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Pure ocular cicatricial pemphigoid
Pure Ocular Cicatricial Pemphigoid A Distinct Immunopathologic Subset of Cicatricial Pemphigoid Thanh Hoang–Xuan, MD,1,2 Herve´ Robin, MD,1,2 Pierre E. Demers, MD, FRCSC,1 Michel Heller, PhD,4 Marianne Toutblanc, MD,3 Louis Dubertret, MD,4 Catherine Prost, MD, PhD4 Objective: To determine whether ocular cicatricial pemphigoid (OCP) may represent a distinct immunopathologic disease when it is pure ocular cicatricial pemphigoid (POCP) (e.g., only confined to the conjunctiva) or when it is associated with skin or extraocular mucous membrane lesions or both (OCP⫹). Design: Prospective, immunologic, and immunopathologic study with special emphasis on direct immunoelectron microscopy. Participants: Six patients with POCP and seven patients with OCP⫹. Intervention: After informed consent was obtained, a conjunctival biopsy was performed in all patients. Skin and extraocular mucosa biopsy specimens were harvested in selected cases only. Main Outcome Measures: Results of direct immunofluorescence and direct immunoelectron microscopy without freezing on conjunctival and skin biopsy specimens, indirect immunofluorescence, and Western immunoblotting analysis were analyzed. Results: Results of direct immunoelectron microscopic examination of the conjunctiva showed the presence of immune deposits in the upper lamina lucida of the basement membrane zone in the six patients with POCP, whereas the immune reactants were located in the lower part of the lamina lucida and in the lamina densa of the basement membrane zone (conjunctiva, buccal mucosa, and skin) in the seven patients with OCP⫹. Direct immunofluorescence was positive in the biopsy specimens of three patients with POCP (50%) and the seven patients with OCP⫹ (100%). Results of indirect immunofluorescence study showed circulating autoantibody levels only in two patients with OCP⫹, and results of Western immunoblot analysis were negative. Conclusions: Results of direct immunoelectron microscopic examination of the conjunctiva support the hypothesis that POCP may be a disease entity distinct from mucocutaneous cicatricial pemphigoid. Ophthalmology 1999;106:355–361 Cicatricial pemphigoid (CP) is a rare subepidermal/subepithelial blistering and scarring autoimmune disorder that affects primarily the mucous membranes, including the conjunctiva (ocular cicatricial pemphigoid [OCP]) and, to a lesser extent, the skin. OCP is characterized by progressive cicatrizing conjunctivitis that leads to fornix foreshortening, symblepharon formation, trichiasis, dry
Originally received: July 1, 1997. Revision accepted: August 5, 1998. Manuscript no. 97355. 1 Department of Ophthalmology, Hoˆpital Bichat-Claude-Bernard, Paris, France. 2 Department of Ophthalmology, Fondation Ophtalmologique Adolphe de Rothschild, Paris, France. 3 Department of Pathology, Hoˆpital Bichat-Claude-Bernard, Paris, France. 4 Department of Dermatology, Hoˆpital Saint-Louis, Paris, France. Presented in part at the Association for Research in Vision and Ophthalmology annual meeting, Fort Lauderdale, Florida, May 1996, and the Joint European Research Meetings in Ophthalmology and Vision annual meeting, Montpellier, France, October 1996. Supported in part by Lions Club, District of Ile-de-France-Est. Reprint requests to Thanh Hoang–Xuan, MD, Bichat-Claude-Bernard Hospital, 46 rue Henri Huchard, Paris, France 75018.
eye syndrome, corneal scars, ankyloblepharon, and blindness. It can be associated with extraocular manifestations of the disease (OCP⫹) or remain isolated. We will call this latter entity “pure ocular cicatricial pemphigoid” (POCP), which is considered by most authors to be a limited form of CP.1–3 With the use of new investigating tools, a great deal of information has been obtained about the molecular mechanisms involved in autoimmune mucocutaneous bullous diseases. Results of studies, including indirect immunofluorescence (IIF), immunoprecipitation, and immunoblot analysis, strongly suggest that these autoimmune disorders belong to a heterogeneous group of diseases that are associated with different target antigens within the basement membrane zone (BMZ).4 –11 Tyagi et al7 recently reported that a 205-kDa protein, the 4 protein of ␣64 integrin, may be one of the BMZ target autoantigens in OCP. Other investigators believe that POCP represents a specific entity because they detected a 45kDa autoantigen4 different from the 180-kDa and 230kDa antigens classically associated with CP and OCP⫹.5 A similar assumption relative to immunologic specificity has also been formulated by Goss et al (Invest Ophthal-
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Ophthalmology Volume 106, Number 2, February 1999 mol Vis Sci 1995;36[suppl]:1025) for pure oral cicatricial pemphigoid, another clinical subset of CP. The immunopathologic hallmark of CP3,12–14 is the in vivo linear deposition of immunoglobulins and complement along the BMZ of the conjunctiva, skin, and/or mucous membranes. Direct immunofluorescence (DIF) is currently the most frequently used immunopathologic technique to show these immunoreactants, but it is inconstantly positive and, moreover, it is not specific for CP.3,15–18 Immunoelectron microscopy is a more sophisticated technique that precisely localizes the level at which immunoreactants deposit within the BMZ. Although relatively complex and time consuming, it is currently almost routinely used for diagnostic purposes in some specialized departments of dermatology. Specific immunoelectron microscopic binding patterns of autoantibodies have been described in the skin of most immune-mediated bullous disorders, including bullous pemphigoid, epidermolysis bullosa acquisita, linear immunoglobulin A (IgA) disease, and CP.19 –23 These findings combined with immunoblot analysis results also confirm that the clinical heterogeneity of this group of diseases may correlate with different target antigens within the BMZ. We have shown in recent studies that direct immunoelectron microscopy (DIEM) applied to the conjunctiva may represent a reliable and useful tool for the diagnosis of autoimmune cicatrizing conjunctivitis, including OCP (Robin et al [Invest Ophthalmol Vis Sci 1994;35(suppl): 2451]) and epidermolysis bullosa acquisita.24 We have shown that the DIEM patterns of immune deposition in the BMZ of these mucocutaneous autoimmune diseases were similar in the conjunctiva, buccal mucosa, and skin of the same patient. In a preliminary report by Robin et al (Invest Ophthalmol Vis Sci 1996;37(suppl):4701), it also has been shown that immunoreactants in POCP had a specific pattern of deposition, in the upper part of the lamina lucida of the BMZ. To support the hypothesis that OCP may include two separate immunologic diseases depending on the presence (OCP⫹) or lack (POCP) of extraocular involvement, we conducted a comparative study using DIEM to localize the immune deposits within the conjunctival BMZ of patients with OCP⫹ and those with POCP.
