Absence of Intercellular Antigens in the DeepLayers of the Epidermis in PemphigusFoliaceus

Jean-Claude Bystryn, Jose Rodriguez

J Clin Invest. 1978;61(2):339-348. https://doi.org/10.1172/JCI108944.

12 patients with pemphigus foliaceus, a form of pemphigus with lesions that arise in theintercellular substance in the superficial layers of the epidermis, and 7 patients withpemphigus vulgaris, where lesions are in the deep layers, were studied byimmunofluorescence. Circulating antibodies to intercellular antigens (IC antibodies) werefound in 11 pemphigus foliaceus and 5 pemphigus vulgaris patients. On directimmunofluorescence of skin lesions 75% (9 of 12), pemphigus foliaceus patients hadintercellular deposits of IgG localized solely or predominantly in the superficial epidermallayers, whereas this was not the case in any of the patients with pemphigus vulgaris. Over70% of the pemphigus foliaceus patients with predominantly superficial IgG deposits lackedin their lesions normal intercellular antigens usually expressed in the deep layers of theepidermis. This was shown by the inability of IC antibodies in autologous or allogeneic serato bind to intercellular antigens in the lower epidermis of patient's skin, even though thesame sera could bind to intercellular antigens in all layers of normal allogeneic skin. Lack ofnormal intercellular antigens deep in the epidermis may result in circulating IC antibodiesbinding to the superficial layers, a site which corresponds to, and thus in some patients mayaccount for, the anatomical location of lesions in pemphigus foliaceus.

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Absence of Intercellular Antigens in the Deep Layers of

the Epidermis in Pemphigus Foliaceus

JEAN-CLAUDE BYSTRYNand JOSE RODRIGUEZ, Department of Dermnatology,New York University School of Medicine, New York 10016

A B S T RA C T 12 patients with pemphigus foliaceus, aform of pemphigus with lesions that arise in theintercellular substance in the superficial layers of theepidermis, and 7 patients with pemphigus vulgaris,where lesions are in the deep layers, were studied byimmunofluoreseence. Circulating antibodies to inter-cellular antigens (IC antibodies) were found in 11pemphigtus foliaceus and 5 pemphigus vulgaris pa-tients. On direct immunofluorescence of skin lesions75% (9 of 12), pemphigus foliaceus patients hadintercellular deposits of IgG localized solely orpredominantly in the superficial epidermal layers,whereas this was not the case in any of the patients withpemphigus vulgaris. Over 70% of the pemphigusfoliaceuis patients with predominantly superficial IgGdeposits lacked in their lesions normal intercellularantigens usually expressed in the deep layers of theepidermis. This was shown by the inability of ICantibodies in autologous or allogeneic sera to bind tointercellular antigens in the lower epidermis ofpatient's skin, even though the same sera could bind tointercellular antigens in all layers of normal allogeneicskin. Lack of normal intercellular antigens deep in theepidermis may result in circulating IC antibodiesbinding to the superficial layers, a site which corre-sponds to, and thus in some patients may account for,the anatonmical location of lesions in pemphigusfoliaceus.

INTRODUCTION

Pemphigus is an uincomnmoni bullouis eruption of theskin characterized by recurrent painful blisters ainderosions in the oral cavity and skin. Untreated, thedisease is usually fatal. There are several forms ofpemplhigus which are differentiated principally by thelevel of lesions in the epidermis. The most commonform of pemphigus, pemphigus vulgaris, is charac-terized by bullae which arise deep in the epidermis just

Receivedfor publication 29 April 1977 atd in revisedfornm26 September 1977.

above the basal cell layer. In pemphigus foliaceus, incontrast, the lesions arise in the superficial epidermis.In all forms of pemphigus, the site of the earliest defectvisualizable by electron microscopy is in the intercellu-lar (IC)' substance (1, 2).

