IMMUNOPATHOLOGY OF CICATRICIAL PEMPHIGOID: STUDIES OF COMPLEMENT DEPOSITION

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1 THE JOURNAl. or I NVE.~TIGATIVE DERMA rolog\, 68: i Copyright@ 1977 by The Williams & Wilkins Co. Vol. 68. No. I Printed tn U..A. IMMUNOPATHOLOGY OF CICATRICIAL PEMPHIGOID: STUDIES OF COMPLEMENT DEPOSITION R. s. ROGERS, III, M.D., H. 0. PERRY, M.D., S. F. BEAN, M.D., AND R. E. JORDON, M.D. Cutaneous Immunopathology Unit and the Departments of Dermatology and Immunology, Mayo Clinic and Mayo Foundation, Rochester, M innesota, and the Diagnostic Clinic of Houston (SFB) Houston, Texas, U.S. A. Im munopathologic investigations were conducted on t he sera and oral mucosal tissue specimens of 23 patients with cicatricial pemphigoid. A linear, continuous basement membrane zone pattern was noted in 83% of oral mucosal biopsy specimens studied. This pattern is indistinguishable from the pattern noted in immunofluorescence studies of bullous pemphigoid, herpes gestationis, and some cases of desquamative gingivitis. Complement studies provided data supportive of classical pathway activation in cicatricial pemphigoid tissue. Deposition oflga with Factor B, properdin, and C3 raised the possibility of alternative pathway activation, a question requiring further study. Circulating antibasement membrane zone antibodies were noted in the sera of two patients with cicatricial pemphigoid. Cicatricial (benjgn mucous membrane) pemphigoid (CP) is a well-defined disease characterized by its chronic course, its scarring nature, and its predilection for the mucosal surfaces. Civatte [1) and Lever [2] separated CP from pemphigus vulgaris by the presence of subepithelial bullae in CP. Lever!3 ], Hardy et al [4), and Shklar and McCarthy!51 reported large series defining the distinct clinical nature of the disease. Lortat-Jacob [6] described a disease. dermatite bulleuse mucosynechante et atrophiante, which is indistinguishable from CP (7]. The clinical and histopathologic characteristics of CP suggest an etiologic relationship between CP and bullous pemphigoid (BP). Several recent investigations [7-14] have demonstrated the presence of tissue-bound immunoglobulin and complement components C3 and C4 at the basement membrane zone (BMZ) in CP. The linear pattern of this BMZ staining is identical to the immunofluorescent (IF) pattern of BP. The identical direct IF findings lend credence to an etiologic relationship between CP and BP. Further support for this relationship is the presence of Manuscript received June 14, 1976; accepted for publication August 15, This investigation was supported in part by Research Grant Al from the. ational Institutes of Health. Public Health Service. and by grants from the Minnesota Chapter of the Arthritis Foundation. Presented in part at the Annual Meeting of the American Federation for Clinical Re!';earch. Atlantic City. New Jersey, May 4, Reprint requests to: Dr. R.. Rogers, Ill. Department of Dermatology, Mayo Clinic. Rochester. Minnesota Abbreviations: BP: bullous pemphigoid BMZ: ba.,ement membrane wne C: complement CP: cicatricial pemphigoid IF: immunofluorescent lg: immunoglobulin 39 circulating anti-bmz antibodies in the sera of several patients with cicatricial pemphigoid [15-18]. We have investigated sera and tissue samples from 23 patients with CP by IF techniques. Two of 21 patients' sera demonstrated cir culating anti BMZ antibodies. Nineteen of 23 patients' tissues demonstrated linear BMZ deposition of immunoglobulins as well as components of both the classical and alternative complement pathways and fibrin. PATIE:-.:T~ A:\0 :\1ETHOD, Twenty-three patients with CP were studied. Four of these patients. whose clinical. histopathologic, and immunopathologic findings have been reported elsewhere [19 ], had their disea e begin as desquamative gingivitis which then e\ olved into CP. Almo t all immunopathologic tests had been conducted since the inception of our Cutaneous Immunopathology Unit in June erum and tissue samples were obtamed by standard procedures and were tested immediateh or were stored at - I0 C until tested. Oral mucosal specimens were obtained in all 23 patients, skin specimens in 2 patients. and vaginal mucosal specimens in l patient. Twenty of 23 of the oral mucosal histopathologic specimens revealed subepithelial bullae. In 3 patients, the histopathologic specimens were nonspecific. Controls consisted of sera and tissues from other patients with oral mucosal inflammatory diseases. the results of which have been reported elsewhere [20 ). Antihuman lgg was prepared in goats, assayed. and conjugated with fluorescein i8othiocyanate by methods pre\ iously reiewed in detail [21]. Labeled antisera to lgm. * lga. * C3," and fibrin.* as well as unlabeled antisera to C4t and labeled goat antirabbit and rabbit antigoat IgG, * were purchased. Hyland Division, Travenol Laboratories. Inc.. Costa Mesa, Calif t Behring Diagnostics, American Hoechst Pharmaceuticals, Sommerville, N. J

