Lacrimal Gland, Tear Film, and Dry Eye Syndromes 3: Basic Science and Clinical Relevance Part B

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Basic Science and Clinical Relevance PartB
Lacrimal Gland, Tear Film, and Dry Eye Syndromes 3
Basic Science and Clinical Relevance PartB
Editorial Board:
IRUN R. COHEN, The Weizmann Institute of Science
RODOLFO PAOLETTI, University of Milan
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Lacrimal Gland, Tear Film, and Dry Eye Syndromes 3 Basic Science and Clinical Relevance Part B
Edited by
David A. Sullivan Schepens E)'e Research Institute and Harvard Medical 5chool Bos/on, Massachusells, USA
Michael E. Stern Al/ergon. Inc. Irvine, Califomia, USA
Kazua Tsubata Tokyo Demal College and Ichikb.wa General Hospital Chiba, Japan
Darlene A. Dartt Schepens Eye Research Institute and Harvard Medical Schoo/ Bos/on, Massachusells, USA
Rose M. Sullivan Schepens Eye Research Institute Boston, Massachusells, USA
B. Britt Bromberg University 0/ New Orleans New Orleans, Louisiana, USA
Springer·Science+Business Media, LLC
Library of Congress Cataloging-in-Publicalion Data
International Conference on the Lacrimal Gland, Tear Film, and Dry Eye Syndromes (3rd: 2000: Maui, Hawaii)
Lacrimal gland, tear film, and dry eye syndromes 3: basic science and clinical relevanceledited by David A. Sullivan ... [eI al.l.
p. ; cm. - (Advances in experimental medicine and biology; v. 506) "Proceedings of the 3rd International Conference on the Lacrimal Gland, Tear Film,
and Dry Eye Syndromes: Basic Science and Clinical Relevance, November 15-18, 2000, Maui, Hawaii" - T.p. verso.
Includes bibliographical references and index.
I. Dry eye syndromes-Congresses. 2. Lacrimal apparatus-Congresses. 3. Tears-Congresses. l. TitIe: Lacrimal gland, tear film, and dry eye syndromes Ihree. 11. Sullivan. David A. III. Tide. IV. Series.
[DNLM: I. Lacrimal Apparatus-Congresses. 2. Dry Eye Syndromes-Congresses. 3. Tears-physiology-Congresses. WW 208 16125 20021 RE216.D78 155 2000 617 .7'64-de21
Proceedings of the 3rd International Conference on the Lacrimal Gland. lear Film and Dry Eye Syndromes: Basic Science and Clinical Relevance, November 15-18, 2000; Maui. Hawaii
ISBN 978-1-4613-5208-2
© 2002 Syrin,ger Science+Business Media New York Originally publishl'd by Klu'wr / I'lrnum I'ublishus, '\'l'\\ \'ork in 2002 Softcover reprint of the hardcover Ist edition 2002
10 9 8 7 6 5 4 3 2 1
A C.I.P. record for Ihis book is a vailable from the Library of Congress
All rights reserved
No part of this boole may be reproduced. stored in a retrieval system, or transmined in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without wrinen permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system. for excJusive use by the purchaser of the worle.
ISBN 978-1-4613-5208-2 ISBN 978-1-4615-07 17-8 (eBook) DOI 10.1007/978-1-4615-0717-8
Immunity and Inflammation I: Influence of Cytokines, Chemokines, Adhesion Molecules and Other Immunoactive Agents
'Laboratory of Immunology
Department of Ophthalmology
Harvard Medical School
Boston, Massachusetts, USA
Dry eye syndromes (DES) represent a common but highly heterogeneous group of
ocular surface disorders (OSD) that affect millions of individuals, in particular women, in
the United States alone.' While efforts to better classify disease categories2.3and determine
relevant diagnostic tests for each disease subtype2.4 continue, common features of DES are
shared by the tear-deficient and non-tear-deficient (evaporative) forms of DES serving as
common denominators of disease. These common denominators include ocular surface
epitheliopathy, tear hyperosmolarity, an unstable preocular tear film and symptoms of dry
eye associated with varying degrees of inflammation.2As such, an increasing number of
investigators contend ocular irritation or discomfort, virtually invariable components of
severe tear-deficient dry eye, are clinical correlates of ocular surface inflammation-which
at the cellular and molecular levels are requisite factors for the pathogenesis of DES.5- 9 In
this brief overview, we will first examine the factors involved in the generation of immune
responsiveness to antigens in the ocular surface and anterior segment. We will then
critically evaluate whether the data generated to date provide a causal link between the
generation of adaptive immunity and OSD in DES, or if the well-described inflammatory
response in the ocular surface could be primarily a consequence of the myriad pathological
Lacrimal Gland. Tear Film. and Dry Eye Syndromes 3 Edited by D. Sullivan el aI., Kluwer Academic/Plenum Publishers, 2002 729
730 M. R. Dana andP. Hamrah
phenomena that characterize all forms of DES. Finally, a hypothetical model is offered that
will attempt to conceptually bridge the gap relating causality and empiric evidence in
relation to immunoinflammatory responses in DES.