Pemphigus is associated with a unique immunologicabnormality. More than 80% of patients with thisdisease develop intercellular antibodies (3-9) toantigens which are normal constituents of the intercel-lular substance in human and mammalian epidermis(10, 11). Immunoglobulins (4, 12-14) together withcomplement (13, 15) are deposited in vivo in theintercellular substance of lesions and normal skin ofpatients with pemphigus. The correlation between theanatomical position of intercellular antigens and theearliest histological lesions of pemphigus, and betweenthe titer of IC antibodies and the severity of the disease(5, 16-18), suggests that IC antibodies play a role in thepathogenesis of the disease. The production of in-traepidermal acantholytic bullae similar to those ofpemphigus by IC antibodies incubated with humanepidermis in organ culture (19) or repeatedly injectedinto the skin (20) or oral mucosa (21) of animals supportsthis concept.

The reasons for the development of bullae indifferent layers of the epidermis in the various forms ofpemphigus are unknown. However, it has been foundthat some patients with pemphigus foliaceus have ICantibodies to antigens present only in the superficialepidermal layers (22), in contrast to the antibodies inother forms of pemphigus which react to intercellularantigens throughout the epidermis. This unusual typeof IC antibodies (subcorneal IC antibodies) has notbeen found in patients with other forms of pemphigusor other bullous diseases and may provide oneexplanation for the superficial location of acantholysisin pemphigus foliaceus.

Wenow report the association of another immunologic

I Abbreviations used in this paper: IC, intercellular; PBS,phosphate-buffered saline.

The Jouirnal of Clinical Investigation Voluime 61 February 1978*339-348 339

abnormality with pemphigus foliaceus, namely theabsence in some patients of intercellular antigensnormally expressed in the lower layers of theepidermis. As a consequence, IC antibodies may onlybe able to bind to the superficial epiderrmis, a site whichcorresponds to (2, 23), and may in some patientsaccount for, the anatomical location of lesions ofpemphigus foliaceus.

METHODS

Patients. All patients in this study had typical pemphigusvulgaris or pemphigus foliaceus by both clinical andhistological criteria. Sera and tissue specimens were collectedfrom all patients during the active phase of their disease andstored at -30°C. Specimens of involved and normal skin wereobtained by punch biopsy and quick-frozen in liquid nitrogen.In one patient, skin specimens were also obtained duringremission of the disease. Normal human skin obtained fromsurgical specimens and monkey and guinea pig esophaguswere used for control studies. Tissue specimens were storedfor no longer than 1 wk and before use were cut into 4 umsections with a cryostat.

Antisera. Fluorescein-labeled conjugates to human IgG,IgM, IgA, and C3 were purchased from Hyland Div., TravenolLaboratories, Inc. (Costa Mesa, Calif.). All sera were tested forspecificity by immunoelectrophoresis. The total proteinconcentrations of the conjugates were, respectively, 10.3, 20.4,20.5, and 10.1 mglml; the specific antibody concentration 2.0,1.9, 1.9, and 1.6 mg/ml; the specific antibody protein-to-fluorescein molar ratio 0.066, 0.028, 0.041, and 0.07; and thefluorescein-to-protein rnolar ratio 3.0, 3.3, 2.3, and 2.3. For use,the conjugates were diluted 1/32, 1/32, 1/32, and 1/64,respectively, in phosphate-buffered saline (PBS), pH 7.3, with4% bovine serum albumin (Schwartz Mann Div., Becton,Dickinson & Co., Orangeburg, N.Y.). These dilutions weredetermined by chessboard titrations (11).

Immunofluorescence studies. Indirect immunofluorescencewas used to test sera for IC antibodies. The tests wereperformed by a standard technique (11), as previouslydescribed (24). Briefly, 0.2 ml of the appropriate dilution ofhuman serum in PBS was incubated for 30 min at 27°C withcryostat-cut sections of frozen normal allogeneic skin andmonkey and guinea pig esophagus. After washing for 10 min inPBS, the specimens were reincubated with 0.2 ml of theappropriate dilution of fluorescein-labeled conjugate for 30min at 27°C, washed for 45 min in PBS, and covered with0.2 ml of mounting medium (90% glycerol in PBS). The slideswere examined with a binocular microscope equipped with amercury vapor lamp and FITC and No. 50 barrier filters. Directimmunofluorescence tests for in vivo deposits of Ig and C3were performed by established methods (11).