Materials and Methods Between November 1994 and June 1996, 13 patients (6 men, 7 women) with clinical evidence of OCP were enrolled in the study at Bichat Hospital, Paris. At the time of presentation, all the patients had active disease (conjunctival hyperemia or progressive fibrosis or both), and none of them were receiving systemic antiinflammatory or immunosuppressive therapy. Patients were excluded from the study if long-term topical medications including antiglaucomatous drugs had been taken before the initiation of the cicatricial process. Other exclusion criteria included autoimmune bullous dermatoses other than cicatricial pemphigoid, Stevens– Johnson syndrome and toxic epidermal necrolysis, atopy, rosacea, systemic diseases known to induce conjunctival fibrosis (scleroderma, Sjo¨gren’s syndrome, sarcoidosis, lupus), and history of conjunctival trauma or burn and of severe infectious conjunctivitis.
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Tissue Biopsy Conjunctival biopsy was performed with the 13 patients and 2 control subjects under local anesthesia after informed consent had been obtained. Specimens measured 7 ⫻ 4 mm and were harvested from clinically uninvolved bulbar conjunctiva adjacent to the superior limbus. Perilesional skin biopsy was performed using a 6-mm punch in patients who had cutaneous lesions. Mucosal biopsy also was performed on affected tissues. All biopsy specimens were then bisected, with the first piece immediately embedded in Tissue-Tek OCT compound (Miles Lab, Elkhart, IN) and snap-frozen for DIF. The second piece was processed immediately for DIEM without freezing.
Direct Immunofluorescence Direct immunofluorescence staining was performed using fluorescein-labeled goat immunoglobulin G (IgG) directed to each of the human immunoglobulin heavy chains (␣, ␥, and ), complement components C3 and C4, and fibrin (Behring, Marburg, Germany). Briefly, 5-m cryosections were mounted on gelatin-coated slides, air-dried, and washed in phosphate-buffered saline. One drop of conjugated goat antiserum diluted (1:10) in phosphate-buffered saline was then applied to cover each section. After a 30-minute incubation at room temperature, the slides were washed in phosphate-buffered saline and cover slipped. The slides were immediately read using a Leitz fluorescence microscope.
Direct Immunoelectron Microscopy We used a technique described previously for the diagnosis of epidermolysis bullosa acquisita associated with cicatrizing conjunctivitis.24 Briefly, the biopsy specimens were freshly cut into 0.7-mm slices with a specially designed hand microtome. The slices were washed under agitation in Hank’s medium, fixed in 4.5% buffered formaldehyde, and washed again in Hank’s medium. The slices then were incubated with goat polyclonal antihuman heavy chains ␣ and ␥ antibodies or rabbit polyclonal antihuman C3 antibodies (Dako Laboratories, Glostrup, Denmark). These antibodies were labeled with horseradish peroxidase (Biosys, Compie`gne, France). The conjugated antihuman immunoglobulins and anti-C3 antibodies were diluted at 1:10 in Hanks’ medium and incubated under agitation for 22 hours at 4° C. The slices were then washed in Hanks’ medium, postfixed in Karnovsky’s fixative, and washed in 0.1-M cacodylate buffer at pH 7.2. Detection of horseradish peroxidase was allowed after incubation of the slices in Graham and Karnovsky medium (0.05% diaminobenzidine and 0.01% hydrogen peroxide) for 1 hour under agitation in the dark at room temperature and postfixation in 1% osmium tetraoxide for 1 hour. The samples were then embedded in epoxy resin. Screening to detect immunoperoxidase staining of the BMZ was performed under light microscopy on semithin sections. Then, ultrathin sections were examined without contrast using a JEOL 3200 EXII electron microscope. Electron-dense material appeared as the result of diaminobenzidine reacting with peroxidase-labeled antibodies attached to the autoantigens. Controls included (1) conjunctiva from a healthy patient undergoing cataract surgery; (2) skin from a healthy patient undergoing abdominoplasty; (3) sections of affected conjunctiva, buccal mucosa, and skin from study patients that were directly incubated in diaminobenzidine for the visualization of potential endogenous peroxidases; and (4) conjunctiva and skin from a patient with proven epidermolysis bullosa acquisita.
Hoang–Xuan et al 䡠 Ocular Cicatricial Pemphigoid Table 1. Patients’ Clinical Characteristics Patient No.
Age (yrs)
1 2 3 4 5 6 7 8 9 10 11 12 13
68 74 75 80 78 70 90 84 81 69 60 66 64
Sex
Initial Organ Involved (duration of symptoms at presentation; yrs)
Time of Ocular Symptoms after Initial Organ Involvement (yrs)
Other Organs Affected
F M F F M M F M M F F M F
Eye (10) Eye (4) Eye (15) Eye (3) Eye (3) Eye (7) Mouth (6) Skin (14) Skin (5) Skin ⫹ Eye (2) Mouth (5) Mouth (3) Skin (13)
NA NA NA NA NA NA 3 11 3 0 4 1 13
— — — — — — Skin ⫹ Gen. — Mouth — — Skin Mouth
NA ⫽ not applicable; Gen ⫽ genitals.
Indirect Immunofluorescence Serum was harvested from the patients at the time of the conjunctival biopsy procedure. Circulating anti-BMZ antibodies were detected by standard IIF technique using rat esophagus, normal human skin, and human salt-split skin.25 Fluorescein-conjugated goat antihuman IgG and IgA antibodies (Dako Laboratories, Glostrup, Denmark) were used. Sera from patients with proven bullous pemphigoid, epidermolysis bullosa acquisita, and from healthy subjects served as control specimens.
Western Immunoblot Analysis Western immunoblot analysis was performed on dermal and epidermal extracts as previously described.26 –29 The same control sera as for IIF study were used.
