Immunophenotypic analysis of the inflammatory infiltrate in ocular cicatricial pemphigoid. Further evidence for a T cell-mediated disease

Ocular cicatricial pemphigoid (OCP) is characterized by the deposition of immunoglobulin and complement along the conjunctival epithelial basement membrane zone (BMZ). In order to further elucidate the cellular populations of the local inflammatory infiltrates, the authors used a panel of monoclonal antibodies in cryostat tissue sections to delineate T cell subsets, B lymphocytes, dendritic cells, and macrophages in six patients with OCP. In comparison with matched controls of the epibulbar conjunctiva, the authors discovered a threefold increase in T lymphocytes within the epithelium and a 20-fold increase within the substantia propria. In contrast with the normal-standing population of conjunctival T lymphocytes, there were activated interleukin 2 receptor (IL-2R)-positive lymphocytes in both the epithelium and the substantia propria. Macrophages were the second most common cells in the substantia propria, accounting for 12.7% of the mononuclear population--a threefold increase over the normal percentage. B cells and plasma cells, normally absent from epibulbar conjunctiva, were the next most prominent populations, constituting 6.9 and 4.6%, respectively, of all mononuclear cells. Dendritic cells which process antigen locally constituted only 1.2% of the mononuclear cell population, but were increased 25-fold over normal controls. By elaborating cytokines that promote fibroplasia, the T cells in OCP may be effector cells along with macrophages and other inflammatory cells in bringing about scarification of the substantia propria, and may furthermore be responsible for an immunoregulatory defect that allows local B lymphocytes to produce autoantibodies to the BMZ.     

Antibasement membrane antibody-mediated experimental conjunctivitis

In ocular cicatricial pemphigoid, the binding of circulating antibodies to conjunctiva is believed to initiate an antibody-mediated cytotoxic response that results in inflammation and tissue damage. To develop a model of antibody-mediated conjunctival inflammation, we examined the effect on conjunctiva of local or systemic administration of a murine monoclonal antibody against basement membrane of stratified squamous epithelium. Neonatal rabbits were given either a single subconjunctival or intraperitoneal injection of the antibody. Eyes were graded clinically for inflammation and conjunctival biopsies were performed. After subconjunctival injection, clinical and histologic inflammation as well as murine antibody and rabbit complement binding to conjunctival basement membrane were detected. With systemic administration there was post-injection clinical inflammation, and conjunctival basement membrane-bound murine antibody was detected. There was no difference observed in conjunctival mitotic rate or goblet cell frequency between treatment groups and controls, following either route of administration. We have created, therefore, a model for antibody-mediated conjunctivitis in rabbits by local or systemic administration of a monoclonal antibody against a component of stratified squamous epithelial basement membrane.     

Lichen planus and cicatrizing conjunctivitis: characterization of five cases

PURPOSE: To report the clinical and immunopathologic features and the response to therapy in a series of six patients with cicatrizing conjunctivitis due to lichen planus. DESIGN: Retrospective case series. METHODS: All six patients were seen in an ocular pemphigoid clinic. Clinical, immunopathologic, and serologic features were evaluated and therapeutic response in each patient was monitored. RESULTS: All six patients had evidence of conjunctival scarring. Five patients had lichen planus of the oral mucosa and gingiva; one patient had involvement of the skin. Histologic findings consisted of thickened epithelium and an interface lymphocytic infiltrate along the lamina propria. In three patients, electron microscopy of the conjunctiva revealed thickening, fragmentation, and duplication of the basement membrane zone. Direct immunofluorescence examination of the conjunctiva and oral mucosa demonstrated linear and shaggy fibrinogen deposition along the basement membrane zone, confirming the diagnosis of lichen planus. All six patients were placed on immunosuppressive therapy with control of the disease. However, only one patient was able to discontinue the anti-inflammatory medication and have the lichen planus remain in remission. CONCLUSIONS: Lichen planus should be included in the differential diagnosis of cicatrizing conjunctivitis. Performing appropriate investigations to distinguish conjunctival lichen planus from other autoimmune diseases such as mucous membrane pemphigoid is critical to managing the patient with cicatrizing conjunctivitis appropriately. Oral cyclosporine effectively controlled the conjunctival lichen planus in four of the six cases.     

