Immunoelectron microscopic study of the conjunctiva in cicatricial pemphigoid

· Background: Immune deposits can be found on the conjunctival basement membrane zone of patients affected by cicatricial pemphigoid using immunofluorescence technique. The purpose of this study was to perform direct immunoelectron microscopy on the conjunctiva of patients with scarring conjunctivitis associated with cicatricial pemphigoid · Methods: Conjunctival and skin biopsies were performed in six patients who presented with presumed autoimmune cicatrizing conjunctivitis associated with cicatricial pemphigoid. Specimens were processed for direct immunofluorescence and direct immunoelectron microscopy. · Results: Direct immunofluorescence was positive in all skin samples and in three of six conjunctival samples. Direct immunoelectron microscopy showed immune deposits in the lamina lucida and the lamina densa of all skin and conjunctival samples. · Conclusions: Direct immunoelectron microscopy can be performed on the conjunctiva. It shows the precise localization of cicatricial pemphigoid target antigens within the conjunctival basement membrane zone. Received: 29 April 1997 Revised version received: 16 February 1998 Accepted: 9 March 1998     

Conjunctival involvement in cicatricial and bullous pemphigoid: a clinical and immunopathological study.

Patients with bullous pemphigoid were found to have significant ocular abnormalities. In a group of 18 patients one had conjunctival shrinkage, and 11 of 15 (73%) had positive linear direct immunofluorescence on conjunctival biopsy from a clinically uninvolved site. Our ocular findings in a group of 14 with cicatricial pemphigoid are also reported and compared with those from a control group of 20. Our findings suggest there is overlap between the pemphigoid groups and raise further questions about the pathogenicity of immunoreactants within the basement membrane zone. Bulbar conjunctival biopsy was simple and well tolerated, and the rate of immunofluorescence positivity of conjunctiva was twice that of skin in both pemphigoid groups.     

The acute manifestations of ocular cicatricial pemphigoid: diagnosis and treatment

Seven patients with ocular cicatricial pemphigoid displayed acute inflammatory activity that could not be attributed to secondary bacterial infections, trichiasis, or lagophthalmos secondary to symblepharon. This acute inflammatory activity was manifested either as a localized conjunctival mound that was ulcerated and intensely hyperemic or as diffuse and intense conjunctival hyperemia and chemosis. Acute disease activity developed shortly after conjunctival biopsy in three patients and appeared spontaneously in the other four patients. Conjunctival biopsy specmens disclosed a heavy infiltrate of polymorphonuclear leucocytes within and beneath the conjunctival epithelium in addition to the chronic inflammatory cells typically found in this condition. The acute manifestations of ocular cicatricial pemphigoid cause rapid shrinkage and scarring of the conjunctiva. Systemic corticosteroids suppressed the acute disease activity and prevented additional scarring in all five patients treated.     

Scanning electron microscopy of conjunctival surfaces in patients with ocular cicatricial pemphigoid

The conjunctival surfaces of ten patients with active, ocular cicatricial pemphigoid, three patients with drug-controlled ocular cicatricial pemphigoid, and six patients with normal conjunctivas were studied using scanning electron microscopy. A homogeneous granular sheet of amorphous mucin-like material was observed covering extensive areas of the conjunctiva in eight of ten patients with active ocular cicatricial pemphigoid. This sheet of amorphous material was absent on drug-controlled ocular cicatricial pemphigoid and normal conjunctival specimens. Our study demonstrates that patients with active ocular cicatricial pemphigoid possess ocular surface mucus that appears thicker and more continuous than normal ocular mucus when observed with scanning electron microscopy. This observation is in agreement with clinical observations of thick mucus strands in the inferior fornix of patients with active ocular cicatricial pemphigoid.     

Eosinophil granule proteins expressed in ocular cicatricial pemphigoid

BACKGROUND—Blister formation and tissue damage in bullous pemphigoid have been attributed to the release of eosinophil granule proteins—namely, to eosinophil derived cationic protein (ECP) and major basic protein (MBP). In the present investigation these eosinophil granule proteins were studied in the conjunctiva of patients with ocular cicatricial pemphigoid (OCP).
METHODS—Conjunctival biopsy specimens obtained from patients with subacute (n=8) or chronic conjunctival disease (n=13) were analysed histologically and immunohistochemically using antibodies directed against EG1 (stored and secreted ECP), EG2 (secreted ECP), MBP, CD45 (common leucocyte antigen), CD3 (pan T cell marker), and HLA-DR (class II antigen).
RESULTS—Subepithelial mononuclear cells, mast cells, and neutrophils were detected in all specimens. The number of mononuclear cells, neutrophils, CD45+ cells, CD3+ cells, and the HLA-DR expression were significantly higher in the subacute than in the chronic disease group. Some eosinophils were found in specimens from five of eight patients with subacute OCP, but in none of the patients with chronic disease. The eosinophil granule proteins (ECP and MBP) were found in the epithelium and substantia propria in patients with subacute conjunctivitis.
CONCLUSIONS—Subepithelial cell infiltration in the conjunctiva greatly differs between subacute and chronic ocular cicatricial pemphigoid specimens. The findings suggest that eosinophil granule proteins may participate in tissue damage in acute phase of inflammation in OCP.