The presence of intercellular antigens in skin specimenswas determined by indirect immunofluorescence. For thispurpose, the specimens were reacted with pemphigus vulgarissera known to have antibodies to intercellular antigens in allepidermal layers. The titer of IC antibodies in these seraranged from 320 to above 1,280. For use, all sera were diluted1/20 in PBS.

In vitro complement binding. The ability of antibodies inpemphigus sera to fix complement when bound to intercellularantigens in human skin was tested by a complement bindingtechnique described by Jordon et al. (25). Briefly, undilutedand 1/5 dilution of patients' sera in PBSwere incubated for 30min at 27°C on cryostat-cut sections of frozen normalallogeneic skin. Each section was washed five times with PBS

and incubated for 30 min with a 1/10 dilution of fresh normalhuman serum in PBS as a complement source. The sectionswere washed five times in PBS and incubated withfluorescein-labeled conjugate to human C3 for 30 min.Thereafter, the slides were washed and read as describedabove for immunofluorescence studies.

RESULTS

Indirect immunofluorescence. During the acutephase of their illness, 11 of the 12 patients withpemphigus foliaceus had in their sera IC antibodies tomonkey and (or) guinea pig esophagus. The titersranged from 80 to above 1,280 (Table I). Subcorneal ICantibodies, reacting solely to superficial intercellularantigens in guinea pig esophagus, were present in onepatient to a titer of 160.

Direct immunofluorescence. Direct immuno-fluorescence studies were performed on skin lesionsof 12 patients with pemphigus foliaceus. Inter-cellular IgG deposits were present in all but one.This patient did not have circulating IC antibodies. Ascan be seen in Table II, the deposits were predomi-

TABLE IDirect Immunofluorescence Studies of Skin for Location

of Intercellular Deposits of IgG Lesions

Location ofintercellular

IgG depositstTiter*

Diagnosis IC antibodies Superficial Deep

Pemphigus foliaceus2844 >1280 ++ -3680 >1280 ++ -3842 > 1280 +++ ±3895 0 - -4009 320 + +4137 160§ ++ -4266 80 - ++4306 160 ++ -4326 320 ++ -4406 >1280 ++ +4443 >1280 ++ -4424 0 ++ ±

Pemphigus vulgaris1830 320 - ++3468 640 - ++3773 80 ++ ++3844 80 + +3864 80 ++ ++4075 0 - -4308 640 ++ ++

* Reciprocal of highest dilution giving + reaction by indirectimmunofluorescence.t By direct immunofluorescence.§ Subcorneal intercellular antibodies.

340 J-C. Bystryn and J. Rodriguez

TABLE IIDirect Immunofluorescence Studies of Skin for Location

of Intercellular Deposits of C3 Lesions

Location of intercellularC3 deposits*

Diagnosis Superficial Deep

Pemphigus foliaceus2844 ++ -3680 - +3842 + ++3895 - -4009 + ++4137 - -4266 - ++4306 - ++4326 ++ +4406 - ++4443 - ++4424 - ++

Pemphigus vulgaris1830 - ++3468 - -3773 - ++3844 - _3864 + ++4075 - -4308 ++ ++

* By clirect immunofluorescence.

nantly in superficial epidermal layers (Fig. IA). Theywere present only in this location in six patients andwere heaviest in this area in an additional three. Thus,in 75% (9 of 12) of patients with pemphigus foliaceus, invivo intercellular deposits of IgG were solely orpredominantly superficial. By contrast, this was not thecase in patients with pemphigus vulgaris. IntercellularIgG deposits were present in lesions of six of sevenpatients with this form of pemphigus. In two patientsthe deposits were only in the deep epidermis, and in theother four they were present throughout the epidermis(see Fig. 2). Intercellular IgM deposits were found in onepemphigus foliaceus patient and in none of those withpemphigus vulgaris. In this patient, the deposits weresuperficial. There were no IgA deposits in any patients.