2 40 ROGERS ET AL Rabbit antisera t.o Clq and to properdin Factor B were prepared and tested by the methods of Morse and Christian [22] and Gotze and Miiller-Eberhard [23), respectively. Goat antiserum to properdin was made after isolating human properdin by the method of Pensky et al [24]. Details of the preparation and use of these antisera (to Clq. Factor B. and properdin) have been published previously in detail [25-27 ]. All antisera were checked for specificity by both double immunodiffussion (Ouchterlony) and immunoelectrophoresis. Units of antisera, fluorescein-protein ratios, antibody protein assays, and dilutions at which the conjugated antisera were used conformed to previously determined standards [21). Serum specimens were tested for circulating antibodies by t he indirect IF staining technique according to established procedures [21) using monkey esophageal mucosal sections. Tissue specimens were tested for fixed immunoglobulins and complement components by both the di.rect and the modified indirect IF procedures as previously reported in detail [21-29]. Direct IF testing of C'P tissue specimens was conducted with antisera to lgg, IgA, lgm, fibrin, and C3 by previously described methods [21-27). The modified indirect IF staining method [25-29] was used to test for the presence of Clq. C4. Factor B, and properdin. Briefly, this method involves treating tissue specimens first with rabbit anti-clq, rabbit anti-c4. or rabbit anti-factor B and then with labeled goat antirabbit lgg. In a similar fashion goat antiproperdin was used first, followed by labeled rabbit antigoat lgg. The specificity controls for the modified indirect IF technique have been reported [25-27]. RESL;LTS The Table is a summary of our immunopathologic observations of the oral mucosal lesions of 23 patients with CP. Both cutaneous lesions and the vaginal mucosal lesion also demonstrated a linear BMZ pattern by direct IF testing with deposition of immunoglobulins and complement components. A continuous, linear BMZ pattern was found in 19 of 23 or 83% of the oral mucosal biopsy specimens by the direct IF test. All 19 tissue specimens had the deposition of one or more TABLE. Summary of immunopathologic obseruations of cicatricial pemphigoid mucosal lesions J\nti~era Linear BMZ panern :--lumber Percent positive lgg 9/ lga 8/20 40 lgm 6/21 29 C3 17/21 81 C!q 7/11 64 C4 2/4 50 Factor B 5/10 50 Properdin" 7/11 64 Fibrin 15/23 65 P ositive BMZ pattern 19/23 83 Modified indirect ff staining employed Vol. 68, No. 1 F1c. l. Linear. continuous deposition of lga at the basement membrane zone of an oral mucosal biopsy specimen. This pattern is typical of the pemphigoid spectrum of disease ( x 250). complement components at the BMZ. Thirteen of the 19 positive tissue specimens had the deposition of 1 or more immunoglobulin at the BMZ. Interestingly, lgg deposition was noted in ouly 9 of 23 tissues tested. lga deposition (Fig. 1) occurred in 8 of 20 and IgM in 6 of 21 tissues tested, respectively. Six had deposition of 2 or more complement components and fibrin at the BMZ. A continuous. linear BMZ pattern similar to the one that we report herein has been seen only in oral mucosal specimens from patients with desquamative gingivitis [19] and BP. Only 2 results (C3 and fibrin in a linear BMZ pattern) in 42 patients with miscellaneous oral mucosal diseases have been noted that could be questionably interpreted as positive linear BMZ staining [20]. Evidence for activation of the classical complement pathway in lesions of CP can be gleaned from the deposition of Clq, one of the subunits of the first component of complement, and C4, another early reacting component of the classical sequence These components were noted in 7 of 11 and 2 of 4 specimens. respectively. lga deposition (Fig. 1) in 8 of 20 tissue specimens studied is noteworthy as this immunoglobulin does not characteristically activate the classical complement pathway but can activate the alternative pathway [23]. Alternative pathway compo-

3 Jan CICATRICIAL PEMPHIGOID 41 nents, Factor B and properdin, were present in a high percentage of lesions studied. in 5 of 10 and 7 of 11 specimens, respectively. In 4 patients' tissues, lga, C3, Factor B, and properdin deposition were apparent at the BMZ in serial sections. As in pemphigus [25] and BP [26}. specific absorption of anti-factor B with Factor B and antiproperdin with properdin resulted in blocking of the IF staining. Circulating anti-bmz antibodies were present in 2 of 23 patients with CP. In both patients t he antibodies were present for a short period of time (1 month and 4 months), after which the antibodies were absent on repeated testing. The titers were 1:320 and 1:80. The substrate for indirect IF testing at the time of the positive tests was guinea-pig esophagus. Both of these patients represent somewhat atypical cases of CP and will be reported elsewhere. No positive indirect IF tests have been noted since June 1973, when monkey esophageal mucosa became our standard substrate. 