73.Reinhold D, Bank U, Tager M et al. DP IV/CD26, APN/CD13 and related enzymes as regu-lators of T cell immunity: implications for experimental encephalomyelitis and multiple sclerosis. Front Biosci. 2008; 13: 2356-63.Sedo A, Duke-Cohan JS, Balaziova E et al. Dipeptidyl peptidase IV activity and/or struc-ture homologs: contributing factors in the pathogenesis of rheumatoid arthritis? Ar-thritis Res.Ther. 2005; 7: 253-69.Wrenger S, Faust J, Friedrich D et al. Attractin, a dipeptidyl peptidase IV/CD26-like en-zyme, is expressed on human peripheral blood monocytes and potentially influencesmonocyte function. J.Leukoc.Biol. 2006; 80: 621-9.Boonacker E, Van Noorden CJ. The multifunctional or moonlighting protein CD26/DP-PIV. Eur.J.Cell Biol. 2003; 82: 53-73.Fleischer B. CD26: a surface protease involved in T-cell activation. Immunol.Today 1994; 15: 180-4.

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Summary and Conclusions

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5.1 Summary and conclusions

Chapter 1, the general introduction, gives an overview of subjects relevant to creating an all-embracing / comprehensive background to consider the observations made in chapters 2 and 3. First, the epidemiology, clinical presentation and course, histopathological features and aspects of the im-munopathogenesis of psoriasis are set out in detail. Subsequently, special reference is made to the expression of CD26 / DPPIV as its structural proper-ties, distribution of expression, multifunctional character and role in auto-immune diseases and inflammation are being addressed. Consequently,the array of available anti-psoriatic treatment modalities are reviewed. In particular, the use of vascular lasers and their application in psoriasis is ad-dressed.Before the investigational approach is reflected, the aims of the presentthesis are presented (as below):

AIM I Assessing clinical observations and interference with T-cell subsets of pathogenesis-based therapeutic interventions

AIM II Defining the expression of CD26 / DPPIV and characterizing itas a potential novel marker in the immunopathogenesis of psoriasis In chapter 2, in four studies several clinical observations are made in co-herence with pathogenic aspects of psoriasis in reference to innovative anti-psoriatic treatments. In the first two studies, two major compartmentsin which T-cells can reside e.g. the cutaneous and peripheral blood com-partment, were evaluated on shifting of T-cells (compartmentalization) in response to immune-targeted therapies. It was concluded that shifts in T-cell subsets between the skin and peripheral blood under influence of immune-targeted therapies, in general, do not represent a prognostic marker for the degree of clinical efficacy. More likely, it is the mode of action which seemsto play a prominent role in the actual compartmentalization of T-cells as a marked difference was detected between primary and secondary T-celltargeting treatments.Secondly, we sought to position vascular lasers in general, and the PDL and Nd:YAG laser in particular, in the array of available anti-psoriatic treatments as the expansion of the superficial psoriatic microvasculature forms an op-

To define the effect of immune-targeted therapies on well-establishedpsoriasis relevant T-cell subsets in the cutaneous and peripheral blood compartment, and to address alleged compartmentalization between them in the context of clinical efficacyTo position vascular laser treatment compared to several established treat-ments in the therapeutic array for psoriasis

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timal target for therapy. Although the PDL proved to be a an effective treat-ment for psoriatic patients with a limited amount of persistent plaques, we also concluded that the combination of PDL and UVB-TL01 therapy, both having divergent modes of action, did not result in synergism with respect to clinical effect as compared to the monotherapy with either one of thetreatments. PDL therapy was positioned next to phototherapy but after the topical therapies and before the systemic therapies on the treatment lad-der for psoriasis. Conversely, the Nd:YAG laser targets the deeper dermal vasculature in psoriasis but in our study did not prove to reduce the clini-cal severity of the treated plaques. Therefore, from these results it could be concluded that targeting more superficial located vasculature in psoriasiswith the PDL is likely to be of stronger importance in achieving remarkable clinical effect than the deeper abnormal vasculature with the Nd:YAG.