     

Ocular cicatricial pemphigoid.

Cicatricial pemphigoid is an inflammatory disease of presumed autoimmune etiology. It most commonly affects the conjunctiva and oral mucosa and less commonly the skin. The ocular manifestations of the disease include bilateral conjunctival shrinkage, xerosis, and corneal opacification. The progression of cicatricial pemphigoid is variable and can be interrupted by periods of remission or by periods of rapid exacerbation. Ocular surgery and topical pharmaceuticals may contribute to the exacerbation of this disease. Current treatment consists of systemic immunosuppressants and systemic corticosteroids.     

Exacerbation of undiagnosed ocular cicatricial pemphigoid after repair of involutional entropion

We report a case of exacerbation of undiagnosed ocular cicatricial pemphigoid after repair of involutional entropion. A lateral tarsal strip was performed to address entropion in the setting of eyelid laxity. No evidence of ocular cicatricial pemphigoid was observed before surgery. Postoperatively the patient developed intense conjunctival inflammation and diffuse symblepharon formation. Conjunctival biopsy demonstrated immunoglobulin and complement deposition at the basement membrane consistent with ocular cicatricial pemphigoid. Clinicians should be aware of the possibility of underlying ocular cicatricial pemphigoid in all patients with entropion, including those without a cicatricial component.     

Ocular cicatricial pemphigoid

Ocular cicatricial pemphigoid is a systemic disease of autoimmune etiology, belonging to the category of acquired oculo-muco-cutaneous bullous dermatosis. Although it is a disease primarily involving the conjunctiva, it frequently affects other mucous membranes (oropharynx, genitalia, anus), and the skin is involved as well in approximately 15% of the cases. The ocular manifestations, during the acute stage, consist of conjunctival bullous, which quickly lead to ulcerous lesions. During the chronic stage the disturbances of the palpebral statics and dynamics, ocular dryness syndrome and corneal lesions will develop. Being a relative less frequent disease, the observation of 3 patients is presented.     

Ocular cicatricial pemphigoid associated with hyperproliferation of the conjunctival epithelium

We determined the mitotic rate, measured by evaluating uptake of tritiated thymidine autoradiographically, and the frequency of goblet cells in conjunctival epithelial biopsy specimens from nine normal subjects and from 11 patients (seven women and four men ranging in age from 50 to 80 years) with ocular cicatricial pemphigoid. The mitotic rate of patients with the disease was significantly higher than that of normal subjects, 7.2 +/- 2.2 vs 1.6 +/- 0.2 labeled cells per 100 basal epithelial cells (P less than .01). The goblet cell frequency, however, was significantly less in patients than in normal subjects. This suggests that ocular cicatricial pemphigoid is associated with hyperproliferation of the conjunctival epithelium, with a concurrent failure of normal conjunctival differentiation.     

Mast cells in conjunctiva affected by cicatricial pemphigoid

Ocular cicatricial pemphigoid (OCP) is characterized by progressive conjunctival subepithelial fibrosis often leading ultimately to corneal blindness. Mast cells have been shown to play a role in several fibrotic disorders, but the role of mast cells in OCP is unknown. The authors compared the mast cell population in conjunctival biopsy specimens from 14 OCP patients and from six controls by using specific histochemical stains for mast cell subsets. The total mast cell number and the ratio of connective tissue mast cells to mucosal mast cells (MMCs) were significantly higher in OCP than in normal conjunctiva (P less than 0.05). This report is the first analysis of mast cell subsets in human ocular tissue. The results suggest that connective tissue mast cells (CTMCs) may play an important role in OCP and that therapy directed toward mast cells and their mediators may be an appropriate avenue for further exploration.     