Amniotic membrane transplantation elicits goblet cell repopulation after conjunctival reconstruction in a case of severe ocular cicatricial pemphigoid

PURPOSE: The surgical procedures used until now to rebuild the conjunctiva in patients affected by ocular cicatricial pemphigoid (OCP) have not demonstrated appreciable anatomical and functional success. This study reports the postoperative clinical and cytological outcomes of a human amniotic membrane transplantation (AMT) to rebuild the conjunctiva in a case of late stage OCP. METHODS: We present a 75-year-old woman with a severe form of pemphigoid with entropion, trichiasis, symblepharon and blunting of the fornices, who underwent excision of the scarred and inflamed tissue covering the ocular surface and AMT. RESULTS: Our data show an improvement of the ocular surface condition, with reacquired fornix depth, reduced inflammation and presence of goblet cells at each follow-up. CONCLUSION: Amniotic membrane transplantation was successful as a first step procedure to reduce inflammation and to rebuild a physiological conjunctival epithelium in late stage OCP.     

Ocular cicatricial pemphigoid

CP is a systemic disease of presumed autoimmune etiology that may involve the skin, conjunctiva, or any mucosal surface. As many patients present with ocular symptoms, ophthalmologists must inquire about signs of potential life-threatening esophageal and laryngeal involvement. Ocular involvement is a bilateral chronic cicatricial conjunctivitis that may result in fornix foreshortening, symblepharon formation, trichiasis, and entropion and keratinization of the eyelids and conjunctiva. These mechanical factors are responsible for progressive scarring and vascularization of the ocular surface. Successful systemic treatment of the autoimmune disease is now available; however, the condition often is not diagnosed until significant structural damage to the ocular adnexa has occurred. Our ability to rehabilitate visually patients with advanced disease by penetrating keratoplasty and associated techniques remains extremely limited. Hence, the focus of treatment in OCP should be on early detection of the disease, including conjunctival biopsy, prompt systemic treatment to prevent disease progression, and early aggressive treatment of secondary eyelid and conjunctival abnormalities to avoid the blinding keratopathy so prevalent in this disease.     

Preliminary serological studies comparing immunofluorescence assay with radioimmunoassay

We assayed serum from 12 patients with untreated cicatricial pemphigoid affecting the conjunctiva for circulating autoantibodies directed against the epithelial basement membrane zone. We employed a conventional indirect immunofluorescence assay, with monkey esophagus and human conjunctiva as substrates, and compared the results with those obtained employing a radioimmunoassay measuring antibasement membrane zone antibody binding to COLO-16 and to SCaBER tumor cell lines. The indirect immunofluorescence assay on normal human conjunctival substrate detected circulating antibodies to conjunctival epithelium in 6 of 12 CP patient serum specimens. Monkey esophagus failed to detect antibodies to the epithelial basement membrane zone. In contrast, autoantibodies were detected in all 12 specimens by the radioimmunoassay. Specificity, as demonstrated by appropriate controls and assay of normal human serum, was 100%. These results demonstrate that radioimmunoassay employing COLO-16 or SCaBER cells is an exquisitely sensitive and specific assay for detection of circulating antibasement membrane antibodies in patients with cicatricial pemphigoid affecting the conjunctiva.     

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.     

Identification of ocular cicatricial pemphigoid antibody binding site(s) in human beta4 integrin

PURPOSE: To identify specific site(s) on human ss4 molecule to which sera from ocular cicatricial pemphigoid (OCP) patients bind and to determine its role in the process of blister formation. METHODS: Clone the fragments representing the extracellular and intracellular domain of ss4 molecule from normal human conjunctival mRNA into an expression vector; map the region to which sera from OCP patients bind by Western blot analysis. Determine the role of the immunodominant region in pathogenesis by demonstrating the ability of the rabbit antibody to the immunodominant region to produce separation of basement membrane zone (BMZ) from the basal epithelial layer when incubated with normal human conjunctiva in an in vitro organ culture model. RESULTS: Majority of the OCP sera tested bound to the C-terminal end of the intracellular domain (IC3.0) of the human ss4 integrin. Further subcloning of IC3.0 demonstrated that a smaller fragment extending from 1489 aa to 1572 aa (IC3.4) was responsible for this binding. This region may have multiple antibody binding sites. Antibody to human IC3.0 and IC3.4 produced in rabbit, resulted in BMZ separation, histologically identical with that observed when normal human conjunctiva was cultured with OCP sera in an human conjunctival organ culture model. CONCLUSIONS: These observations identify IC3.4 as the antibody binding site for sera of OCP patients and suggest a possible role for it in blister formation. Indirectly it highlights certain important aspects of the structural and functional dynamics of the biology of the hemidesmosomes and basement membranes.     