In chapter 3, the expression of CD26 / DPPIV in psoriasis was investigated, especially in relation to two main key players in the pathogenesis of pso-riasis: keratinocytes and T-cells. CD26 / DPPIV proved to be upregulated on keratinocytes and T-cells in the psoriatic (epi)dermis compared to skin of healthy volunteers. The observed pattern of expression on keratinocytes topographically cohered with the enzymatic activity of CD26 / DPPIV. The expression and enzyme activity of CD26 / DPPIV in uninvolved skin of pso-riatic patients showed to precede epidermal hyperproliferation and the ac-tual presence of an overt psoriatic lesion. Furthermore, the expression of CD26 / DPPIV on peripheral blood T-cells was analyzed in psoriatic patients and healthy volunteers. We concluded that the CD8CD26bright T-cell pop-ulation was markedly decreased compared to healthy volunteers. Finally, the dynamics in regulation of CD26 / DPPIV under influence of treatmentwith an anti-TNFα agent (infliximab) and thus its potential as a marker cor-relating with disease activity, was further investigated. It was demonstrated that the expression of CD26 / DPPIV on keratinocytes on the one hand, and on T-cell subsets in the peripheral blood of psoriatic patients on the other, showed to be independent of the major (and rapid) clinical effect of treat-ment with infliximab. The potential of the expression of CD26 / DPPIV asa marker for clinical efficacy could not convincingly be substantiated norrefuted. Future research could lead to clarification on this.

In chapter 4, the general discussion portrays the results of the various in-vestigations of the present thesis in light of relevant literature. The aims for-mulated in chapter 1 are being addressed. Subsequently, concluding com-ments and future outlooks for further research are outlined which bring the current thesis to an end.

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5.2 Samenvatting en conclusies

Allereerst wordt in hoofdstuk 1, de algemene introductie, een overzicht gegeven van onderwerpen die relevant zijn als allesomvattende achter-grondinformatie om vervolgens de bevindingen in de hoofdstukken 2 en 3 te kunnen interpreteren en plaatsen. Hiertoe worden de epidemiologie, klinische presentatie en beloop, histopathologische kenmerken en di-verse aspecten (van de immunopathogenese) van psoriasis nauwkeurig uiteengezet. Vervolgens wordt de expressie van CD26 / DPPIV specifiek be-licht te weten de structurele eigenschappen, de distributie van de expres-sie, het multifunctionele karakter en de rol ervan in de context van auto-im-muun aandoeningen en inflammatie. Aansluitend volgt een overzicht vande beschikbare behandelingsmogelijkheden voor psoriasis.Alvorens de toegepaste onderzoeksmethoden in dit eerste hoofdstuk worden toegelicht, zijn de doelstellingen van het proefschrift uiteengezet zoals hierna nog eens kort weergegeven:

Doel I Inzicht krijgen in het klinisch effect en de beïnvloeding van T-celsubsets in respons op therapeutische interventies welke specifiek ge-baseerd zijn op de pathogenese van psoriasis

Doel II De expressie van CD26 / DPPIV te definiëren en te karakteri-seren als een nieuwe potentiële marker in de immunopathogenese van psoriasis

In hoofdstuk 2 worden, in vier uiteenlopende studies, klinische waarnemingen gedaan betreffende pathogenetische aspecten van pso-riasis in relatie tot innovatieve behandelingen voor psoriasis. In de eerste twee studies worden de twee belangrijkste compartimenten waar T-cellen zich kunnen bevinden, te weten het cutane en het perifere bloed comparti-ment, nader bekeken op het shiften van die T-cellen (zgn. compartimen-talisatie) in respons op therapieën die aangrijpen op het immuunsysteem. Deze studies lieten zien dat het shiften van T-cel subsets tussen de huid en het perifere bloed onder invloed van immuun-gemedieerde thera-

Het effect van immuun-gemedieerde therapieën te bepalen op speci-fieke T-cel subsets (welke geassocieerd worden met psoriasis) in ener-zijds de huid en anderzijds het perifere bloed van psoriasis patiënten, om zodoende de veronderstelde compartimentalisatie van deze T-cel subsets tussen de huid en het perifere bloed in de context van klinische effectiviteit van deze therapieën te analyserenHet positioneren van vasculaire laserbehandeling ten opzichte van enkele gevestigde behandelingen in het therapeutisch arsenaal voor psoriasis

