The T cell receptor in normal and inflamed human conjunctiva.

The majority of human peripheral blood and lymphoid tissue T cells express the TCR alpha/beta heterodimer, while the TCR gamma/delta is expressed on only a small subset of T lymphocytes. However, the majority of murine intraepithelial lymphocytes and most Thy-1+ murine dendritic epidermal cells express the TCR gamma/delta. Selective homing of avian TCR gamma/delta bearing lymphocytes to the intestinal epithelium also has been shown. These findings have suggested that these cells play a role against transformation and infection. More recently, a role in autoimmunity also has been proposed. We examined normal human conjunctiva and inflamed conjunctiva from patients with ocular cicatricial pemphigoid (OCP), an autoimmune disorder, and atopic keratoconjunctivitis (AKC). The majority of T cells in the epithelium and substantia propria of normal conjunctiva expressed the TCR alpha/beta. Tropism of TCR gamma/delta-expressing lymphocytes to normal human conjunctiva was not present. However, in OCP, there was a statistically significant increase in the absolute number of TCR gamma/delta cells/mm2 (epithelium, 33.9 +/- 10.5 [mean +/- standard error of the mean] vs. 159.9 +/- 51.5, P = less than 0.0008; substantia propria, 4.1 +/- 0.9 vs. 240.1 +/- 191.3, P less than 0.002) and TCR gamma/delta cells as a percentage of CD3+ cells (epithelium, 0.18 +/- 0.06 vs. 0.39 +/- 0.07, P = less than 0.02; substantia propria, 0.10 +/- 0.05 vs 0.33 +/- 0.08, P = less than 0.03). This was not the case for AKC. These findings suggest that TCR gamma/delta lymphocytes play a specific but undefined role in certain conjunctival inflammatory conditions and may be important in autoimmunity.     

Ocular rosacea. A histologic and immunopathologic study

Acne rosacea is an idiopathic dermatologic disease that frequently produces conjunctival inflammation. The authors studied the histology and immunopathology of epibulbar conjunctival biopsy specimens from eight patients with ocular rosacea and compared the findings with those from conjunctiva from 13 normal individuals. The conjunctival epithelium in ocular rosacea was attenuated and infiltrated by inflammatory cells, mainly T-helper/inducer (CD4) cells, phagocytic cells, and antigen-presenting (CD14, Mac-1) cells. The difference between the normal control group and the rosacea group in the number of mononuclear cells forming these populations was statistically significant (P less than 0.01). The substantia propria of the rosacea specimens contained large subepithelial infiltrates of chronic inflammatory cells, and in some cases frank granuloma formation was evident. There was an overall mean increase of nearly all cell types, but especially of T-helper cells in the rosacea specimens compared with the controls. Interestingly, T-helper/inducer (CD4) cells, which were outnumbered by the T-suppressor (CD8) cells in the normal conjunctival epithelium (CD4/CD8 = 0.85), outnumbered the CD8-positive cells in the rosacea specimens (CD4/CD8 = 1.6). There also was a 3.5-fold increase of the CD4/CD8 ratio in the rosacea conjunctival stroma compared with the normal specimens. The mechanism involved in rosacea conjunctival inflammation resembles a type IV hypersensitivity reaction.     

Characterisation of the Normal Conjunctival Leukocyte Population

Lymphoid cells are present in normal mucosal tissue as scattered cells or organised into MALT. Knowledge of the non-pathological conjunctival lymphoid tissue is a vital basis for the further study of ocular surface inflammatory disorders. Therefore, we used immunohistochemistry to examine the leukocyte population of tarsal and bulbar conjunctiva from normal patients with no ocular or systemic inflammatory disease.CD3⁺T cells were the most frequently occurring, and macrophages the second most frequently occurring, conjunctival cell type and were seen in the epithelium and substantia propria. There were greater numbers of leukocytes in the bulbar than in the tarsal area and this reached statistical significance for CD3⁺T cells and CD57⁺NK cells. In both bulbar and tarsal conjunctiva, B cells and neutrophils were seen in the epithelium and substantia propria, but plasma cells, NK cells and mast cells were present only in the substantia propria. No eosinophils were seen. CD8⁺cells outnumbered CD4⁺cells in the epithelium (CD4:CD8 0.3) but this was reversed in the substantia propria (CD4:CD8 1.3 tarsal, 2.0 bulbar). Most epithelial T cells and half the stromal T cells were CD45Ro⁺. IL-2R (CD25) staining was infrequent in the tarsal but not the bulbar area. The greater number and activation of T cells in the bulbar region may relate to greater antigen exposure. HLA-DR⁺cells were mainly macrophages, but also included dendritic cells in the epithelium and a few epithelial cells. The conjunctival lymphocytes do have certain features of MALT cells, apart from mucosal recirculation, homing and promotion of sIgA production. Conjunctival IEL, like gut IEL, are predominantly CD8⁺, are found in the basal epithelium, are HML-1⁺, and have very high expression of CD45Ro. Substantia propria lymphocytes have more equal CD4 and CD8 numbers and frequently express CD45Ro. We found only one example of organised conjunctival lymphoid tissue in our study and suggest that the presence of CALT in humans is not universal.     