01. CUSS I O~ Our findings are consistent with the reports of others [7-13 ] who have noted deposition of immunoglobulins C3 and C4 at the BMZ of CP lesions. Nineteen of 23 (83%) of the oral mucosal biopsy specimens studied were positive. Thirteen of the 19 specimens had the deposition of 1 or more immunoglobulin with or without 1 or more complement components at the BMZ in a continuous, linear pat tern. The presence of 2 or more complement components in the oral mucosal specimens, even in the absence of immunoglobulin deposition, increased the number of positive results from 13 of 23 to 19 of 23 patients. A few of our patients had negative results on the first oral mucosal biopsy specimen. onl~, to have a positive result when the study was repeated on " better" tissue. The mucosal surfaces of CP are prone to separation from the submucosa due to the nature of the subepithelial bullous process. With the loss of the mucosal-submucosal interface. specificity of immunopathologic interpretation is lost. Also, tissue which has been ulcerated or eroded is inadequate for interpretation. For these reasons, we have selected the areas of perilesional erythema as the site of our biopsy specimens. With " better" tissue selection positive results are much more frequent and add an important diagnostic method to t he study of CP. The IF pattern reported here (Figs. 1. 2) is a linear. continuous deposition of immunoreactive proteins at the BMZ. This pattern is distinctive for the pemphigoid spectrum of diseases which now appears to include CP, BP, herpes gestationis [27,28,30 j, and perhaps desquamative gingivitis [19]. The Jack of lgg deposition in the majority of tissues tested is somewhat puzzling, however, in FIG. 2. Linear, continuous deposition of C3 at the basement membrane zone of an oral mucosal biopsy specimen (Y. 250). light of the other reports [8,10, 12). Only 9 of 23 CP biopsies tested by us demonstrated IgG deposition to the BMZ. The explanation for this finding is not clear at the present time, although similar findings have recently been noted in both BP (31] and herpes gestations [27]. Deposition of C3. a pivotal protein in the complement sequence which appears at the interface of both the classical and alternative pathways, appears to be a more sensitive. specific immunopathologic finding in all of these diseases. Provost and Tomasi [28,29] and Jordon et al [261 have reported deposition of Clq, C4, and C3, in addition to IgG and IgM at the BMZ of cutaneous lesions of BP by IF techniques. These studies provide immunopathologic evidence for activation of the classical pathway in CP. Immunoglobulins G and M characteristically acti, ate the classical pathway and were present in 11 of 23 patients. Early classical pathway components. Clq and C4. were present in the majority of tissue specimens so tested, and C3 was present in 17 of 21 tissue specimens. On the other hand, one cannot state unequivocally that the classical pathway is activated on the basis of immunopathologic observations alone. although our data would support this contention. The finding of lga deposition in about one-half of CP tissue specimens tested is also worthy of note. Alt hough all immunoglobulins when aggregated or altered may activate the alternative path-

4 42 ROGERS ET AL way,:): lga may activate complement only by that pathway [23 ]. Factor B and properdin, two components of the alternative pathway, were found bound to the BMZ in 5 of 10 and 7 of 11 specimens, respectively. In addition, 4 of our patients' tissues demonstrated IgA, C3, Factor B, and properdin at the BMZ in serial sections. Similar findings have been noted in a few patients with dermatitis herpetiformis [32] where IgA deposition characteristically occurs. Binding of Factor B and properdin in tissues, however, may not be indicative of true alternative pathway activation. Recently Schreiber et al [33] and Fearon and Austen {34] have demonstrated that Factor B and properdin can bind to C3b, thus stabilizing the ''C3b-dependent C3 convertase." Classical pathway activation of C3, which was apparent in all specimens with Factor B and properdin deposition (i.e., Clq and C4 deposition as well), produces C3b which presumably binds to the BMZ with other immunoreactive proteins. Thus, deposition of Factor B and properdin can be explained by their binding to C3b at the BMZ. Similar findings in BP [26,28,29] and. more recently, in herpes gestationis [27,30] are also best explained by this phenomenon. The question of true alternative pathway activation, independent of generation of the C3b-dependent C3 convertase, in all of these diseases requires further study. The presence of circulating anti-bmz antibodies in the sera of 2 of these patients was a transient phenomenon lasting only 1 and 4 months until consistently negative results were obtained. The disease characteristics and substrate specificities have been discussed by other investigators [15-18]. These two patients and a third with circulating anti-bmz antibodies will be reported in a separate communication. REFERENCES 1. Civatte A: Le diagnostic des dermatoses butleuses an laboratorie. Arch Belg Dermatol Syphiligr 5: Lever WF: Pemphigus: a histopathologic study. 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