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pieën over het algemeen niet van prognostische waarde is voor de mate van klinische effectiviteit van de desbetreffende therapie. Het is eerder hetwerkingsmechanisme van de therapieën welke een prominente rol lijkt te spelen in het feitelijke compartimentaliseren van T-cel subsets, aangezien een duidelijk verschil werd gevonden tussen therapieën die primair ofwel secundair op de T-cel aangrijpen.Daarnaast is gepoogd vasculaire lasers in het algemeen en de Pulsed Dye Laser (PDL) en Nd:YAG laser specifiek, een plek te geven in de reeks vanbeschikbare behandelingen voor psoriasis aangezien de uitbreiding van de oppervlakkig gelegen microvasculatuur in psoriasis een optimaal doelwit vormt als aangrijpingspunt. Alhoewel werd gevonden dat de PDL een effec-tieve behandeling is voor juist die patiënten die maar een beperkt aantal hardnekkige psoriasis plekken hebben, konden we ook concluderen dat de combinatie van PDL met UVB-TL01 lichttherapie, allebei met uiteenlopende aangrijpingspunten in de pathogenese, niet uitmondde in synergisme met betrekking tot klinisch effect of in vergelijking tot het afzonderlijk gevenvan deze twee therapieën. In de stapsgewijze benadering van de behande-ling van psoriasis werd behandeling middels de PDL naast lichttherapie ge-plaatst, dus nà de topicale behandelingen en vóór de systemische behan-delingen. De Nd:YAG laser grijpt daartegenover juist aan op de meer dieper gelegen dermale vasculatuur in psoriasis. Echter, de resultaten van onze studie lieten niet zien dat de klinische ernst van de behandelde plaques afnam na behandeling met de Nd:YAG laser. Zodoende werd uit deze twee laserstudies afgeleid dat het richten op de meer oppervlakkig gelegen vas-culatuur in psoriasis door middel van de PDL waarschijnlijk belangrijker is in het bereiken van een goed klinisch effect dan het richten op de diepergelegen abnormale vasculatuur door middel van de Nd:YAG laser.

In hoofdstuk 3 werd de expressie van CD26 / DPPIV in psoriasis bestudeerd, met name in relatie tot de twee voornaamste participanten in de patho-genese van psoriasis: de keratinocyten en T-cellen. CD26 / DPPIV bleek toegenomen tot expressie te komen op keratinocyten en T-cellen in de psoriatisch aangedane (epi)dermis in vergelijking tot de huid van gezonde vrijwilligers. Het waargenomen patroon van expressie op keratinocyten cor-releerde topografisch met de enzymactiviteit van CD26 / DPPIV. Daarnaast bleek de expressie en enzymactiviteit van CD26 / DPPIV in niet-aangedane huid van psoriasispatiënten aanwezig en tevens vooraf te gaan aan enige mate van epidermale hyperproliferatie en het ontwikkelen van een feitelijke psoriasis plaque. Ook werd de expressie van CD26 / DPPIV geanalyseerd opT-cellen in het perifere bloed van psoriasis patiënten en gezonde vrijwilligers. Uit deze analyse konden we concluderen dat de CD8CD26bright T-cel po-pulatie aanzienlijk verlaagd was ten opzichte van gezonde vrijwilligers. Als laatste, werd de dynamiek in de regulatie van de expressie van CD26 / DPPIV

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onder invloed van een anti-TNFα behandeling (infliximab) en de potentieervan als marker voor ziekte-activiteit nader onderzocht. Aangetoond werd dat enerzijds de expressie van CD26 / DPPIV op keratinocyten en anderzijds op T-cel subsets in het perifere bloed van psoriasis patiënten, onafhankelijk was in relatie tot het snelle en aanzienlijke klinische effect van behandelingmet infliximab. De mogelijke potentie van de expressie van CD26 / DPPIVals een marker voor klinische effectiviteit kon niet afdoende worden aange-toond danwel ontkracht. Verder onderzoek in de toekomst zou uitsluitsel kunnen geven hierover.

In hoofdstuk 4, de algemene discussie, worden de resultaten van de di-verse onderzoeken weergegeven en gebundeld in het licht van relevante literatuur. De doelstellingen zoals geformuleerd in hoofdstuk 1 worden nader toegelicht. Tot besluit worden enkele slotopmerkingen en sugges-ties gedaan voor verder onderzoek.

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Reduced CD26 bright expression of peripheral blood CD8+ T-cell sub-sets in psoriatic patientsRG van Lingen, PCM van de Kerkhof, EMGJ de Jong, MMB Seyger, JBM Boezeman, PEJ van Erp.Exp Dermatol. 2008 Apr; 17 (4): 343-48

Distribution of dipeptidyl-peptidase IV on keratinocytes in the margin zone of a psoriatic lesion: a comparison with hyperproliferation and aberrant differentiation markersRG van Lingen, MKP Poll, MMB Seyger, EMGJ de Jong, PCM van de Kerkhof, PEJ van Erp.Arch Derm Res 2008; Online early at http://dx.doi.org/10.1007/s00403-008-0862-1.