Intravenous immunoglobulin therapy for ocular cicatricial pemphigoid: a preliminary study

OBJECTIVE: To report the effects of intravenous immunoglobulin treatment of ten patients with progressive ocular cicatricial pemphigoid who did not respond to conventional immunomodulatory regimens. DESIGN: Noncomparative, interventional case series. PARTICIPANTS: Ten patients with biopsy-proven progressive cicatricial pemphigoid affecting the eyes who did not respond adequately to other local and systemic immunosuppressive treatment regimens. INTERVENTION: Intravenous infusions of pooled human immunoglobulin, 2 to 3 g/kg body weight/cycle, divided over 3 days, and repeated every 2 to 6 weeks. MAIN OUTCOME MEASURES: Reduction in conjunctival inflammation, prevention of progression of subepithelial conjunctival fibrosis, improvement in ocular symptoms (discomfort, photophobia), improved visual acuity, reduction in extraocular mucosal lesions. RESULTS: Clinical deterioration was arrested and resolution of chronic conjunctivitis was documented in all ten patients. Maximum therapeutic effect was observed and maintained after a minimum of 4 cycles of therapy; three patients required 12 cycles before disease control. The duration of therapy in these ten patients has been 16 to 23 months (mean, 19.3 months) with no treatment-induced side effects. Extraocular mucosal lesion resolution has occurred in all but one patient, Visual acuity has stabilized or improved in all ten patients, and subjective complaints of discomfort and photophobia have decreased in all patients. CONCLUSIONS: Intravenous immunoglobulin immunomodulatory therapy can be a safe and effective therapy for otherwise treatment-resistant ocular cicatricial pemphigoid.     

Two cases of cicatricial pemphigoid showing atypical immunofluorescent findings.

We describe two patients with the clinical symptoms of cicatricial pemphigoid (CP). Biopsy specimens of the conjunctiva were taken. Histologic examination revealed subepidermal bullae and infiltration of inflammatory mononuclear cells. Direct immunofluorescent study showed immunoglobulins bound to the basement membrane zone (BMZ) in these patients. The patients also had intercellular immunoglobulin deposition in the conjunctival epithelium. No circulating anti-BMZ antibodies were detected, but one patient had a circulating antiintercellular antibody. Rare cases of CP with atypical immunofluorescent findings are reported.     

Role of enhanced expression of m-CSF in conjunctiva affected by cicatricial pemphigoid

PURPOSE: Local proliferation of macrophages has been reported to augment the inflammatory response in various human and experimental diseases. Macrophage accumulation in the submucosa is also an important feature in the pathogenesis of ocular cicatricial pemphigoid (OCP). In the present study, the role of local proliferation of macrophages in conjunctiva affected by OCP and the relationship between local proliferation of macrophages and expression of macrophage-colony-stimulating factor (m-CSF) in such conjunctiva were examined. METHODS: Biopsy specimens from the conjunctiva of 10 untreated patients with active OCP and from 5 normal subjects were studied for the expression of m-CSF, macrophages, and proliferating cell nuclear antigen (PCNA), a cell cycle protein, by immunohistochemistry. Dual staining for CD68 (a cell surface marker for macrophages) and PCNA was also performed to identify proliferating macrophages. In addition, fibroblasts isolated from conjunctiva of normal individuals and from patients with OCP were studied for the expression of m-CSF by immunostaining and real-time PCR. To identify the factors that induce m-CSF in conjunctival fibroblasts, the fibroblasts were incubated with different concentrations of interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha, and the levels of m-CSF mRNA were determined by real-time PCR and the amount of m-CSF produced was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Normal conjunctiva showed weak expression of m-CSF in the conjunctival epithelial cells and stroma. Conjunctival expression of m-CSF protein was significantly (P < 0.0001) increased in conjunctival biopsy specimens from patients with OCP. m-CSF was detected in the infiltrating macrophages, stromal cells (presumably fibroblasts), and conjunctival epithelial cells. Compared with normal control conjunctival tissue, a 1.2-fold increase in the expression of mRNA for m-CSF was detected by real-time PCR in the conjunctival tissue obtained from patients with OCP. Increased expression of m-CSF correlated significantly (P < 0.0004) with an increased stromal accumulation of macrophages in conjunctival biopsy specimens of patients with OCP. A number of these accumulated macrophages (CD68-positive) were found to be proliferating (PCNA-positive). In addition, fibroblasts isolated and cultured from conjunctiva of patients with OCP showed significantly increased (1.7-fold) expression of m-CSF compared with normal conjunctival fibroblasts. When conjunctival fibroblasts were treated with IL-1alpha or TNF-alpha, real-time PCR and ELISA detected an increased level of m-CSF. CONCLUSIONS: An increased expression of m-CSF was observed in conjunctiva from patients with active OCP. There was a positive correlation between expression of m-CSF and accumulation of macrophages in conjunctival biopsy sections obtained from patients with OCP. Increased expression of m-CSF, mainly by conjunctival fibroblasts and infiltrating inflammatory cells, may play an important role in the regulation of local proliferation of macrophages in OCP. In the conjunctiva of patients with OCP, this process could augment or enhance the local inflammatory response and tissue injury consequent to it.     