Conjunctival amyloidosis: report of six cases and review of the literature

Conjunctival amyloidosis is an uncommon condition that occasionally is associated with systemic involvement. The clinical presentations of conjunctival amyloidosis are diverse. We present six patients with conjunctival amyloidosis who were referred to us with the suspicion of another conjunctival lesion. The conjunctival lesion was circumscribed in two patients and diffuse in four patients. The lesion color was yellow in one patient and yellow-pink in five patients. The associated features were intrinsic vascularization (six patients), recurrent subconjunctival hemorrhage (four patients) and blepharoptosis that involved the palpebral conjunctiva (two patients). Systemic evaluation revealed primary systemic amyloidosis in one patient and no related systemic abnormalities in five patients. Management consisted of complete excisional biopsy for the two circumscribed lesions and incisional biopsy for the four diffuse lesions. Two patients with diffuse involvement showed progressive involvement of the conjunctiva over 3 years following incisional biopsy and the other four patients remained stable. Additionally, there was no systemic involvement in five patients. In conclusion, conjunctival amyloidosis generally manifests as a yellowish-pink, hemorrhagic mass deep to the epithelium. Most patients show no evidence of systemic amyloidosis.     

Role of collagen-binding heat shock protein 47 and transforming growth factor-beta1 in conjunctival scarring in ocular cicatricial pemphigoid

PURPOSE: Submucosal fibrosis due to excessive accumulation of collagens is an important histologic feature in the pathogenesis of ocular cicatricial pemphigoid (OCP). Heat shock protein 47 (HSP47), a collagen-binding protein, plays an important role in the biosynthesis of procollagens. In the present study, we examined the role of HSP47 in conjunctival scarring in patients with OCP. METHODS: Biopsy specimens of the conjunctiva of 15 patients with OCP and 5 normal subjects were studied for the expression of HSP47, transforming growth factor (TGF)-beta1, type I collagen, and type III collagen. The role of TGF-beta1 on the induction of HSP47 and type I collagen by conjunctival fibroblasts was studied by immunostaining, Western blot analysis, and quantitative real-time PCR. RESULTS: Compared with the control, increased accumulations of type I and type III collagens were detected by immunohistochemistry in fibrotic conjunctiva of patients with OCP. Weak and sparse expression of HSP47 was detected in the epithelial cells and stromal fibroblasts in control conjunctival tissues. In contrast to the control, the expression of HSP47 was markedly increased in the stromal fibroblasts in conjunctival tissues obtained from patients with OCP, as detected by immunohistochemistry. By quantitative real-time PCR, compared with control conjunctival tissues, a 3.4-fold increase in the expression of HSP47 was noted in the conjunctival tissues obtained from patients with OCP. Similar to conjunctival tissues, fibroblasts isolated from conjunctiva of patients with OCP exhibited 4.8-fold increase in the expression of HSP47, compared with control fibroblasts. When conjunctival fibroblasts were treated with various concentration of TGF-beta1, upregulation in the expression of HSP47 and type I collagen was detected. CONCLUSIONS: This study demonstrated increased expression of HSP47 and TGF-beta1 by conjunctival fibroblasts in biopsy specimens obtained from patients with OCP. TGF-beta1 induced the expression of HSP47 and type I collagen by conjunctival fibroblasts. Increased levels of TGF-beta1 and HSP47 may regulate increased synthesis, assembly, and production of collagens and thereby could significantly contribute to the process of conjunctival scarring in patients with OCP.     