Intercellular deposits of C3 were present in lesions of10 of 12 (83%) of the patients with pemphigus foliaceusand in 4 of 7 (57%) of those with pemphigus vulgaris. Inboth diseases, the deposits were predominantly in thedeep layer of the epidermis. They were exclusively inthis area in six of the pemphigus foliaceus patients andheaviest in this area in another two. Thus, in 80%of thepemphigus foliaceus patients, C3 was deposited pre-dominantly in the deep epidermis. As implicated bythese findings, there was no correlation between thesite of IgG and C3 deposits.

Three possibilities were considered to account forthe superficial localization of intercellular deposits ofIgG in pemphigus foliaceus: (a) Circulating IC an-tibodies in this disease may be of restricted specificityand not directed, and thus unable to bind, to deepintercellular antigens. (b) Some intercellular antigensmay not be expressed in the lower epidermis in lesionsof pemphigus foliaceus. (c) IgG may be blockedfrom binding to deep intercellular antigens by otherclasses of Ig or by complement already deposited atthese sites.

Specificity of circulating IC antibodies. The spec-ificity of IC antibodies in the 10 pemphigus foliaceuspatients with diminished or absent deposits of IgG intheir deep epidermis was studied by reacting their seraat a 1/20 dilution with guinea pig and monkeyesophagus and normal allogeneic skin. As can be seenin Table III, only one patient had subcorneal ICantibodies which reacted only to superficial intercellu-lar antigens in guinea pig esophagus. Two patients didnot have IC antibodies. The remaining seven patientshad IC antibodies reactive to intercellular antigens inall layers of allogeneic skin and zenogeneic esophagus.This finding indicates that restricted specificity of ICantibodies cannot account for the superficial location ofin vivo bound IgG in the majority of patients with thisphenomenon.

Expression of intercellular antigens in deep epider-mal layers of lesions. The presence of intercellularantigens in lesions of six pemphigus foliaceus patientswithout intercellular deposits of IgG in the lowerepidermis and one patient with very faint deposits wasstudied as described in Methods. Allogeneic IC anti-bodies in pemphigus vulgaris sera were unable to bindto deep epidermal intercellular antigens in five of theseven (71%) patients (see Table IV). Autologous ICantibodies were unable to bind to the lower epidermisin four of five (80%) patients (Figs. lB and 3A). Theremaining two patients were not studied because theylacked IC antibodies. One patient (3842) lacked deepintercellular antigens reactive to autologous but not toallogeneic IC antibodies (see Figs. 3A-C) whereas theconverse was true in another patient (4306). In all cases,IC antibodies in pemphigus foliaceus and pemphigusvulgaris sera were able to bind to intercellular antigensin the lower layers of normal allogeneic epidermis (seeTables III and IV, and Figs. 1C and 3C), indicating thatthe lack of binding to pemphigus foliaceus lesions wasdue to the absence of appropriate antigens in this area.Overall, deep intercellular antigens reactive to autolo-gous sera were not expressed in lesions of 33% ofpemphigus foliaceus patients and in 80% of those whodid not have in vivo intercellular deposits of IgG in thislocation. In a somewhat smaller proportion of patients,IC antigens reactive to allogeneic antibodies were alsoabsent.

Absence of Deep Intracellular Antigens in Pemphigus Foliaceus 341

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TABLE IIISpecificity of IC Antibodies in Patients with Pemphigus

Foliaceus and Absent or Diminished DeepIntercellular Deposits of IgG

Specificity of IC antibodies

EsophagusPemphigus Allogeneic normal skin

foliaceus Guineasera pig Monkev 4453 4437 4528 4546

2844 G G G* G G G*3680 G G G G G G3842 G G G G* G G*3895 -

4137 U -4306 G G G G G G4326 G G G NS G G4406 G G G U G G4443 G G NS G G G4424 - NS

NS, not studied; U, antibodies directed to IC aintigens inupper layer of epidermis only; L, antibodies directed to ICantigens in lower layer of epidermis only; G, antibodiesdirected to IC antigens in all layers.* Reactive predominately to U-IC.