Results Table 1 summarizes the patients’ clinical characteristics. Group 1 (POCP) included six patients (patients 1– 6) with pure conjunctival
involvement (POCP), and group 2 (OCP⫹) included seven patients (patients 7–13) with conjunctival lesions associated with extraocular mucosal (five patients) or cutaneous (two patients) involvement or both (four patients). The patients’ mean age at the time of the initial visit was 74.2 years (range, 68 – 80 years) and 73.4 years (range, 60 –90 years) in groups 1 and 2, respectively. Average duration between initial symptoms and presentation was 7 years (range, 3–15 years) and 6.8 years (range, 2–13 years) in groups 1 and 2, respectively. No statistical differences were found in these parameters. In group 2, initial symptoms involved the mouth in three patients and the skin in four patients. In patient 10, the first lesions simultaneously involved conjunctiva and skin. In six patients, ocular involvement followed initial symptoms by an average of 5.8 years (range, 1–13 years). At presentation, all patients had uncontrolled progressive and inflammatory cicatrizing conjunctivitis, including fornix foreshortening and symblepharon formation (stages II and III according to Tauber’s classification30), with no distinctive clinical ocular features between groups 1 and 2. Table 2 summarizes the immunopathologic findings including DIF and DIEM study results. In group 1, results of DIF examination of the conjunctiva showed immunoreactants, mainly IgG and C3, at the BMZ of three (50%) patients with POCP. In group 2,
Table 2. Direct Immunofluorescence and Direct Immunoelectron Microscopic Studies of Conjunctival, Skin, and Buccal Mucosa Biopsies Patient No.
Diagnosis
1 2 3 4 5 6 7 8 9 10 11 12 13
POCP POCP POCP POCP POCP POCP OCP⫹ OCP⫹ OCP⫹ OCP⫹ OCP⫹ OCP⫹ OCP⫹
DIF Conjunctiva IgA, IgG, — — IgA, IgG, IgG, C3 — IgA, IgG C3 IgA, IgG, IgA, IgG, IgA, C3 IgA, C3 IgA
C3 C3
C3 C3
DIF Skin and/or BM ND ND ND ND ND ND IgA, IgG, IgA, IgG, IgA, IgG IgA,
IgG C3 IgG, C3 C3 IgG, C3 IgG
DIEM Conjunctiva HD and upper LL HD and upper LL HD and upper LL HD and upper LL HD and upper LL HD and upper LL Lower LL and LD Lower LL and LD Lower LL and LD Lower LL and LD Lower LL and LD Lower LL and LD Lower LL and LD
DIEM Skin and/or BM ND ND ND ND ND ND Lower Lower Lower Lower Lower Lower Lower
LL LL LL LL LL LL LL
and and and and and and and
LD LD LD LD LD LD LD
DIF ⫽ direct immunofluorescence; DIEM ⫽ direct immunoelectron microscopy; OCP⫹ ⫽ ocular cicatricial pemphigoid associated with skin and/or extraocular mucosal involvement; POCP ⫽ pure ocular cicatricial pemphigoid; BM ⫽ buccal mucosa; ND ⫽ not done; LL ⫽ lamina lucida; LD ⫽ lamina densa; HD ⫽ hemidesmosomes.
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Figure 3. Direct immunoelectron microscopy on conjunctival biopsy (OCP⫹ patient 12). Immune deposits (thick arrow) are thick and are located in the lamina densa (LD) and partly obscure the lamina lucida. Ep, epithelium; star, stroma (original magnification, ⫻10,000; bar, 1 mm). Figure 1. Direct immunoelectron microscopy on conjunctival biopsy (pure ocular cicatricial pemphigoid patient 3). Immune deposits (thick arrow) are thin and discontinuous and are located in the upper part of the lamina lucida (LL). Ep, epithelium; star, stroma (original magnification, ⫻12,000; bar, 1 mm).
DIF was positive on the BMZ of the conjunctiva and the skin or buccal mucosa or both of all the seven (100%) patients with OCP⫹. The immune deposits included mainly IgA and C3 in the conjunctiva and IgG in the skin and buccal mucosa. Results of light microscopy screening of semithin sections from all biopsy specimens (perilesional skin and buccal mucosa from patients with OCP⫹ and conjunctiva from patients with POCP and those with OCP⫹) processed for DIEM showed positive immunoperoxidase staining that appeared as a continuous linear brown underlining of the BMZ, corresponding to immune deposition. Electron microscopic examination of the ultrathin sections allowed us to precisely locate these deposits within the BMZ. Results of DIEM examination of the conjunctival biopsy specimens from the six patients with POCP showed thin and discontinuous immune deposition located only in the upper part of the lamina lucida (Fig 1), associated with thicker deposits opposite the hemidesmosomes (Fig 2). In contrast, in the seven patients with OCP⫹, the deposits
Figure 2. Direct immunoelectron microscopy on conjunctival biopsy (pure ocular cicatricial pemphigoid patient 1). Immune deposits (thick arrow) are thick and span the whole area of the lamina lucida (LL) opposite the hemidesmosomes (Hd). Ep, epithelium; star, stroma (original magnification, ⫻8000; bar, 1 mm).
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were thick and discontinuous and were seen in both the lower part of the lamina lucida and in the lamina densa of the BMZ of the conjunctiva (Figs 3 and 4A), skin, and buccal mucosa (Fig 4B). Results of DIEM examination of the negative controls (1), (2), and (3) confirmed the absence of BMZ staining (Fig 5). Direct immunoelectron microscopic examination of the positive controls (4) (skin and conjunctiva from a patient with proven epidermolysis bullosa acquisita) disclosed the immune deposition pattern specific for epidermolysis bullosa acquisita (e.g., deposits in the anchoring fibril zone) just beneath the lamina densa of the BMZ (Fig 6).24 Results of IIF technique using rat esophagus and normal human skin as substrates showed absence of circulating autoantibodies in all 13 patients. However, when human salt-split skin was used as a substrate, circulating autoantibodies could be detected in the roof of the cleavage in two patients with OCP⫹ (patients 7 and 13). Results of Western immunoblot analysis using human epidermal and dermal extracts showed absence of reactivity in the sera of all 13 patients, while all control sera reacted with their respective protein bands.