"Adaptive immunity," whether (T) cell-mediated or humoral (antibody-mediated),
represents that arm of the immune response generated de novo or secondarily to specific
antigen(s). As such, it is characterized by a delayed clonal response specific to certain
epitopes. In contrast, "innate immunity" refers to that segment of the immune response
whose receptors and effector elements are fixed in the genome, and hence the reaction is
immediate and non-clonal. 1O The latter is typified by neutrophils and macrophages and
their released proteins and peptides generated in response to local perturbations-whether
traumatic (e.g., as occurs in wound healing), infectious or immune (involving new antigens
or epitopes). Since "immune-mediated inflammation" is increasingly being cited as a
primary process in the pathogenesis of both autoimmune (e.g., Sj0gren's) and non­
autoimmune (e.g., primary sicca syndrome or evaporative DES secondary to meibomian
gland dysfunction) dry eye, it is important to first provide a brief outline of the processes
that mediate immune responses in tissues.
The first step in generation of inflammation is an inciting stimulus. This may be
microbial, traumatic or due to introduction of novel antigens (e.g., as occurs in tissue
allotransplantation). These stimuli may lead to the release of proinflammatory cytokines,
nucleic acid fragments, heat shock proteins and various other mediators that, in the aggregate, signal the host that normal physiology and the microenvironment have been
violated. In response to these signals, the second step in this cascade of events occurs when local (resident) tissue cells activate signal transduction pathways (e.g., NFK-B) that
augment or downmodulate expression of cellular cytokine genes and/or cytokine receptor
genes that, in turn, dictate the response of these resident cells to paracrine signals in the
microenvironment by other cells in proximity. These responses are not limited to classic
immunoinflammatory mediators (e.g., interleukins and interferons) but also include other
molecular classes such as growth factors, chemokines and adhesion factors ll acting in a
coordinated fashion to regulate the immune/inflammatory response in the tissue. Mild
stimuli (e.g., a minor abrasive injury to an epithelialized tissue) may not generate a
response beyond this step. However, generation of primary adaptive immune responses,
mediated by stimulation of antigen-specific cells in lymphoid organs, requires recruitment
and activation of bone marrow-derived cells. The latter is accomplished through the
function of two classes of immunoactive agents-eell adhesion molecules (CAMs) and
CAMs represent a heterogeneous group of molecules composed of members of the
immunoglobulin superfamily (e.g., ICAM-l), selectins (e.g., E-selectin) and integrins that
act in concert to activate leukocytes and enhance cell-cell interactions. 12 These cell-cell
interactions are critical in increasing leukocyte adhesion to vascular endothelial cells
Role of Immunity and Inflammation in Corneal and Ocular Surface Disease 731
(VEC) and thereby allow them to "roll" on the VEC prior to integrin-mediated activation
and finally transendothelial migration. Additionally, certain CAMs such as ICAM-I are
important as costimulatory factors that provide na"ive T-cells with the requisite second (T­
cell receptor-independent) signal for sensitization. In corneal immune and inflammatory
diseases, the central role of ICAM-I is now well established. 13 • 14
Once the process of leukocyte activation and transendothelial migration is
accomplished, leukocytes, whether antigen-presenting cells (APC) or effectors, require
recruitment to the primary site of inflammation. If APC (e.g., dendritic cells or
macrophages), they initiate the process of antigen uptake and processing prior to T-cell
priming. However, whereas transendothelial migration from the intravascular compartment
to the tissue compartment is a requisite step for leukocyte function in the periphery, it is
insufficient since leukocytes do not have the capacity for independent homing. This
directionality is provided to leukocytes by chemotactic cytokines, also known as
chemokines, which provide a chemotactic gradient for leukocytes. To date, nearly 50
chemokine species (ligands) and nearly two dozen chemokine receptors have been
characterized, 15 and chemokines are now recognized as important mediators of ocular
surface and corneal disease including ocular allergy, 16 corneal graft rejection,17.18 microbial
keratitis l9.20 and possibly DES. 21 Examples of chemokines implicated in ocular immune
disorders include RANTES, MIP-lalpha and IL-8, just to name a few. Hence, the
coordinated and differential expression of select chemokines and CAMs leads to
recruitment of APCs to sites of inflammation. These requisite steps in the generation of
antigen-specific adaptive immunity are in turn related to upregulation in the activity of
proinflammatory cytokines such as interleukin-l (IL-I). For example, in the cornea and
ocular surface the expression of ICAM-I appears to be largely regulated by IL-I
expression. 13 Moreover, IL-I in concert with tumor necrosis factor-alpha (TNF-a) leads to
upregulation of select chemokines that in turn lead to activation and recruitment of ocular
dendritic (e.g., Langerhans) cells. 22
The role of "master molecules" such as IL-I and TNF-a that are capable of (1)
augmenting inflammation and innate immunity (e.g., IL-I, IL-6, TNF-a); (2)
overexpressing factors involved in T helper cell differentiation, activation (e.g., IL-12,
CD40) and proliferation (e.g., IL-2) and (3) APC maturation and recruitment is getting
increasing attention in the corneal immunology literature/2.25 because these molecules link
innate and adaptive immune responses and may serve as therapeutic targets in a host of
immune disorders.