FIGURE 2 Patien1t 3773. Direct immunofluorescence ofpemiiphigus vulgaris lesion. Intercellular deposits of IgG arepresenit in all layers of the epidermis. X228.

Effect of il viv(o deposits of Ig or complement onibitnditng of itntercellular anitibodies. The possibilitythat Ig or complement bound in vivo to intercellularanitigens in the deep epidermis blocked the bindiing ofother IC antibodies was unlikely because of thefollowinig observations: (a) There were no in vivodeposits of IgG, IgM, or IgA in the deep epidermallayers of the five patients lacking IC antigens in this

location. IgE and IgD were not tested for, since theirconcentration in serum is very low and no abnormaldeposits of these classes have been reported inpemphigus. (b) There were no deep in vivo deposits ofC3 in two of these patients (2844 and 4137), indicatingthat in at least these cases, blocking by complement wasnot a factor. (c) In the two patients (3842 and 4306) inwhich it could be tested, in vivo deposits of C3 in thedeep epidermis did not prevent the subsequent in vitrobinding of antibodies to IC antigens in this location.

Expression of deep intercellular antigens in unin-volved skin of patients with pemphigus foliaceuis. Todetermine whether the loss of intercellular antigens inpemphigus foliaceus was restricted to lesions or was amore general phenomenon, we examined uninvolvedskin in five patients for in vivo deposits of IgG and for thepresence of intercellular antigens in the lower epider-mis. As can be seen in Table V, intercellular deposits ofIgG were present in uninvolved skin in three of five

FIGURE 1 Patient 3680. (A) Direct immunofluorescence of pemphigus foliaceus lesion. Inter-celltilar deposits of IgG are present only in the superficial layers of the epidermis. There areniO deposits in deeper layers. In this and all subsequent figures, the dermal-epidermal junctionis incdicatecl by an arrow. (B) Indirect immunofluorescence. Autologous serum reacted withlesion, indicatinig lack of binding of IC antibodies to deep epidermal layers. Similar resultswere obtained when the same lesion was reacted with allogeneic pemphigus vulgaris sera.(C) Indirect immnlunofluorescence. Patient's serum reacted with normal human skin, demonstratingthat its IC antibodies can react to intercellular antigens in deep layers of allogeneic epidermis.x217.

Absence of Deep Intracellular Antigens in Pemphigus Foliaceus 343

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TABLE IVPresence of Deep Intercellular Antigens in Lesions of Pemnphigus Foliaceus

and Normal Allogeneic Skini

Binding of IC antibodies to deep intercellular antigens*

NornialPenmphiguis vulgaris serum serumii

AutologousSkin specimen serum 2103 2600 2730 2832 2932 3573 1 2

Pemphigus foliaceuslesions

2844 - - - -

3680 - -

3842 - + + - -3895t NS + - - -4137t NS - - - -4306 + - - - - -4443

Normal allogeneic1 + + + + + + + _ _2 + + + + + + + _ _3 + + +4 + + +5 + + +6 + + +7 + + +8 + + +

NS, not studied.* All sera tested at 1/20 dilution.

Sera of patients 3895 and 4137 were negative for

patients, b)ut in only one were these exclusively in thesuperficial layers. Intercellular IgG deposits werepresent in lesions of four of the five patients, but bycontrast, were exclusively localized to the upperepidermis in all cases. The presence of intercellularantigens in the lower epidermis of lesions anduninvolved skin was tested by reacting these tissueswith autologous and pemphigus vulgaris sera aspreviously described. In uninvolved skin, as can beseen in Table VI, deep intercellular antigens reactive toautologous sera in vivo or in vitro or to allogeneic sera invitro were present in four of five patients. In lesions ofthe same patients, deep intercellular antigens werepresent in only one patient and were weakly expressed

IC antil)odies to autologous or allogeneic skin.