Discussion Cicatricial pemphigoid belongs to the family of autoimmune mucocutaneous subepithelial vesiculobullous diseases. It is characterized by chronic inflammation of the mucosal surfaces and the skin, leading to contraction and scarring of tissues. Some clinical variants of CP have been described, including Brunsting–Perry CP (skin lesions confined to the head and neck),31 localized oral CP,32 disseminated CP,33 and OCP.3 CP-associated target autoantigens not only differ from the antigens of other subepithelial autoimmune diseases including bullous pemphigoid and epidermolysis bullosa acquisita, but they also may differ between the clinical subsets of CP themselves.4 –11 However, the BMZ antigen responsible for antibody binding in CP is still controversial. Depending on the studies, OCP patients sera have recognized a variety of proteins including 45-, 85-, 180-, 230, and 205-kDa proteins.4,5,7,8 Putative target antigens for OCP could be epiligrin or 4-integrin.6,7 In our study, results of IIF and Western immunoblot analysis in group 1 (POCP) showed absence of circulating anti-BMZ antibodies. They were consistent with the find-
Hoang–Xuan et al 䡠 Ocular Cicatricial Pemphigoid
Figure 4. Direct immunoelectron microscopy on conjunctival (A) and oral mucosa (B) biopsies (OCP⫹ patient 11). Binding patterns in ocular and buccal tissue show analogous thick immune deposits (thick arrow) located mainly in the lamina densa (LD) and lamina lucida. Ep, epithelium; star, stroma (A, original magnification, ⫻5000; bar, 1 mm; B, original magnification, ⫻10,000; bar, 1 mm).
ings of Chan et al,34 who believe that absence of detectable circulating anti-BMZ antibodies is characteristic of POCP. In group 2 (OCP⫹), two of seven patients had circulating autoantibodies detected by IIF. Both patients had severe and multiple extraocular lesions. It has already been suggested that the yield of successful detection of circulating autoantibody levels correlated with the extent of the disease.3,13,35 In our study, Western immunoblot analysis was negative in all the cases and thus was unhelpful to demonstrate whether POCP and OCP⫹ differ by antigenic specificities of autoantibodies. This negative result was probably not only caused by the paucity of circulating antibodies, but also by the types of substrate we used. In the future, human conjunctiva as a substrate may be more appropriate than human skin or rat esophagus.13,36
Figure 5. Direct immunoelectron microscopy on conjunctival tissue taken from a patient having undergone cataract surgery (negative control). Note the lack of immunodense material in the lamina lucida and lamina densa (LD). Hemidesmosomes (Hd) are seen as a discrete thickening of the cytoplasmic membrane of basal epithelial cells. The dark dots in the stroma are artifacts. Ep, epithelium, star, stroma (original magnification, ⫻12,000; bar, 1 mm).
In the past 10 years, a great deal of information about the classification of autoimmune bullous dermatoses including CP, bullous pemphigoid, epidermal bullosa acquisita, and linear IgA dermatosis has been obtained with the use of immunoelectron microscopy.19 –23 Direct immunoelectron microscopy is an immunopathologic technique based on the electron microscopic detection of peroxidase-labeled antibodies that are attached to the autoantigens in the affected tissues and that react with diaminobenzidine, hydrogen peroxide, and osmium tetraoxide to form electron-dense material. Thus, not only is it more sensitive than conventional direct immunofluorescence (DIF)3,13,14 and immunoenzymatic techniques,12 but it also precisely shows the immune deposits within the BMZ at the ultrastructural level. In our study, DIF was positive in 10 (77%) of 13 patients, whereas immune reactants were detected by DIEM in all 13 patients
Figure 6. Direct immunoelectron microscopy on conjunctival biopsy of a patient with epidermolysis bullosa acquisita (positive control). Immune deposits (thick arrow) are very thick and are distributed below the lamina densa (LD) in the anchoring fibril zone. Ep, epithelium, star, stroma (original magnification, ⫻10,000; bar, 1 mm).
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Ophthalmology Volume 106, Number 2, February 1999 (100%). The fact that the three DIF-negative patients all belonged to the POCP group could not be correlated with any distinctive feature in their ocular manifestations compared with that of the other patients. Unlike Fine et al,37 who used frozen biopsy specimens to perform the first DIEM study on the conjunctiva of a patient with OCP and reported conflicting results, we used a preembedding technique without freezing on our conjunctival samples. This technique, which has already been well tested by dermatologists for perilesional skin biopsies,19 –23 may best preserve the ultrastructural integrity of the tissues, allow better immunoglobulin penetration, and prevent alteration of their antigenicity. We showed in this study that patients with POCP and those with OCP⫹ had different immunolocations of their target antigens. In all patients with POCP, the immune deposits were located in the upper lamina lucida of the conjunctival BMZ. They were thin and discontinuous, except for those located opposite to the hemidesmosomes, which appeared thicker. These ultrastructural pictures may represent binding of autoantibodies to different target antigens within the lamina lucida. One can hypothesize that the autoantigen located next to the hemidesmosomes may be the 4 integrin identified by Tyagi et al.7 Indeed, this molecule is thought to be located close to hemidesmosomes and to be of importance in maintaining attachment of epithelial cells to the BMZ.38 In all patients of OCP⫹ group, immunoreactant deposition appeared in the lower part of the lamina lucida and in the lamina densa of the BMZ of the skin, buccal mucosa, and conjunctiva. This pattern was identical to that we reported earlier (Robin et al. Invest Ophthalmol Vis Sci 1994; 35[suppl]:2451), and its consistency in the conjunctiva and the other biopsy sites of a same OCP⫹ patient confirmed the reliability of our conjunctival processing technique. Similarly, in another study, we also have shown consistent DIEM patterns in the conjunctiva and skin of patients with epidermolysis bullosa acquisita.24 In conclusion, this study strongly supports the idea that POCP may be a unique clinical and immunopathologic disease distinct from OCP⫹. But POCP may also simply represent an early stage of OCP⫹. The fact that ocular inflammation and fibrosis were equally severe in both groups of patients does not favor this hypothesis. However, longer follow-up of patients with POCP is needed, as extraocular signs may become manifest years after initial ocular involvement. Whether or not the distinction between POCP and OCP⫹ is of prognostic significance and/or implies more specific therapeutic strategies needs to be proved.
5. 6. 7. 8.
9. 10. 11.
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13. 14. 15. 16. 17. 18. 19.
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21.