A comparative study on the efficacy of treatment with 585 nm PulsedDye Laser and Ultraviolet B-TL01 in plaque type psoriasisJ de Leeuw, R van Lingen, H Both, B Tank, T Nijsten, M Neumann.Derm Surg. 2008; In Press.

CD26 / dipeptidyl-peptidase IV in psoriatic skin: upregulation and top-ographical changesRG van Lingen, PCM van de Kerkhof, MMB Seyger, EMGJ de Jong, DWA van Rens, MKP Poll, PLJM Zeeuwen, PEJ van Erp.Br J Dermatol. 2008; 158(6): 1264-72.

Relevance of compartmentalization of T-cell subsets for clinical im-provement in psoriasis: effect of immune-targeted anti-psoriatic ther-apiesRG van Lingen, JEM Körver, PCM van de Kerkhof, MAM Berends, DWA van Rens, AM Langewouters, JBM Boezeman, MMB Seyger, EMGJ de Jong.Br J Dermatol. 2008; 159: 91-96.

Good clinical response to anti-psoriatic treatment with adalimumab and methotrexate does not inflict a direct effect on compartmentaliza-tion of T-cell subsets: A pilot studyRG van Lingen, EMGJ de Jong, MAM Berends, MMB Seyger, PEJ van Erp, PCM van de Kerkhof.J Dermatol Treatment. 2008; Apr 29: 1-6.

LIST OF PUBLICATIONS

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Nd: YAG laser (1064 nm) fails to improve localized plaque type psoria-sis: a clinical and immunohistochemical pilot studyRG van Lingen, EMGJ de Jong, PEJ van Erp, WSEC van Meeteren, PCM van de Kerkhof, MMB Seyger.Eur J Dermatol 2008; In Press.

Persistent expression of CD26 / DPPIV after treatment with infliximabin psoriasis despite clinical improvementRG van Lingen, PEJ van Erp, MMB Seyger, EMGJ de Jong, RT de Boer-van Huizen, RJB Driessen, PCM van de Kerkhof.Submitted.

Validation of clinical and image skin scoring systems for a single chron-ic discoid lupus erythematosus lesionAE Erceg, EMGJ de Jong, RG van Lingen, T de Boo, PCM van de Kerkhof, MMB Seyger.J Dermatol Treatment 2008; Accepted.

The universal detection of antigens from one skin biopsy specimenHMJ van der Velden PCM van de Kerkhof, MC Pasch, RT de Boer-van Huizen, RG van Lingen, PEJ van Erp.J Cutan Pathol 2009; Accepted

Human orf complicated by mucous membrane pemphigoidRG van Lingen, RG Frank, RJ Koopman, MF Jonkman.Clin Exp Dermatol. 2006 Sep; 31(5): 711-2.

De effecten van pulsed-dye laser (PDL) in vergelijking met UVB-TL-01behandeling in gelokaliseerde chronische plaque psoriasisE Racz, J de Leeuw, R van Lingen, A. van Tuyll van Serooskerken, H. Both, E.P. Prens, L. van der Fits.Nederlands Tijdschrift voor Dermatologie & Venereologie 2006; 16 (1): 11-13.

Het syndroom van Wells op kinderleeftijdRG van Lingen, M Bol, WAM Blokx, WHPM Vissers, PGM van der Valk, MMB Seyger.Nederlands Tijdschrift voor Dermatologie & Venereologie 2005; 15(8): 360-63.