Ocular involvement in anti-epiligrin cicatricial pemphigoid

PURPOSE: To report an anti-epiligrin cicatricial pemphigoid (AECP) patient with severe ocular involvement and to provide a practical approach to distinguishing AECP patients from those with other subepidermal blistering diseases. METHODS: Techniques included direct and indirect immunofluorescence microscopy, Western blot and immunoprecipitation studies, as well as interdisciplinary examinations of mucous membranes and skin. RESULTS: This study describes a patient with clinical features of cicatricial pemphigoid, circulating anti-basement membrane zone IgG antibodies, and subepidermal blisters. Histopathology and immunofluorescence analysis suggested the diagnosis of a cicatricial pemphigoid-like type of epidermolysis bullosa acquisita. However, Western blot and immunoprecipitation studies demonstrated that the patient's serum contained autoantibodies against laminin 5 alpha3 subunit, leading to the diagnosis of an AECP. CONCLUSION: Since patients with AECP have an increased relative risk for malignant tumors, it is important to distinguish this entity within the spectrum of cicatricial pemphigoid patients by additional studies such as Western blot or immunoprecipitation.     

Immunopathology of cicatricial pemphigoid affecting the conjunctiva

Conjunctival biopsy specimens from 13 patients with cicatricial pemphigoid and from 13 age-matched healthy individuals undergoing cataract surgery were analyzed by light microscopy and immunohistochemical techniques, including a panel of monoclonal antibodies used to characterize inflammatory mononuclear cell phenotypes. Results of histologic examination of cicatricial pemphigoid specimens showed typical squamous metaplasia, vasculopathy, increased numbers of mast cells, and abundant plasma cells. All cicatricial pemphigoid specimens demonstrated immunoreactants at the epithelial basement membrane zone (BMZ). Epithelium of cicatricial pemphigoid conjunctiva showed significantly more T-helper cells (CD4+), dendritic cells (CD1+), and macrophages (CD14+), and a significantly higher helper/suppressor ratio than did controls. In the substantia propria, pemphigoid specimens showed dramatically increased inflammatory infiltrate with significantly more cells staining, in order of frequency, for T cells (CD3+, CD5+), T-helper cells (CD4+), T-suppressor cells (CD8+), macrophages (CD14+, Mac-1+), and dendritic cells (CD1+, HLA-DR+). Ten percent of these cells expressed interleukin-2 receptor protein (CD25+), indicating T-cell activation.     

Detection of ocular mucus in normal human conjunctiva and conjunctiva from patients with cicatricial pemphigoid using lectin probes and histochemical techniques

Conjunctival biopsies from patients with cicatricial pemphigoid affecting the conjunctiva and patients undergoing cataract surgery (normal conjunctiva) were snap-frozen, cryostat sectioned and incubated with fluorescein-conjugated lectins; peanut agglutinin (PNA), Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and succinylated wheat germ agglutinin (S-WGA). Controls consisted of preincubating the lectins with the appropriate blocking sugars before applying the lectins to the sections. PNA and HPA stained the mucus granules contained in the conjunctival goblet cells but did not stain mucus or glycocalyx at the ocular surface distal to the goblet cells. Native WGA and S-WGA had high affinities for conjunctival goblet cells and the apical epithelial cell layers. Native WGA stained mucus and glycocalyx at the ocular surface. This staining of the ocular surface by WGA was confirmed at the transmission electron microscopic level using WGA conjugated to ferritin. Cicatricial pemphigoid patients in this study had reduced numbers of goblet cells; however, those goblet cells which were observed in cicatricial pemphigoid conjunctiva stained positively with HPA, PNA, WGA, and SWGA as did goblet cells in normal conjunctiva.     