TABLE VDirect Immunofluorescence in Pemphigus Foliaceus

Lesions and Uninvolved Skin

Location of intercellular deposits of IgG

Lesion Uninvolved skinPemphigus foliaceus

patients Stuperficial Deep Superficial Deep

2844 ++ - ++ -3895 - - - -4137 ++ - - -4306 ++ - ++ ++4443 ++ - ++ ++

FIGuRE 3 Patient 3842. (A) Indirect immunofluorescence. Autologous serum reacted withpatient's lesion, demonstrating absence of deep intercellular antigens reactive to autologous ICantibodies. (B) Indirect immunofluorescence. Pemphigus vulgaris serum reacted with samelesion, demonstrating that tissue retains intercellular antigens in lower epidermis reactive toallogeneic IC antibodies. (C) Indirect immunofluorescence. Patient's serum reacted with normalhuman skin, demonstrating that its IC antibodies can react to intercellular antigens inlower layers of epidermis. x215.

Absence of Deep Intracellular Antigens in Pemphigus Foliaceus 345

TABLE VIPresence of Deep Intercellular Antigens in Pemphigus

Foliaceus Lesions and Uninvolved Skin

Binding of IC antibodies to deep intercellular antigens

Lesions Uninvolved skin

Pemphigus PemphigusAutologous vulgaris Autologous vulgaris

Patient serum serum serum serum

28443895* NS + NS +4137* NS - NS +4306 + - NS4443 - - + NS

NS, not studied.* Antibodies to G-IC lacking in autologous sera.t In vivo.

in a second. These findings indicate that the absence ofintercellular antigens in the lower layers of theepidermis in pemphigus foliaceus is predominantlyassociated with lesions of this disease, though occa-sionally the process may involve uninvolved skin aswell.

Specificity of deep intercellular antigens. Thepatterns of cross-reactivity observed when pemphigusfoliaceus and vulgaris sera were reacted with differentspecimens of skin indicate that there are severaldifferent intercellular antigens in the lower layers of theepidermis. As can be seen in Table III, tissue specimen4437 lacked deep intercellular antigens reactive withsera 4406, but had deep intercellular antigens reactiveto other sera. In Table IV, lesion 3842 had deep inter-cellular antigens reactive to allogeneic but not toautologous sera, whereas the reverse was true for le-sion 4306. These findings suggest that there may beindividual and group-specific intercellular antigens inthe lower epidermis, either or both of which may belost in pemphigus foliaceus.

In vitro complement binding by pemphigus sera.After their interaction with normal allogeneic humanskin, none of the pemphigus foliaceus or vulgaris serawere able to fix C3 in vitro.

DISCUSSION

This study shows that pemphigus foliaceus may in someinstances be associated with a loss of intercellularantigens normally present in the deep layers of theepidermis.

It was found that patients with pemphigus foliaceushad IgG fixed in vivo exclusively or predominantly tothe intercellular substance in the uppermost layers ofthe epidermis. In contrast, in pemphigus vulgaris, IgGwas deposited in the deeper layers as well. Several

possibilities were entertained to account for thisfinding. The first was that the specificity of ICantibodies in patients with pemphigus foliaceus wasrestricted to superficial intercellular antigens. Sub-corneal IC antibodies reactive only to superficialantigens occur in a few patients with pemphigusfoliaceus (22). In the present study, subcorneal ICantibodies were found in only 1 of 12 patients. Theantibodies in the other patients were capable ofreacting to intercellular antigens in all layers ofnormal allogeneic or xenogeneic stratified squamousepithelium, thus excluding this possibility from con-sideration. The second possibility was that somenormal intercellular antigens were not expressed in thelower epidermis of the patients. This appeared to be thecase in several patients, as evidenced by the inability ofautologous or allogeneic IC antibodies to bind to theintercellular substance in deep layers of their epider-mis. This was due to a lack of intercellular antigensnormally expressed in this location, since the same seracould bind to deep intercellular antigens in normalallogeneic skin. It was unlikely that C3 or other classesof immunoglobulin bound in vivo to deep intercellularantigens were preventing other antibodies from bind-ing to the same site, leading to an erroneous interpreta-tion of antigen loss. None of the five patients lackingdeep IC antigens had in vivo deposits of IgG, IgM, orIgA in this location, and in two patients C3 was absent aswell. Furthermore, in the cases which could be studied,intercellular deposits of C3 did not block the sub-sequent binding of IC antibodies to the same sites. IgEand IgD were not looked for, since no abnormaldeposits of these classes of immunoglobulin have beenreported in pemphigus. Overall, deep intercellularantigens reactive to autologous sera were not expressedin lesions of 33% of the pemphigus foliaceus patientsand in 80% of those lacking deep intercellular depositsof IgG in vivo.