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CURRICULUM VITAE

Op 26 juli 1979 werd Rosanne Gwendolyn van Lingen geboren in ’s-Graven-hage. Al snel verhuisde de familie van Lingen naar Halsteren te Noord-Bra-bant. Tussen 1983 en 1987 bezocht ze daar de katholieke basisschool ‘De Biezenhof’. Een volgende verhuizing bracht haar naar Bergen te Noord-Hol-land alwaar zij haar tienerjaren aan de Europese School doorbracht.Na het Europees Baccalaureaatsexamen in 1997 meldde zij zich tot tweemaal toe aan voor de studie Geneeskunde en werd even zo veel keer uitgeloot. Als “tweede keus” werd met de studie Psychologie aan de Rijksuniversiteit Groningen aangevangen en het jaar daarop in 1998 met de studie Bedrijfs-kunde eveneens in Groningen. Gelukkig was het in 1999 raak, werd drie-maal scheepsrecht beloond, en begon zij aan de studie Geneeskunde ook in Groningen. Tijdens de coschappen in het Medisch Spectrum Twente te Enschede, werd haar interesse voor het vakgebied van de dermatologie ge-prikkeld, waarna zij gedurende een half jaar een wetenschappelijke stage vervulde op de afdeling Dermatologie en Venereologie van het Erasmus MC te Rotterdam. Haar interesse in de Dermatologie werd bevestigd en spoorde haar aan om ook haar 3-maanden keuze coschap te vervullen in het specialisme van de Dermatologie en Venereologie, maar nu in het UMC St. Radboud te Nijmegen. Na het behalen van haar artsexamen in 2005 werd zij aangenomen als junior-onderzoeker aan de afdeling Dermatolo-gie en Venereologie van het UMC St. Radboud en werd aangevangen met het verrichten van onderzoek onder begeleiding van professor dr. dr. PCM van de Kerkhof, dr. EMGJ de Jong, dr. PEJ van Erp en dr. MMB Seyger. Het proefschrift dat voor u ligt is hier het resultaat van.Sinds 1 januari 2008 is zij in opleiding tot dermatoloog in het UMC St. Rad-boud te Nijmegen.Op 23 juni 2007 trouwde zij met Joep van Roermund.

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DANKWOORD “YOU MADE THE DIFFERENCE!!”

Mijn onderzoeksperiode was één groot avontuur en jullie hebben het leuk gemaakt! Na vele PASI’s, CRF’s en CRA’s maar ook na veel pipetteren, flowen, over-leggen, afstemmen, denken, schrijven en liters thee kon het samenbunde-len van de data gaan beginnen naar het eindproduct in de vorm van het proefschrift dat voor u ligt. Een eindproduct waar velen een bijdrage aan geleverd hebben. Daar hoort een plek in de schijnwerpers bij!

Allereerst ben ik veel dank verschuldigd aan mijn promotor, prof.dr.dr. PCM van de Kerkhof. Beste Professor, het was altijd een feest om bij u langs te komen. Door uw enthousiaste en creatieve inbreng tijdens onze besprekin-gen, leerde u mij in de loop der tijd een wetenschappelijke feeling te krij-gen en uw enthousiasme werkte aanstekelijk…! Onderzoek doen in hèt grote psoriasiscentrum van Nederland was dan ook een groot genot. De aanwezigheid van veel expertise en kennis, maar ook de fijne samenwer-king en gemoedelijke sfeer onderling maakt het hier tot een echt weten-schappelijk Eldorado. Bedankt voor deze ervaring. Het is een geweldige tijd geweest, ik had het niet willen missen.

Daarnaast wil ik mijn copromotoren dr. MMB Seyger en dr. PEJ van Erp bedanken. Marieke, waar zal ik beginnen. Waar wij begonnen zijn…? In de periode van mijn oudste coschap 2005 tijdens het schrijven van een stuk voor het NTvDV. Door jouw juiste zet op het juiste moment is mijn promotietraject op de rol gekomen. Tja, wat moet je dan nog zeggen in een dankwoord… Simpelweg “bedankt” is wat droog, “ik ben je eeuwig dankbaar” veel te dramatisch. Maar je snapt dat het daar wel dichtbij ligt. Je betrokkenheid, enthousiasme en luisterend oor zijn een grote steun geweest, je was letter-lijk en figuurlijk altijd met 2 stappen in de buurt.Piet, voor jouw adviezen en begeleiding hoefde ik ook altijd maar een deurtje verder, en die stond altijd open! Als brein achter CD26 / DPPIV heb je me vanaf ‘scratch’ ingewijd in de wondere wereld van het lab. Erg waardevol waren je praktische adviezen en kritische blik naar manuscripten. Dat heb ik bij-zonder gewaardeerd. Dank voor alles, het was fijn samenwerken met je.

In de volgende plaats wil ik dr. EMGJ de Jong enorm bedanken! Mijn schaduw-copromotor! Elke, met raad en daad heb je me bijgestaan ge-durende mijn hele promotietraject. Je niet aflatende optimisme, betrokken-heid en steun zijn voor mij erg belangrijk geweest in het plezier wat ik heb ervaren tijdens mijn onderzoek. Hoe jij in oplossingen en mogelijkheden kan denken, daar kan menigeen nog veel van leren!