Transplantation of amniotic membrane for patients with bullous keratopathy and chemical and thermal burns

The aim of this paper is, to evaluate the efficacy of amniotic membrane transplantation for ocular surface reconstruction in patients with bullous keratopathy and chemical and thermal burns of cornea and conjunctiva. Amniotic membrane is a thin, semitransparent tissue forming an innermost layer of the fetal membrane, which contains a thick basement membrane with a single layer of epithelium and avascular matrix. This transplantation promotes normal conjunctival epithelization while suppressing fibrosis formation. Amniotic membrane transplant may be considered as an alternative method for treating ocular surface reconstruction in patients with thermal and chemical burns. Authors suggest that this method of treatment is not efficient in patients with bullous keratopathy.     

Inferior retractor plication surgery for lower lid entropion with trichiasis in ocular cicatricial pemphigoid.

AIMS--To assess the outcome of inferior retractor plication surgery for lower lid entropion in patients with ocular cicatricial pemphigoid (OCP). This technique avoids surgery on the conjunctiva that can result in exacerbations of disease activity. METHODS--This prospective study assessed the outcomes of a standard 'Jones' type plication in 14 lids of 10 patients with OCP. Seven patients were taking systemic immunosuppression and no patients had conjunctival inflammation for the 4 months before surgery. RESULTS--Life table analysis showed a 77% chance of anatomical success at 2 years and a 54% chance of completely preventing lash-globe touch. The surgery did not cause clinical activation of conjunctival inflammation or other complications. Anatomical failure was primary (n = 2) and due to late cicatrisation (n = 1). Three further cases had restoration of normal anatomy but the patients had persistently misdirected lashes that touched the globe. CONCLUSION--This technique gives good anatomical success over long periods and is particularly safe when there is no conjunctival inflammation present before surgery.     

Role of amniotic membrane transplantation for conjunctival reconstruction in ocular-cicatricial pemphigoid

PURPOSE: To evaluate the role and the effectiveness over time of amniotic membrane transplantation (AMT) as a first-step procedure to treat conjunctival reconstruction in late-stage ocular-cicatricial pemphigoid (OCP). DESIGN: Prospective interventional noncomparative case series. PARTICIPANTS: Nine eyes (9 patients) with advanced OCP. METHODS: Preoperatively, the ocular surface conditions were evaluated by immunohistochemistry of conjunctival biopsy and impression cytology specimens. The amniotic membrane was obtained during cesarean section from women who were 39 weeks pregnant and seronegative for human immunodeficiency virus, hepatitis B and C, and syphilis; it was processed, histologically tested, and stored at -80 degrees C. After scar tissue was removed, the preserved amniotic membrane was placed over the cornea, the bulbar, and tarsal conjunctiva, and was secured with 8-0 Vicryl sutures to the conjunctival edges and the deep fornices with double-armed 6-0 silk sutures. In 2 cases a double layer of amniotic membrane was transplanted. All patients received immunosuppressive systemic therapy and preservative-free tear substitutes and steroids topically for at least 6 months. During follow-up (average, 48 weeks; range, 28-96 weeks), a new standardized method was used to evaluate the fornix depth, and impression cytology testing was performed and conjunctival inflammation recorded and used as parameters for monitoring disease activity. MAIN OUTCOME MEASURES: Symblepharon, increased inferior fornix depth, presence of conjunctival goblet cells, and the degree of conjunctival inflammation. RESULTS: The conjunctival surface was free from symblepharon in all subjects for the first 16 weeks. At the week 28 examination, a small area of symblepharon was present in four eyes (44.4%). The depth of the fornix was significantly (P < 0.0001, analysis of variance) improved at weeks 4, 16, and 28. The normal conjunctival epithelium with goblet cells was restored in 6 of 9 eyes (66.7%) at the week 4 examination and in 4 eyes (44.4%) at the week 28 examination. Conjunctival inflammation was clinically but not statistically reduced. The visual acuity improved in 5 subjects. CONCLUSIONS: AMT can be a first-step procedure for ocular surface reconstruction in OCP, but its effectiveness deteriorates slightly over time.     