The causes of antigen loss were not determined. Itwas more commonin lesions than in uninvolved skin ofthe same patient. While possibly due to tissue damageresulting from the disease, this was unlikely to be thesole explanation, since intercellular antigens wereretained in the superficial layers of the epidermis atsites of greatest tissue damage. Furthermore, deepintercellular antigens were absent in the uninvolvedskin of at least one patient. The predominant associa-tion of antigen loss with skin lesions suggests theintriguing possibility that regional variations in antigendistribution may account for the distinctive distributionof pemphigus lesions.

Chessboard titrations performed by reacting a panelof IC antibodies with a number of skin specimenssuggest that there are intercellular antigens of at leasttwo differing specificities in human skin. These can bedistinguished by their reactivity with autologous or

346 J-C. Bystryn and J. Rodriguez

allogeneic IC antibodies and may represent individualand group-specific antigens, respectively. Either orboth of these antigens may be lost in pemphigusfoliaceus.

The absence of intercellular antigens in the deeplayers of the epidermis may in part account forsuperficial location of lesions in some patients withpemphigus foliaceus. As a consequence, IC antibodiesmay only be able to react to superficial antigens, a sitewhich corresponds to the anatomical location of lesions.

Wehave previously reported that another immunemechanism which may account for superficial lesions inpemphigus foliaceus is subcorneal IC antibodies,which react only to antigens in the superficial layers ofthe epidermis (22). Such antibodies are extremely rarein patients without pemphigus foliaceus, having beenfound in only 0.1% of over 1,000 patients with unrelatedskin diseases. Subcorneal IC antibodies were presentin one patient in this study.

Thus, at least two different mechanisms may accountfor the location of bullae in the upper layers of theepidermis in pemphigus foliaceus: (a) the presence ofIC antibodies directed only to superficial antigens, and(b) the absence of normal deep intercellular antigens.These mechanisms are not mutually exclusive, andeither or both may be operative in an individual patient.In both cases, IC antibodies would bind only to thesuperficial layers of the skin at sites which correspondto, and thus may account for, the superficial locations oflesions in this form of pemphigus.

The ultimate mechanisms producing acantholysis inpemphigus are unknown. Complement activation issuggested by the binding of complement componentsto the intercellular substance of the epidermis and thepresence of reaction products of complement activationin blister fluid of pemphigus lesions (13, 26). However,our study indicates that there is often a discrepancybetween the site of C3 deposition, which is predomi-nantly restricted to the deeper layers ofthe epidermis inpemphigus foliaceus, and that of lesions of the disease,which are always superficial. Furthermore, circulatingIC antibodies were unable to fix complement whenbound to skin in vitro. These findings, together with theobservation that pemphigus serunm can cause acan-tholysis in vitro in the absence of complement (19),suggests that complement activation is a result ratherthan a cause of the initial injury. Viewed in this light,the predominant location of C3 in the deep epidermismight be due to diffusion restraints imposed by thelarge molecular weight of C3. An alternate mechanismof acantholysis is that binding of intercellular anti-bodies to the surface of epidermal cells causesactivation or release of epidermal proteolytic enzymes(27) or of other factors which in turn lead toacantholysis and activation of complement. Coating ofkeratinocyte surfaces by intercellular antibodies might

also contribute to acantholysis by interfering with theadhesive properties of the cells.

ACKNOWLEDGMENTSWewish to thank Mr. Robert Bonomo and Ms. Beverly Smithfor their technical assistance.

This research was supported, in part, by funds from theDermatology Department at New York University School ofMedicine and the Chernow Fund.

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