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Maar er waren nog meer die het verschil hebben gemaakt afgelopen jaren…Jaap de Leeuw, jij was degene die me als eerste liet kennismaken met het opzetten en uitvoeren van een klinisch onderzoek in een dermatologische setting. Uiteindelijk heeft het geleid tot één van de artikelen dat is opge-nomen in dit proefschrift! Dank voor de fijne en gezellige samenwerkingin Rotterdam. Ik kijk er met veel plezier op terug en… kijk uit naar jouw promotie!

Matthijs Poll en Arjan Bergsma, dank voor jullie inzet tijdens jullie weten-schappelijke stages over CD26 / DPPIV. Het was leuk om samen met jullie het pad verder te verkennen rondom de expressie van dit o-zo-veelzijdige eiwitje. Het heeft zeker zijn vruchten afgeworpen!

Marisol Kooijmans-Otero, onze one-and-only onderzoeksverpleegkundige. Dank voor de hulp en gezellige samenwerking! Samen met Rieke vormden we het infliximab- trio waarin jij als -i- de onmisbare schakel vormde tus-sen inflix (Rieke) en mab (ikke). Altijd in voor een geintje, ik heb erg met jegelachen.

Ook mijn collega onderzoekers / ex-labgenoten wil ik danken voor de hulp en goede sfeer: Tsing, Patrick2, David, Mieke, Sandra, Marijke, Judith B, Maartje, John, Jeanette, Michelle en Haike. In het bijzonder Ivonne, Desiree, Diana en Roelie voor de ondersteuning bij het verwerken van biopten en “bloedjes”. Zonder jullie was het nooit zo vlot gegaan.

Mijn collega assistenten in opleiding en de leden van de staf wil ik bedan-ken voor hun steun en collegialiteit. Specifiek mijn kamergenoten Maartje,Jorn, Tim en Haike; het was erg belangrijk en bijdragend om met elkaar vooral eens nìet over dat “onderzoek doen” te praten...! Alle secretaresses, de fotografen, iedereen van de administratie en verplegend personeel: dank voor jullie hulp en interesse. “De mensen van het CHL”: dank voor jul-lie ondersteuning met de flow cytometer. Zonder patiënten was er boven-dien niets terecht gekomen van dit proefschrift, dus ook naar hen gaat een woord van dank uit.

Jan Boezeman, je adviezen hebben mij geholpen wegwijs te worden in de wir-war van statistische toetsen. Dank daarvoor. Matthijs Poll, 2x genoemd worden in een dankwoord, dan moet je toch wel een verschil hebben ge-maakt…! Dank voor het realiseren van de fraaie lay-out. We hebben er wat moois van gemaakt.

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Mijn vrienden en collega-dr-in-spé’s, drs. RJB Driessen en drs. HM Kroon. Niet voor niks heb ik jullie gevraagd voor de ceremoniële taak van een para-nimf. Rieke, mijn onderzoeksmaat, het was erg gezellig om met elkaar in hetzelfde schuitje te zitten. We vaarden er wel bij en vormden een goed team. Bedankt voor alle adviezen maar ook gezellige mailtjes en klets-praatjes als collega en vriendin. Hidde, mijn studiemaat, beiden meermalig uitgeloot en dan beginnen aan een studie geneeskunde. Van begin tot eind hebben we elkaar gevolgd door de studie heen, en nu ook erna. Ik waar-deer je vriendschap enorm en ben blij dat je weer “op tijd” terug in NL bent om mijn paranimf te zijn.

Dan natuurlijk, wil ik mijn dierbare (schoon)familie, Wouter, Matthia, vrien-den en vriendinnen ontzettend bedanken voor hun belangstelling, morele boosts en niet aflatende steun. Misschien dat de Nederlandse samenvattingenigszins helder heeft gemaakt wat ik nu afgelopen jaren heb gedaan…! Voor het kritisch doorlezen van mijn manuscript was ik overigens aan het juiste adres bij “de Harry’s”. Dank voor jullie aanvullingen en opmerkingen! Pauline, Willem, dank voor jullie betrokkenheid en bemoedigende woorden. Helaas hebben jullie dit eindproduct niet meer uit mijn handen kunnen ontvangen, maar ik weet dat jullie daarboven mee aan het glunderen zijn en het fantastisch zouden vinden. Lief sissie, besef dat ik net zo trots op jou ben. Lieve ouders, dank voor jullie niet aflatende vertrouwen, steun en de moge-lijkheden die jullie tot op de dag van vandaag bieden en hebben geboden, en dat al bijna 30 jaar! Ik had me geen liever koppel kunnen wensen om onvoorwaardelijk op terug te kunnen vallen. Dit eindproduct dat voor jullie ligt, moet dan ook als een product van jullie gezien worden.