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32. Rogers RS III, Sheridan PJ, Nightingale SH. Desquamative gingivitis: clinical, histopathologic, immunopathologic, and therapeutic observations. J Am Acad Dermatol 1982;7:729 – 35. 33. Hausser I, Fartasch M, Schleiermacher E, Anton–Lamprecht I. Disseminated cicatricial pemphigoid in a child and in an adult. Ultrastructural diagnostic criteria and differential diagnosis with special reference to acquired epidermolysis bullosa. Arch Dermatol Res 1987;279:357– 65. 34. Chan LS, Yancey KB, Hammerberg C, et al. Immunemediated subepithelial blistering diseases of mucous membranes. Pure ocular cicatricial pemphigoid is a unique clinical and immunopathological entity distinct from bullous pemphigoid and other subsets identified by antigenic specificity of autoantibodies. Arch Dermatol 1993; 129:448 –55. 35. Laskaris G, Angelopoulos A. Cicatricial pemphigoid: direct and indirect immunofluorescent studies. Oral Surg Oral Med Oral Pathol 1981;51:48 –54. 36. Franklin RM, Fitzmorris CT. Antibodies against conjunctival basement membrane zone. Occurrence in cicatricial pemphigoid. Arch Ophthalmol 1983;101:1611–3. 37. Fine JD, Neises GR, Katz SI. Immunofluorescence and immunoelectron microscopic studies in cicatricial pemphigoid. J Invest Dermatol 1984;82:39 – 43. 38. Sonnenberg A, Calafat J, Janssen H, et al. Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol 1991;113:907–17.
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Originally received: July 1, 1997. Revision accepted: August 5, 1998. Manuscript no. 97355. 1 Department of Ophthalmology, Hoˆpital Bichat-Claude-Bernard, Paris, France. 2 Department of Ophthalmology, Fondation Ophtalmologique Adolphe de Rothschild, Paris, France. 3 Department of Pathology, Hoˆpital Bichat-Claude-Bernard, Paris, France. 4 Department of Dermatology, Hoˆpital Saint-Louis, Paris, France. Presented in part at the Association for Research in Vision and Ophthalmology annual meeting, Fort Lauderdale, Florida, May 1996, and the Joint European Research Meetings in Ophthalmology and Vision annual meeting, Montpellier, France, October 1996. Supported in part by Lions Club, District of Ile-de-France-Est. Reprint requests to Thanh Hoang–Xuan, MD, Bichat-Claude-Bernard Hospital, 46 rue Henri Huchard, Paris, France 75018.
eye syndrome, corneal scars, ankyloblepharon, and blindness. It can be associated with extraocular manifestations of the disease (OCP⫹) or remain isolated. We will call this latter entity “pure ocular cicatricial pemphigoid” (POCP), which is considered by most authors to be a limited form of CP.1–3 With the use of new investigating tools, a great deal of information has been obtained about the molecular mechanisms involved in autoimmune mucocutaneous bullous diseases. Results of studies, including indirect immunofluorescence (IIF), immunoprecipitation, and immunoblot analysis, strongly suggest that these autoimmune disorders belong to a heterogeneous group of diseases that are associated with different target antigens within the basement membrane zone (BMZ).4 –11 Tyagi et al7 recently reported that a 205-kDa protein, the 4 protein of ␣64 integrin, may be one of the BMZ target autoantigens in OCP. Other investigators believe that POCP represents a specific entity because they detected a 45kDa autoantigen4 different from the 180-kDa and 230kDa antigens classically associated with CP and OCP⫹.5 A similar assumption relative to immunologic specificity has also been formulated by Goss et al (Invest Ophthal-
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Ophthalmology Volume 106, Number 2, February 1999 mol Vis Sci 1995;36[suppl]:1025) for pure oral cicatricial pemphigoid, another clinical subset of CP. The immunopathologic hallmark of CP3,12–14 is the in vivo linear deposition of immunoglobulins and complement along the BMZ of the conjunctiva, skin, and/or mucous membranes. Direct immunofluorescence (DIF) is currently the most frequently used immunopathologic technique to show these immunoreactants, but it is inconstantly positive and, moreover, it is not specific for CP.3,15–18 Immunoelectron microscopy is a more sophisticated technique that precisely localizes the level at which immunoreactants deposit within the BMZ. Although relatively complex and time consuming, it is currently almost routinely used for diagnostic purposes in some specialized departments of dermatology. Specific immunoelectron microscopic binding patterns of autoantibodies have been described in the skin of most immune-mediated bullous disorders, including bullous pemphigoid, epidermolysis bullosa acquisita, linear immunoglobulin A (IgA) disease, and CP.19 –23 These findings combined with immunoblot analysis results also confirm that the clinical heterogeneity of this group of diseases may correlate with different target antigens within the BMZ. We have shown in recent studies that direct immunoelectron microscopy (DIEM) applied to the conjunctiva may represent a reliable and useful tool for the diagnosis of autoimmune cicatrizing conjunctivitis, including OCP (Robin et al [Invest Ophthalmol Vis Sci 1994;35(suppl): 2451]) and epidermolysis bullosa acquisita.24 We have shown that the DIEM patterns of immune deposition in the BMZ of these mucocutaneous autoimmune diseases were similar in the conjunctiva, buccal mucosa, and skin of the same patient. In a preliminary report by Robin et al (Invest Ophthalmol Vis Sci 1996;37(suppl):4701), it also has been shown that immunoreactants in POCP had a specific pattern of deposition, in the upper part of the lamina lucida of the BMZ. To support the hypothesis that OCP may include two separate immunologic diseases depending on the presence (OCP⫹) or lack (POCP) of extraocular involvement, we conducted a comparative study using DIEM to localize the immune deposits within the conjunctival BMZ of patients with OCP⫹ and those with POCP.
Materials and Methods Between November 1994 and June 1996, 13 patients (6 men, 7 women) with clinical evidence of OCP were enrolled in the study at Bichat Hospital, Paris. At the time of presentation, all the patients had active disease (conjunctival hyperemia or progressive fibrosis or both), and none of them were receiving systemic antiinflammatory or immunosuppressive therapy. Patients were excluded from the study if long-term topical medications including antiglaucomatous drugs had been taken before the initiation of the cicatricial process. Other exclusion criteria included autoimmune bullous dermatoses other than cicatricial pemphigoid, Stevens– Johnson syndrome and toxic epidermal necrolysis, atopy, rosacea, systemic diseases known to induce conjunctival fibrosis (scleroderma, Sjo¨gren’s syndrome, sarcoidosis, lupus), and history of conjunctival trauma or burn and of severe infectious conjunctivitis.