A large body of evidence suggests clinically significant, and especially (but
importantly not exclusively) tear-deficient, DES is associated with variable degrees of
ocular surface inflammation. The hallmark of the inflamed and irritated eye is "red eye,"
characterized by vascular engorgement and variable degrees of matrix (stromal) edema.
Nonspecific features of this tissue inflammation include extravasation of protein and fluid
732 M. R. Dana andP. Hamrah
from "leaky" vessels with impaired barrier function at the level of their VEC, loss of
epithelial barrier function and infiltration of leukocytes into the tissue matrix. At the
molecular level, ample histological and flow cytometric data demonstrate enhanced
expression of proinflammatory cytokine (e.g., IL-I, IL-6, IL-8, TNF-a) mRNA and protein
by the ocular surface epithelium or tear film.5.21.26-31 Conversely, treatment of DES, either
clinically or in animal models of keratoconjunctivitis sicca, with anti-inflammatory agents
(e.g., cyclosporin A) has been associated with improved disease endpoints either clinically6.9.32-36 or at the level of the tissue.5.26.37 In addition to these findings, DES­
associated ocular surface epitheliopathy has been associated with a significantly enhanced
level of class II major histocompatibility complex (MHC) antigen (HLA DR) expression
by resident ocular surface epithelial cells.5 Since presentation of processed (foreign or
autoantigen) peptides in the context of class II MHC molecules is a classic pathway for
priming of CD4-positive T-cells, it has been postulated but not proven that this
phenomenon enables these epithelial cells to serve as "non-professional" (since they are
not bone marrow-derived) APC,5.38 as has been postulated for lacrimal gland epithelia.39
The fact the expression of CD40 and its ligand (CD40L or CD154) is likewise significantly
enhanced in OSD related to ocular sicca has similarly provided indirect support, but again
no proof, these epithelial cells may have all the requisite phenotypic markers for
stimulating a primary T-cell response.5
In summary, the association between OSD in DES with immunoinflammatory
markers on the one hand, and the subsequent downmodulation of these factors when dry
eye is treated with anti-inflammatory/immunomodulatory treatments on the other, has
provided indirect correlative evidence for the involvement of immunoactive agents in the
pathophysiology of DES.
As noted above, the substantial empirical evidence linking DES with
immunoinflammatory molecular and cellular markers in the ocular surface is sufficient to
demonstrate a significant role for these mediators in the pathophysiology of OSD in dry
eyes. The question, however, is not whether these mediators playa role, but rather whether
this role is causal. Moreover, is the site of putative T-cell activation the ocular surface? If
so, how would these molecular features fit with the paradigm of immunity described
above, and with the various features of pathobiology described thus far in DES?