α/β- andγ/δ-T cell-receptor-positive lymphocytes in healthy and inflamed human conjunctiva

• Background: The possible existence and distribution patterns of α/β- and γ/δ-TCR⁺ cells, which are important constituents of immune surveillance and act via the CD3⁺ cell complex have not yet been elucidated in the healthy and inflamed conjunctiva. • Materials and methods: Paraffin-embedded conjunctival specimens included 18 from 18 patients with ocular cicatricial pemphigoid (OCP), 20 from 20 healthy controls, 6 from 6 patients with lye burns, and 6 from 2 patients with Stevens-Johnson syndrome; all were worked up by histology and immunohistochemistry. • Results: α/β-TCR⁺ cells were visualized in the conjunctival epithelium and stroma of healthy persons, OCP, lye burns and Stevens-Johnson syndrome. α/β-TCR⁺ cells and a small number of γ/δ-TCR⁺ cells were observed in the corneal epithelium and stroma of patients who have failing corneal grafts. After ileal mucosa transplantation to the epibulbar conjunctiva, membrane staining changes to nuclear and cytoplasmic staining. Treatment with systemic cytotoxic drugs abolishes all α/β-TCR⁺ and γ/δ-TCR⁺ cells. • Conclusions: α/β-TCRu+ cells can be found in the non-infected epithelium and stroma of the healthy and inflamed (OCP, lye burns, and Stevens-Johnson syndrome) conjunctiva, as well as in the corneal epithelium and stroma of failing corneal grafts, whereas γ/δ-TCR⁺ cells are absent. A small number of γ/δ-TCR⁺ cells are present in the corneal stroma and adjacent conjunctival epithelium of patients with chronic corneal graft rejection or after transplantation of gut tissue. Further investigations may establish the role, if any, of these T-cell subsets in immune surveillance of the non-infected outer eye and in corneal graft rejection.     

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.     

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.     

Role of connective tissue growth factor in the pathogenesis of conjunctival scarring in ocular cicatricial pemphigoid

PURPOSE: Conjunctival fibrosis due to excessive accumulation of collagens is an important histologic feature in ocular cicatricial pemphigoid (OCP). Studies have suggested a role of transforming growth factor (TGF)-beta1 in conjunctival fibrosis in patients with OCP. Connective tissue growth factor (CTGF) is an important downstream mediator of TGF-beta1-induced collagen synthesis. CTGF usually acts synergistically with TGF-beta1 during the process of fibrosis in various organs. Hence, studying the mechanism by which CTGF influences TGF-beta1-induced synthesis of collagen in conjunctiva of patients with OCP would provide insight into the mechanism of conjunctival fibrosis in patients with OCP. METHODS: Biopsy specimens from conjunctiva of 10 patients with OCP and 5 normal subjects, were studied, with immunohistochemistry and real-time PCR, for the expression of CTGF and interstitial type I collagen. Using fibroblasts cultured from conjunctival biopsies we determined the effects of TGF-beta1 on the induction of CTGF and type I collagen by immunostaining, and quantitative real-time PCR. The effects of blocking the bioactivity of TGF-beta1 on the expression of CTGF and type I collagen were determined in TGF-beta1-stimulated fibroblasts, before and after treatment with type II receptor neutralizing antibody. RESULTS: An increased stromal accumulation of interstitial type I collagen with an increased expression of CTGF was observed in biopsy sections of patients with OCP, compared with the control. By quantitative real-time PCR, a 3.2-fold increase in the expression of CTGF was detected in conjunctival tissues obtained from patients with OCP, compared with control conjunctiva. Fibroblasts isolated from conjunctiva of patients with OCP expressed 4.4-fold more CTGF, compared with control conjunctival fibroblasts, by real-time PCR. When these cultured fibroblasts were immunostained, an increased expression of CTGF was detected in fibroblasts isolated from patients with OCP, compared with control. Furthermore, when conjunctival fibroblasts were treated with TGF-beta1, an approximately ninefold increase in the expression of CTGF and an approximately threefold increase in the expression of type I collagen were detected by real-time PCR, compared with unstimulated fibroblasts. Finally, when antibody to TGF-beta type II receptor was added before TGF-beta1 treatment of these fibroblasts, the expression of type I collagen and CTGF was significantly reduced. CONCLUSIONS: In the present study, an increased expression of CTGF was recorded in conjunctiva of patients with OCP. TGF-beta1 can induce production of CTGF and type I collagen by fibroblasts obtained from conjunctiva in OCP. This induction of CTGF by TGF-beta1 can be blocked by antibody to TGF-beta type II receptors. The findings lead to the conclusion that CTGF is one of the molecules involved in the pathogenesis of conjunctival fibrosis in patients with OCP.     