Tja lieverd, en dan moet ik woorden zien te vinden om jou te bedanken. Voor mij heb jij hét verschil gemaakt. Relativeringsvermogen en opbeurende woorden kwamen onuitputtelijk van jouw kant ondanks een roerige tijd. Daar heb ik je ontzettend lief om, en om nog veel meer.

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COLOUR ILLUSTRATIONS

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I. Chapter 1, paragraph 1.1.2, p. 21

Figure 1 Typical clinical presentation of psoriasis vulgaris with sharply delineated erythematous plaques covered by silvery white scales.

II. Chapter 1, paragraph 1.1.2, p. 22

Figure 2 Clinical pictures of patients with psoriasis unguium, psoriasis pustulosis (palmo)plantaris and psoriasis inversa.

III. Chapter 2, paragraph 1.2, p. 25

Figure 3 Histopathological characteristics of psoriasis.

Top left: normal skin of healthy volunteer, Top right: psoriatic skin (both HE-staining). Bottom left: T-cell infiltrate in psoriasis (CD3; red stained),Bottom right: Prominent vasculature in psoriasis (CD31; red stained).

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IV. Chapter 1, paragraph 1.3.2, p. 33

Figure 5 Five steps in skin homing of leukocytes.

VI. Chapter 1, paragraph 1.4.5, p. 44

Figure 7 Hypothesized position of CD26 / DPPIV in psoriasis.

V. Chapter 1, paragraph 1.4.2, p. 38

Figure 6 Various characteristics of T-cells in peripheral blood expressing high levels of CD26 on their surface, the CD26bright T-cells.

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VII.a Chapter 1, paragraph 1.5.3, p. 52

Figure 9 Sites of action of major biologics.

Interactions between APCs and T-cells are crucial in the pathogenesis of psoriasis. Several immunomodulatory biological therapies can target the molecules expressed on the surface of both cells.

VII.b

Figure 9 Modes of action of major biologics.

Alefacept blocks the interaction between LFA-3 on APCs and CD2 onT-lymphocytes. Moreover, alefacept functions as a bridging molecule be-tween CD2 that is upregulated on CD45RO+ T-lymphocytes and the Fc y III receptor (CD16) on NK cells which induces apoptosis of T-lymphocytes through the release of granzyme by NK cells. Efalizumab blocks the in-teraction between LFA-1 and ICAM-1 thereby inhibiting the T-cell acti-vation and migration into the skin. Etanercept antagonizes the effectsof endogenous TNF by competitively inhibiting its interactions with cell-surface receptors. Infliximab binds to and inhibits the activity of trans-membrane-bound and soluble TNF-α with high specificity, affinity andactivity. Finally, adalimumab is directed against TNF-α as well and blocks its interaction with the p55 and p75 cell-surface receptors thereby lysing the cells that express surface TNF-α.

197

IX. Chapter 1, paragraph 1.5.4, p. 61

Figure 12 Depth of penetration by different medical lasers.KTP=potassium titanyl phosphate; Nd= neodymium; PDL= pulsed dye laser; YAG= yttrium-aluminium-garnet

VIII. Chapter 1, paragraph 1.5.4, p. 61

Figure 11 Absorption spectra of tissue chromophores with wavelengths of medical lasers.Nd:YAG=neodymium:yttrium-aluminium-garnet; PDL= pulsed dye laser; Er:YAG= erbium:YAG

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X. Chapter 2, paragraph 2.1, p. 93

Figure 1 Fluctuations in T-cell subset counts in response to treatment with efalizumab or etanercept. (a) Significant changes in CD markers and peripheral blood lymphocytesin response to a 12-week treatment with efalizumab and corresponding Psoriasis Area and Severity Index (PASI) reduction (%). (b) Changes in CD markers and peripheral blood lymphocytes in response to efalizumab through the first 4 weeks of treatment. (c) Changes in CD markers and peripheral blood lymphocytes in response to efalizumab between 4 and 12 weeks of treatment. (d) Changes in CD markers and peripheral blood lymphocytes in response to a 12-week treatment with etanercept and corresponding PASI reduction (%). Data are shown in mean percentag-es ± SEM. ED, epidermis; D, dermis; BL, peripheral blood; *, significantlychanged (P < 0,05).