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Tissue Biopsy Conjunctival biopsy was performed with the 13 patients and 2 control subjects under local anesthesia after informed consent had been obtained. Specimens measured 7 ⫻ 4 mm and were harvested from clinically uninvolved bulbar conjunctiva adjacent to the superior limbus. Perilesional skin biopsy was performed using a 6-mm punch in patients who had cutaneous lesions. Mucosal biopsy also was performed on affected tissues. All biopsy specimens were then bisected, with the first piece immediately embedded in Tissue-Tek OCT compound (Miles Lab, Elkhart, IN) and snap-frozen for DIF. The second piece was processed immediately for DIEM without freezing.
Direct Immunofluorescence Direct immunofluorescence staining was performed using fluorescein-labeled goat immunoglobulin G (IgG) directed to each of the human immunoglobulin heavy chains (␣, ␥, and ), complement components C3 and C4, and fibrin (Behring, Marburg, Germany). Briefly, 5-m cryosections were mounted on gelatin-coated slides, air-dried, and washed in phosphate-buffered saline. One drop of conjugated goat antiserum diluted (1:10) in phosphate-buffered saline was then applied to cover each section. After a 30-minute incubation at room temperature, the slides were washed in phosphate-buffered saline and cover slipped. The slides were immediately read using a Leitz fluorescence microscope.
Direct Immunoelectron Microscopy We used a technique described previously for the diagnosis of epidermolysis bullosa acquisita associated with cicatrizing conjunctivitis.24 Briefly, the biopsy specimens were freshly cut into 0.7-mm slices with a specially designed hand microtome. The slices were washed under agitation in Hank’s medium, fixed in 4.5% buffered formaldehyde, and washed again in Hank’s medium. The slices then were incubated with goat polyclonal antihuman heavy chains ␣ and ␥ antibodies or rabbit polyclonal antihuman C3 antibodies (Dako Laboratories, Glostrup, Denmark). These antibodies were labeled with horseradish peroxidase (Biosys, Compie`gne, France). The conjugated antihuman immunoglobulins and anti-C3 antibodies were diluted at 1:10 in Hanks’ medium and incubated under agitation for 22 hours at 4° C. The slices were then washed in Hanks’ medium, postfixed in Karnovsky’s fixative, and washed in 0.1-M cacodylate buffer at pH 7.2. Detection of horseradish peroxidase was allowed after incubation of the slices in Graham and Karnovsky medium (0.05% diaminobenzidine and 0.01% hydrogen peroxide) for 1 hour under agitation in the dark at room temperature and postfixation in 1% osmium tetraoxide for 1 hour. The samples were then embedded in epoxy resin. Screening to detect immunoperoxidase staining of the BMZ was performed under light microscopy on semithin sections. Then, ultrathin sections were examined without contrast using a JEOL 3200 EXII electron microscope. Electron-dense material appeared as the result of diaminobenzidine reacting with peroxidase-labeled antibodies attached to the autoantigens. Controls included (1) conjunctiva from a healthy patient undergoing cataract surgery; (2) skin from a healthy patient undergoing abdominoplasty; (3) sections of affected conjunctiva, buccal mucosa, and skin from study patients that were directly incubated in diaminobenzidine for the visualization of potential endogenous peroxidases; and (4) conjunctiva and skin from a patient with proven epidermolysis bullosa acquisita.
Hoang–Xuan et al 䡠 Ocular Cicatricial Pemphigoid Table 1. Patients’ Clinical Characteristics Patient No.
Age (yrs)
1 2 3 4 5 6 7 8 9 10 11 12 13
68 74 75 80 78 70 90 84 81 69 60 66 64
Sex
Initial Organ Involved (duration of symptoms at presentation; yrs)
Time of Ocular Symptoms after Initial Organ Involvement (yrs)
Other Organs Affected
F M F F M M F M M F F M F
Eye (10) Eye (4) Eye (15) Eye (3) Eye (3) Eye (7) Mouth (6) Skin (14) Skin (5) Skin ⫹ Eye (2) Mouth (5) Mouth (3) Skin (13)
NA NA NA NA NA NA 3 11 3 0 4 1 13
— — — — — — Skin ⫹ Gen. — Mouth — — Skin Mouth
NA ⫽ not applicable; Gen ⫽ genitals.
Indirect Immunofluorescence Serum was harvested from the patients at the time of the conjunctival biopsy procedure. Circulating anti-BMZ antibodies were detected by standard IIF technique using rat esophagus, normal human skin, and human salt-split skin.25 Fluorescein-conjugated goat antihuman IgG and IgA antibodies (Dako Laboratories, Glostrup, Denmark) were used. Sera from patients with proven bullous pemphigoid, epidermolysis bullosa acquisita, and from healthy subjects served as control specimens.
Western Immunoblot Analysis Western immunoblot analysis was performed on dermal and epidermal extracts as previously described.26 –29 The same control sera as for IIF study were used.
Results Table 1 summarizes the patients’ clinical characteristics. Group 1 (POCP) included six patients (patients 1– 6) with pure conjunctival
involvement (POCP), and group 2 (OCP⫹) included seven patients (patients 7–13) with conjunctival lesions associated with extraocular mucosal (five patients) or cutaneous (two patients) involvement or both (four patients). The patients’ mean age at the time of the initial visit was 74.2 years (range, 68 – 80 years) and 73.4 years (range, 60 –90 years) in groups 1 and 2, respectively. Average duration between initial symptoms and presentation was 7 years (range, 3–15 years) and 6.8 years (range, 2–13 years) in groups 1 and 2, respectively. No statistical differences were found in these parameters. In group 2, initial symptoms involved the mouth in three patients and the skin in four patients. In patient 10, the first lesions simultaneously involved conjunctiva and skin. In six patients, ocular involvement followed initial symptoms by an average of 5.8 years (range, 1–13 years). At presentation, all patients had uncontrolled progressive and inflammatory cicatrizing conjunctivitis, including fornix foreshortening and symblepharon formation (stages II and III according to Tauber’s classification30), with no distinctive clinical ocular features between groups 1 and 2. Table 2 summarizes the immunopathologic findings including DIF and DIEM study results. In group 1, results of DIF examination of the conjunctiva showed immunoreactants, mainly IgG and C3, at the BMZ of three (50%) patients with POCP. In group 2,
Table 2. Direct Immunofluorescence and Direct Immunoelectron Microscopic Studies of Conjunctival, Skin, and Buccal Mucosa Biopsies Patient No.