Subconjunctival mitomycin C for the treatment of ocular cicatricial pemphigoid

PurposeThe authors performed a prospective evaluation of the efficacy of treating ocular cicatricial pemphigoid (OCP) with subconjunctival mitomycin C.DesignUnmasked, prospective, internally controlled case series.MethodsPatients were eligible for treatment with subconjunctival mitomycin C under three criteria: (1) significant complications of systemic immunosuppressant therapy; (2) markedly asymmetric conjunctival disease; and (3) end-stage OCP. All patients received monocular subconjunctival injections of 0.25 ml of 0.2 mg/ml mitomycin C to both the superior and inferior bulbar conjunctivae in the eye with the more severe disease.ResultsNine eyes of nine patients (mean age, 74 years) were treated with subconjunctival mitomycin C to the more-involved eye and were followed for a mean of 23.5 months (range, 12–40 months). Eight of nine patients showed quiescence of their OCP in the treated eye based on serial evaluation of conjunctival cicatrization and grading of conjunctival erythema. Five of the nine untreated eyes showed progression of the conjunctival disease. One patient required concomitant systemic immunosuppressive therapy after subconjunctival mitomycin C. Two patients underwent successful visual rehabilitative surgery in the mitomycin C-treated eye.ConclusionThe use of subconjunctival mitomycin C may be effective in preventing progression of conjunctival cicatrization and erythema in patients with OCP. No complications of mitomycin C treatment were noted. Long-term follow-up and further investigation into the efficacy of subconjunctival mitomycin C in the management of OCP is warranted.     

Cataract surgery in ocular cicatricial pemphigoid

The authors report the results of their experience with cataract surgery in 20 patients (26 eyes) with biopsy-proven cicatricial pemphigoid. All patients were on systemic immunosuppression at the time of surgery (dapsone, azathioprine, cyclophosphamide, or combinations) and were treated with perioperative oral corticosteroids. Patients were evaluated pre- and postoperatively for conjunctival inflammation, conjunctival cicatrization, degree of keratopathy, and disease stage. No patient progressed in disease stage. Vision improved an average of 3.5 Snellen lines (-3 to +8). Worse outcome was associated with chemotherapy intolerance or the presence of any preoperative conjunctival inflammation. Thirteen patients remained on immunosuppressives for the entire study. Corneal ulcers developed postoperatively in three patients in whom continued immunosuppression was not tolerated. Possible mechanisms for inflammatory exacerbation after surgery are discussed. Results indicate that after successful abolition of all conjunctival inflammation through chemotherapy, cataract surgery may be safely performed in patients with cicatricial pemphigoid.     

Corneal ulcer as an atypical presentation of ocular cicatricial pemphigoid

PURPOSE: To describe three patients, each presenting noninfective corneal epithelial damage as first manifestation of ocular cicatricial pemphigoid (OCP). METHODS: Case report. RESULTS: Patients 1 and 2 were referred to the authors' clinic for corneal ulcer while Patient 3 for relapsed epithelial defects. All patients had negative history for systemic diseases and microbiological tests were negative. Topical steroid treatment induced the complete resolution of corneal damage. During the follow-up period, the onset of mild conjunctival fibrosis in the lower fornix allowed the authors to suspect OCP, confirmed by conjunctival biopsy. CONCLUSIONS: In the three patients corneal damage was an early sign of OCP, in the absence of typical signs of conjunctival fibrosis. The authors thus suggest considering conjunctival biopsy as a useful additional test in the management of idiopathic corneal ulcers.     

Diagnostics and pharmacological treatment of ocular cicatrical pemphigoid

Ocular cicatricial pemphigoid (OCP) is an autoimmune disease characterize by mucous membrane fibrosis and skin changes resulting with scarring. The pathogenic mechanisms of ocular cicatricial pemphigoid are incompletely understood. Antibasement membrane antibodies which lead to subepithelial blistering, granulation tissue and inflammatory infiltrate formation in the substantia propria are thought to be the main pathophysiological mechanisms in cicatricial pemphigoid. It has been found eosinophils and increased collagen type I and III. Human leukocyte antigen HLA-DR2, HLA-DR4 and DQw7 genotypes have been identified as conferring increased susceptibility to the development of this disease. Ocular cicatrical pemphigoid (OCP) is one of the forms of bullous pemphigoid. Initial symptoms of ocular pemfigoid are not characteristic. Conjunctival fibrosis may cause severe entropion, trichiasis, symblepharon, dry eye syndrome, corneal epithelial erosions or ulceration. Secondary glaucoma is one of the most frequent complications. Ocular cicatricial pemphigoid may be chronic, acute, or subacute disease with periodic exacerbation of conjunctival inflammation. The treatment in this disease are topical drops or ointment (lubricants, corticosteroids, antibiotics, antiglaucomatous). Oral dapsone and corticosteroids may control the activity of the disease. In other progressive cases immunosuppressive drugs must be used (azathioprine, cyclophosphamide, methotrexate, mycophenolan mofetil, daclizumab, intravenous immunoglobulin therapy). To make an early diagnosis of ocular cicatricial pemphigoid, biopsy and immunohistochemical analysis of conjunctiva should be performed in every case of persistent conjunctival inflammation.