XII. Chapter 2, paragraph 2.3, p. 110

Figure 7 For each therapy, the dis-tribution of the degree of change in physician’s global assessment score at week 13. Proportion of plaques (%) (n = 27).

XI Chapter 2, paragraph 2.3, p. 109

Figure 6 For each therapy, the distribution of physician’s global assessment-scores at baseline (T0) and after 13 weeks (T13) of treatment.

201

XIII Chapter 3, paragraph 3.1, p. 129

Figure 2 Equal topographical immunohistochemical and enzyme histo-cytometric staining of psoriatic skin vs. normal skin with CD26/dipepti-dylpeptidase IV (DPP-IV).

(a) Immunohistochemical staining of normal skin with CD26/DPP-IV; (b) in psoriatic skin a clearly distinguishable column-like staining pattern in the suprabasal compartment in the rete ridges was observed; and (c) a patchy suprapapillary staining of the psoriatic epidermis extending from the stratum basale up to several keratinocyte layers of the stratum spino-sum was observed. (d) In addition, a distinct focal honeycomb-like struc-ture in the suprabasal layers of the psoriatic epidermis was noted. (e) The dermal reactivity for CD26/DPP-IV shows a complex staining pattern not suitable for a clear-cut analysis of T-cell bound CD26/DPP-IV expres-sion in the dermis. (f,g,i) Enzyme histocytometric staining of psoriatic skin vs. normal skin (h) with CD26/DPP-IV (clone BA-5) revealed an equal topographical staining pattern compared with the immunohistochemi-cal staining pattern of CD26/DPP-IV (a–e). (j) Enzyme histocytometric re-activity was absent in serial sections using the DPP-IV inhibitor diprotin A. (k, l) Frozen sections of murine kidney were stained with CD26/DPP-IV as a positive control (m) and with diprotin A.

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XIV. Chapter 3, paragraph 3.1, p. 131

Figure 3 Phenotyping of CD26+ T cells in lesional psoriatic (epi)dermis using the Zenon immunofluorescence labelling technique.

Sections of lesional psoriatic skin were double stained using direct im-munofluorescence labelling for CD3 and CD26/dipeptidyl-peptidase IV(DPP-IV). (a) Costaining of CD3 (green) and CD26 (red) in lesional psori-atic skin revealed colocalization of these two proteins (merge; yellow) in the dermis (inset). (b) Monoclonal anti-CD26 antibody. (c) Monoclonal anti-CD3 antibody. (d) DAPI counterstaining of nuclei. (e, f) Colocaliza-tion of CD3 (green) and CD26 (red) was also evident in lesional psoriatic epidermis (merge; yellow).

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XVI. Chapter 3, paragraph 3.2, p. 138

Figure 1 Expression of DPPIV protein expression, enzyme activity, Kera-tin 16 and Ki-67 throughout the margin zone of a psoriatic lesion com-pared to normal skin. Plaque centre, inner margin, clinically uninvolved skin (UIS): x200 magni-fication. Healthy volunteers skin (HV): x100 magnification. Noticeably, inthe skin of healthy volunteers the DPPIV enzymeactivity staining creates a brownish background staining.

XVII. Chapter 3, paragraph 3.4, p. 154

Figure 2 Phenotyping of CD8+CD26+ T-cells in lesional psoriatic epider-mis using the Zenon immunofluorescence double-labeling technique.

(Top panel) Costaining of CD8 (green) and CD26 (red) in lesional psoriatic skin revealed colocalization of these two proteins (merge; yellow) in the epidermis (white arrows). (Panel below) Overview of mean changes in re-spectively the percentage of (epi)dermal double-labeled CD3+CD26+ T-cells, and the percentage of (epi)dermal double-labeled CD8+CD26+ T-cells during treatment with infliximab. T= timepoint in weeks, ED=epidermis,D=dermis. Error bars indicate standard error of the mean.

XV. Chapter 2, paragraph 2.4, p. 118

Figure 2: A) Results for the clinical efficacy of treatment with CBD-oint-ment and the Nd:YAG laser compared to no treatment (No Tx) reflected inmean SUM-scores. B) Results for experienced pain during Nd:YAG treatment with differentspotsizes reflected in mean VAS-scores. The VAS-scores at the differentpoints in time are taken together. Error bars indicate the Standard Error of the Mean. * = significantly decreased compared to baseline (p<0.05).** = significantly increased compared to week 12 (p<0.05).

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PDF hosted at the Radboud Repository of the Radboud ...The Netherlands, with summary in Dutch; 208...

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