Diagnosis
1 2 3 4 5 6 7 8 9 10 11 12 13
POCP POCP POCP POCP POCP POCP OCP⫹ OCP⫹ OCP⫹ OCP⫹ OCP⫹ OCP⫹ OCP⫹
DIF Conjunctiva IgA, IgG, — — IgA, IgG, IgG, C3 — IgA, IgG C3 IgA, IgG, IgA, IgG, IgA, C3 IgA, C3 IgA
C3 C3
C3 C3
DIF Skin and/or BM ND ND ND ND ND ND IgA, IgG, IgA, IgG, IgA, IgG IgA,
IgG C3 IgG, C3 C3 IgG, C3 IgG
DIEM Conjunctiva HD and upper LL HD and upper LL HD and upper LL HD and upper LL HD and upper LL HD and upper LL Lower LL and LD Lower LL and LD Lower LL and LD Lower LL and LD Lower LL and LD Lower LL and LD Lower LL and LD
DIEM Skin and/or BM ND ND ND ND ND ND Lower Lower Lower Lower Lower Lower Lower
LL LL LL LL LL LL LL
and and and and and and and
LD LD LD LD LD LD LD
DIF ⫽ direct immunofluorescence; DIEM ⫽ direct immunoelectron microscopy; OCP⫹ ⫽ ocular cicatricial pemphigoid associated with skin and/or extraocular mucosal involvement; POCP ⫽ pure ocular cicatricial pemphigoid; BM ⫽ buccal mucosa; ND ⫽ not done; LL ⫽ lamina lucida; LD ⫽ lamina densa; HD ⫽ hemidesmosomes.
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Figure 3. Direct immunoelectron microscopy on conjunctival biopsy (OCP⫹ patient 12). Immune deposits (thick arrow) are thick and are located in the lamina densa (LD) and partly obscure the lamina lucida. Ep, epithelium; star, stroma (original magnification, ⫻10,000; bar, 1 mm). Figure 1. Direct immunoelectron microscopy on conjunctival biopsy (pure ocular cicatricial pemphigoid patient 3). Immune deposits (thick arrow) are thin and discontinuous and are located in the upper part of the lamina lucida (LL). Ep, epithelium; star, stroma (original magnification, ⫻12,000; bar, 1 mm).
DIF was positive on the BMZ of the conjunctiva and the skin or buccal mucosa or both of all the seven (100%) patients with OCP⫹. The immune deposits included mainly IgA and C3 in the conjunctiva and IgG in the skin and buccal mucosa. Results of light microscopy screening of semithin sections from all biopsy specimens (perilesional skin and buccal mucosa from patients with OCP⫹ and conjunctiva from patients with POCP and those with OCP⫹) processed for DIEM showed positive immunoperoxidase staining that appeared as a continuous linear brown underlining of the BMZ, corresponding to immune deposition. Electron microscopic examination of the ultrathin sections allowed us to precisely locate these deposits within the BMZ. Results of DIEM examination of the conjunctival biopsy specimens from the six patients with POCP showed thin and discontinuous immune deposition located only in the upper part of the lamina lucida (Fig 1), associated with thicker deposits opposite the hemidesmosomes (Fig 2). In contrast, in the seven patients with OCP⫹, the deposits
Figure 2. Direct immunoelectron microscopy on conjunctival biopsy (pure ocular cicatricial pemphigoid patient 1). Immune deposits (thick arrow) are thick and span the whole area of the lamina lucida (LL) opposite the hemidesmosomes (Hd). Ep, epithelium; star, stroma (original magnification, ⫻8000; bar, 1 mm).
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were thick and discontinuous and were seen in both the lower part of the lamina lucida and in the lamina densa of the BMZ of the conjunctiva (Figs 3 and 4A), skin, and buccal mucosa (Fig 4B). Results of DIEM examination of the negative controls (1), (2), and (3) confirmed the absence of BMZ staining (Fig 5). Direct immunoelectron microscopic examination of the positive controls (4) (skin and conjunctiva from a patient with proven epidermolysis bullosa acquisita) disclosed the immune deposition pattern specific for epidermolysis bullosa acquisita (e.g., deposits in the anchoring fibril zone) just beneath the lamina densa of the BMZ (Fig 6).24 Results of IIF technique using rat esophagus and normal human skin as substrates showed absence of circulating autoantibodies in all 13 patients. However, when human salt-split skin was used as a substrate, circulating autoantibodies could be detected in the roof of the cleavage in two patients with OCP⫹ (patients 7 and 13). Results of Western immunoblot analysis using human epidermal and dermal extracts showed absence of reactivity in the sera of all 13 patients, while all control sera reacted with their respective protein bands.
Discussion Cicatricial pemphigoid belongs to the family of autoimmune mucocutaneous subepithelial vesiculobullous diseases. It is characterized by chronic inflammation of the mucosal surfaces and the skin, leading to contraction and scarring of tissues. Some clinical variants of CP have been described, including Brunsting–Perry CP (skin lesions confined to the head and neck),31 localized oral CP,32 disseminated CP,33 and OCP.3 CP-associated target autoantigens not only differ from the antigens of other subepithelial autoimmune diseases including bullous pemphigoid and epidermolysis bullosa acquisita, but they also may differ between the clinical subsets of CP themselves.4 –11 However, the BMZ antigen responsible for antibody binding in CP is still controversial. Depending on the studies, OCP patients sera have recognized a variety of proteins including 45-, 85-, 180-, 230, and 205-kDa proteins.4,5,7,8 Putative target antigens for OCP could be epiligrin or 4-integrin.6,7 In our study, results of IIF and Western immunoblot analysis in group 1 (POCP) showed absence of circulating anti-BMZ antibodies. They were consistent with the find-
Hoang–Xuan et al 䡠 Ocular Cicatricial Pemphigoid
Figure 4. Direct immunoelectron microscopy on conjunctival (A) and oral mucosa (B) biopsies (OCP⫹ patient 11). Binding patterns in ocular and buccal tissue show analogous thick immune deposits (thick arrow) located mainly in the lamina densa (LD) and lamina lucida. Ep, epithelium; star, stroma (A, original magnification, ⫻5000; bar, 1 mm; B, original magnification, ⫻10,000; bar, 1 mm).