External ocular findings in lupus erythematosus: a clinical and immunopathological study.

A selected group of 18 patients with systemic lupus erythematosus (SLE) and 30 patients with chronic cutaneous lupus erythematosus (CCLE) showed an unexpectedly high incidence of problems involving the globe or eyelids. Five SLE patients had recurrent episcleritis, two CCLE patients had lower tarsal plaques, and two further CCLE patients had erosion of the lower lid margins associated with conjunctival scarring and symblepharon. This association has not previously been reported. There was an unexpectedly high incidence of deposition of immunoreactants in a linear pattern at the basement membrane zone in normal bulbar conjunctiva, which occurred in both SLE (42%) and CCLE (50%). The significance of these findings is discussed. We believe surface ocular problems in lupus erythematosus to be under-reported and that direct immunofluorescence of bulbar conjunctival biopsy might be helpful in diagnosis.     

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.     

Role of macrophage migration inhibitory factor in conjunctival pathology in ocular cicatricial pemphigoid

PURPOSE: Macrophage migration inhibitory factor (MIF) is a pleiotropic, proinflammatory cytokine that mediates various immunoinflammatory processes. Ocular cicatricial pemphigoid (OCP) is an autoimmune disease in which affected conjunctivae show features of an immunoinflammatory disease. In this study, the role of MIF in the pathogenesis of OCP was examined. METHODS: The expression of MIF in conjunctival tissues of patients with OCP (n = 10) and normal subjects (n = 5) was studied by quantitative real-time PCR and immunohistochemistry. The production of MIF by conjunctival fibroblasts of normal control subjects and patients with OCP was determined, by using quantitative real-time PCR and enzyme-linked immunosorbent assay (ELISA). In addition, the effects of interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and transforming growth factor (TGF)-beta1 on the induction of MIF by conjunctival fibroblasts were studied by quantitative real-time PCR. To determine the relationship between conjunctival expression of MIF and accumulation of macrophages, in patients with OCP, a correlation study was performed. RESULTS: An increased conjunctival expression of MIF was detected in patients with OCP, both at the mRNA (by real-time PCR) and protein level (by immunohistochemistry), compared with normal control patients. The expression of MIF was detected in the epithelial cells and occasionally in the stromal cells in control conjunctival tissues, by immunohistochemistry. In contrast, a statistically significant increase (P < 0.0001) in the expression of MIF was detected in the stromal cells of conjunctival tissues obtained from patients with OCP (control: 4.89 +/- 0.5; OCP: 19.82 +/- 1.34). By quantitative real-time PCR, compared with control conjunctiva, an increase in the expression of MIF was detected in the conjunctiva obtained from patients with OCP. A similar increase in the expression of MIF was also detected in conjunctival fibroblasts of patients with OCP, compared with control fibroblasts, by quantitative real-time PCR. A significantly increased (P < 0.001) level of MIF was also detected in supernatant collected from conjunctival fibroblasts of patients with OCP (186 +/- 5.4), compared with supernatant collected from control fibroblasts (9.3 +/- 7.6). Moreover, IL-1, TNF-alpha, and TGF-beta1, known factors involved in the pathogenesis of OCP, were found to induce the expression of MIF by conjunctival fibroblasts. A statistically significant correlation (P < 0.0001, r(2) = 0.4465) was observed between the expression of MIF and accumulation of CD68-positive macrophages in conjunctiva of patients with OCP. CONCLUSIONS: This study demonstrated an increased conjunctival expression of MIF in patients with OCP. MIF may be actively involved in the pathogenesis of OCP, possibly regulating the inflammatory events of the disease process.