We simply do not have adequate knowledge to answer any of these questions
definitively. For example, the immune (HLA DR, CD40 overexpression) and inflammatory
(IL-I, TNF-a, ICAM-I overexpression) features of DES may similarly be applied to non­
sicca-related ocular conditions such as atopic disease,8 allograft rejection,23.40 cicatrizing
conjunctivitis,41 ocular surface burns42 and ocular graft-versus-host disease post-allogeneic
Role of Immunity and Inflammation in Corneal and Ocular Surface Disease 733
bone marrow transplantation,7 just to name a few. Hence, the overexpression of these
factors is by no means specific to DES. In addition, reasons exist why some putative
"immune" features of the OSD observed in DES may be non-specific consequences of
inflammatory responses. For example, increased expression of HLA DR has been
demonstrated in autoimmune (Sj0gren's) and non-autoimmune DES, and as has been
argued by Baudouin and colleagues,S may well represent a nonspecific marker of
inflammation, since proinflammatory cytokines such as interferon-gamma (IFN-y) and
TNF-a may lead to class II MHC overexpression.31 .43 Increased expression of CD40, a
member of the TNF superfamily of receptors, which is expressed near-ubiquitously by
nucleated cells, may likewise be secondary to inflammation44 and not necessarily as a facet
of APC-T-cell interaction. In fact, for the specific case of DES, central features of its
pathology, namely alterations in the availability of lacrimal trophic factors and neural (and
possibly endocrine) dysregulation, may amplify ocular surface inflammatory changes.45.46
We propose that while significant work remains in elucidating the molecular
mechanisms of ocular surface inflammation in the pathophysiology of DES, it is unlikely a
simple linear and highly reductionist cascade of events will emerge to explain all the
pathological features of the different types of DES. However, ample scientific evidence
exists from the ophthalmic and non-ophthalmic fields that can allow us to propose a model
that could explain ocular surface inflammation as a cause and consequence of OSD in
DES. In so doing, we hope to refrain from selective emphasis or de-emphasis of empiric
evidence and provide investigators with a model system whose parts can be independently
This model (Fig. 1) proposes that lacrimal gland disease, which itself has multifaceted
dimensions (immune, apoptotic, neurendocrine), leads to OSD through various
mechanisms-alterations in trophic factor availability, lacrimal insufficiency leading to
secondary hyperosmolarity, etc. These features of the ocular surface pathology, potentially
compounded by microabrasive effects of blinking in the dry eye state or other anatomic
alterations, lead to upregulation of proinflammatory cytokines such as IL-l, IL-6 and TNF­
a. 36 However, these cytokines are not simply "passive" markers of disease. Rather, these
molecules are critical in amplifying the disease process in the local microenvironment of
the cornea and conjunctiva. IL-l, for example, can lead to epithelial cell proliferation,
keratinization and angiogenesis47 and thereby possibly link OSD with lid margin
disease-a commonly seen association. Additionally, IL-I may lead to upregulation of
matrix proteases,48 including collagenases and therefore exacerbate stromal pathology as
well as alter the paracrine effect of other cytokines on resident fibroblasts and epithelial
cells. Inflammatory cytokines have also been implicated in the regulation of epithelial
mucin expression49 and may be relevant in the mucin alterations previously described in
DES.50.51 Ocular surface changes as a result of the cytokine microenvironment may lead to
relative hypesthesia and changes in blink rate, a common feature of chronic ocular surface
inflammatory disease, that may in turn exacerbate disease by altering the feedback to the
734 M. R. Dana andP. Hamrah
lacrimal gland. This non-reductionist view of cytokine biology in the ocular surface relates
disease parameters to an ocular-lacrimal gland "functional unit" as proposed by Stern and
Paradigm for Dry Eyes
Many critical questions should be answered in regard to the pathophysiology of OSD
in DES. One critical area for further study is the activation and function of ocular surface
APC in dry eye. Our laboratory has recently discovered that the dogma stating the cornea
is devoid of any APC53-60 is woefully inadequate. Interestingly, we found the normal
cornea has a significant endowment of class II MHC- but CD45+CDllc+ dendritic cells
in the stroma and epithelium. These cells do not express B7 (CD80/CD86) costimulatory
markers in the normal state and are hence "immature.,,61 However, on exposure to
inflammatory stimuli associated with inflammatory cytokine (IL-l, TNF-a) upregulation,
they significantly alter their phenotype and express CD80, CD86 and class II MHC
antigens (Fig. 2). We recently determined these cells are capable, at least in the transplant
setting, of efficiently migrating to draining lymph nodes where they normally congregate
in T-cell-rich areas in the parafollicular areas of the node.62 Since the interaction of APC
and T-cells in lymphoid organs, as opposed to peripherally, is far more efficient in
inducing T-cell activation,63 it is critical to determine how corneal and ocular surface APC
function and trafficking is altered in DES, if indeed adaptive immune responses are
germane to the disease mechanisms of OSD in DES.
Role of Immunity and Inflammation in Corneal and Ocular Surface Disease 735
Figure 2. The center of the normal cornea is endowed with many CDllc· dendritic cells (A). Double
staining with CD80 (B) shows these cells do not express this maturation marker. In addition, these dendritic
cells are MHC class II and CD86 negative. In the inflamed cornea, the number of CDllc· dendritic cells
increases (C). Moreover, significant change occurs in the phenotype of these cells and they upregulate CD80
(D), CD86 and MHC class II. Magnification (A-D) 400X.
Finally, it would be critical to determine if and to what extent the inflammatory
changes observed in the ocular surface in severe DES are, at least in part, a protective and
not solely deleterious mechanism. Accordingly, it is important to underscore the fact that
immune responses are critical in host defense against pathogens. lO The ocular surface
epithelium relies on two main sources, the…