ings of Chan et al,34 who believe that absence of detectable circulating anti-BMZ antibodies is characteristic of POCP. In group 2 (OCP⫹), two of seven patients had circulating autoantibodies detected by IIF. Both patients had severe and multiple extraocular lesions. It has already been suggested that the yield of successful detection of circulating autoantibody levels correlated with the extent of the disease.3,13,35 In our study, Western immunoblot analysis was negative in all the cases and thus was unhelpful to demonstrate whether POCP and OCP⫹ differ by antigenic specificities of autoantibodies. This negative result was probably not only caused by the paucity of circulating antibodies, but also by the types of substrate we used. In the future, human conjunctiva as a substrate may be more appropriate than human skin or rat esophagus.13,36
Figure 5. Direct immunoelectron microscopy on conjunctival tissue taken from a patient having undergone cataract surgery (negative control). Note the lack of immunodense material in the lamina lucida and lamina densa (LD). Hemidesmosomes (Hd) are seen as a discrete thickening of the cytoplasmic membrane of basal epithelial cells. The dark dots in the stroma are artifacts. Ep, epithelium, star, stroma (original magnification, ⫻12,000; bar, 1 mm).
In the past 10 years, a great deal of information about the classification of autoimmune bullous dermatoses including CP, bullous pemphigoid, epidermal bullosa acquisita, and linear IgA dermatosis has been obtained with the use of immunoelectron microscopy.19 –23 Direct immunoelectron microscopy is an immunopathologic technique based on the electron microscopic detection of peroxidase-labeled antibodies that are attached to the autoantigens in the affected tissues and that react with diaminobenzidine, hydrogen peroxide, and osmium tetraoxide to form electron-dense material. Thus, not only is it more sensitive than conventional direct immunofluorescence (DIF)3,13,14 and immunoenzymatic techniques,12 but it also precisely shows the immune deposits within the BMZ at the ultrastructural level. In our study, DIF was positive in 10 (77%) of 13 patients, whereas immune reactants were detected by DIEM in all 13 patients
Figure 6. Direct immunoelectron microscopy on conjunctival biopsy of a patient with epidermolysis bullosa acquisita (positive control). Immune deposits (thick arrow) are very thick and are distributed below the lamina densa (LD) in the anchoring fibril zone. Ep, epithelium, star, stroma (original magnification, ⫻10,000; bar, 1 mm).
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Ophthalmology Volume 106, Number 2, February 1999 (100%). The fact that the three DIF-negative patients all belonged to the POCP group could not be correlated with any distinctive feature in their ocular manifestations compared with that of the other patients. Unlike Fine et al,37 who used frozen biopsy specimens to perform the first DIEM study on the conjunctiva of a patient with OCP and reported conflicting results, we used a preembedding technique without freezing on our conjunctival samples. This technique, which has already been well tested by dermatologists for perilesional skin biopsies,19 –23 may best preserve the ultrastructural integrity of the tissues, allow better immunoglobulin penetration, and prevent alteration of their antigenicity. We showed in this study that patients with POCP and those with OCP⫹ had different immunolocations of their target antigens. In all patients with POCP, the immune deposits were located in the upper lamina lucida of the conjunctival BMZ. They were thin and discontinuous, except for those located opposite to the hemidesmosomes, which appeared thicker. These ultrastructural pictures may represent binding of autoantibodies to different target antigens within the lamina lucida. One can hypothesize that the autoantigen located next to the hemidesmosomes may be the 4 integrin identified by Tyagi et al.7 Indeed, this molecule is thought to be located close to hemidesmosomes and to be of importance in maintaining attachment of epithelial cells to the BMZ.38 In all patients of OCP⫹ group, immunoreactant deposition appeared in the lower part of the lamina lucida and in the lamina densa of the BMZ of the skin, buccal mucosa, and conjunctiva. This pattern was identical to that we reported earlier (Robin et al. Invest Ophthalmol Vis Sci 1994; 35[suppl]:2451), and its consistency in the conjunctiva and the other biopsy sites of a same OCP⫹ patient confirmed the reliability of our conjunctival processing technique. Similarly, in another study, we also have shown consistent DIEM patterns in the conjunctiva and skin of patients with epidermolysis bullosa acquisita.24 In conclusion, this study strongly supports the idea that POCP may be a unique clinical and immunopathologic disease distinct from OCP⫹. But POCP may also simply represent an early stage of OCP⫹. The fact that ocular inflammation and fibrosis were equally severe in both groups of patients does not favor this hypothesis. However, longer follow-up of patients with POCP is needed, as extraocular signs may become manifest years after initial ocular involvement. Whether or not the distinction between POCP and OCP⫹ is of prognostic significance and/or implies more specific therapeutic strategies needs to be proved.
5. 6. 7. 8.
9. 10. 11.
12.
13. 14. 15. 16. 17. 18. 19.
20.
21.
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32. Rogers RS III, Sheridan PJ, Nightingale SH. Desquamative gingivitis: clinical, histopathologic, immunopathologic, and therapeutic observations. J Am Acad Dermatol 1982;7:729 – 35. 33. Hausser I, Fartasch M, Schleiermacher E, Anton–Lamprecht I. Disseminated cicatricial pemphigoid in a child and in an adult. Ultrastructural diagnostic criteria and differential diagnosis with special reference to acquired epidermolysis bullosa. Arch Dermatol Res 1987;279:357– 65. 34. Chan LS, Yancey KB, Hammerberg C, et al. Immunemediated subepithelial blistering diseases of mucous membranes. Pure ocular cicatricial pemphigoid is a unique clinical and immunopathological entity distinct from bullous pemphigoid and other subsets identified by antigenic specificity of autoantibodies. Arch Dermatol 1993; 129:448 –55. 35. Laskaris G, Angelopoulos A. Cicatricial pemphigoid: direct and indirect immunofluorescent studies. Oral Surg Oral Med Oral Pathol 1981;51:48 –54. 36. Franklin RM, Fitzmorris CT. Antibodies against conjunctival basement membrane zone. Occurrence in cicatricial pemphigoid. Arch Ophthalmol 1983;101:1611–3. 37. Fine JD, Neises GR, Katz SI. Immunofluorescence and immunoelectron microscopic studies in cicatricial pemphigoid. J Invest Dermatol 1984;82:39 – 43. 38. Sonnenberg A, Calafat J, Janssen H, et al. Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol 1991;113:907–17.
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