Lymphocytic subpopulations in the normal human conjunctiva. A monoclonal antibody study

Monoclonal antibodies were used to analyze the lymphocytic subpopulations in frozen tissue sections of normal human conjunctiva (epibulbar, tarsal, and forniceal). The overwhelming majority of lymphocytes were T-cells (Leu1+ and OKT8+). In the epithelium, the predominant cell type was the OKT8+ cytotoxic/suppressor cell, whereas in the substantia propria, helper T-cells (Leu3a/3b+) equalled suppressor T-cells. T-cells outnumbered B-cells 20-fold; the letter cells were detected only in the substantial propria, particularly in the fornices, and not in the epithelium. Plasma cells, as identified by OKT10 staining, were completely absent except in the accessory lacrimal glands of Krause. Langerhans' cells, identified by OKT6 and HLA-DR (la) staining, were observed in the epithelium of all conjunctival regions. Our findings suggest that the reactivity of B-lymphocytes and plasma cells is heavily damped down by T-lymphocytes, thus allying the conjunctiva to other mucosal membranes and the skin as heavily immunoregulated tissues.     

Immunopathology of atopic keratoconjunctivitis

Conjunctival biopsies from 11 patients with atopic keratoconjunctivitis (AKC) and from 13 age-matched healthy individuals undergoing cataract surgery were analyzed by light microscopy and immunohistochemical techniques. Histology of AKC specimens showed goblet cell proliferation, epithelial pseudotubular formation, eosinophil and mast cell invasion of the epithelium, and pronounced mononuclear cell infiltration of the substantia propria, often with frank granuloma formation. Epithelium of AKC conjunctiva showed significantly more T cells (CD3+, CD5+), T-helper cells (CD4+), macrophages (Mac-1+, CD14+), activated T cells, (CD25+), and dendritic cells (CD1+), and a higher helper/suppressor ratio than did control subjects. In the substantia propria, AKC specimens showed dramatically increased inflammatory cell infiltration with significantly more cells staining, in order of frequency, for T-cells (CD3+, CD5+), T-helper cells (CD4+), T-suppressor/cytotoxic cells (CD8+), macrophages (CD14+, Mac-1+) activated T cells (CD25+), B cells (CD22+), and dendritic cells (CD1+, HLA-DR+). Fifty-three percent of T cells in the substantia propria expressed the interleukin-2 receptor protein (CD25+). These findings indicate that the chronic conjunctivitis of AKC is complex, with activated T-cells and macrophages dramatically participating in the process. Successful long-term control of the potentially binding conjunctival inflammation of this disease is likely to require therapeutic strategies directed toward more than just the mast cell component of the process.     

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.     

Immunopathology of scleritis.

Conjunctival and scleral biopsies from 25 patients with necrotizing scleritis and 5 patients with recurrent nonnecrotizing scleritis were studied by histopathologic, immunofluorescence, and immunoperoxidase techniques. Vasculitis with fibrinoid necrosis and neutrophil invasion of the vessel wall was present in 75% of the scleral and 52% of the conjunctival specimens. Vascular immunodeposits were found in 93% of the scleral and 79% of the conjunctival tissue tested by immunofluorescence techniques. A dramatic increase in the number of inflammatory cells over normal controls was detected in both tissues by immunoperoxidase techniques. In the conjunctival epithelium, there were significantly more T-helpers, macrophages, and B cells. In the conjunctival substantia propria, there were significantly more T cells of all types, macrophages, and B cells. Likewise, scleral specimens showed an increase over controls of T cells of all types and macrophages. HLA-DR expression was dramatically increased in both tissues. Immune-complex-mediated vasculitis plays a pivotal role in the pathogenesis of necrotizing scleritis and recurrent nonnecrotizing scleritis. Induced HLA-DR expression on ocular nonimmune cells and T cell controlled responses also may participate.     

T cells and trachoma. Their role in cicatricial disease

Frozen sections of tarsoconjunctival biopsies with trachomatous scarring from 14 black adults undergoing corrective surgery for trichiasis, and "normal" tissue from three postmortem controls, were immunohistochemically stained for the major T- and B-cell subsets, and for macrophages and monocytes. T cells outnumbered B cells by 2 to 17 times, and macrophages and monocytes by approximately 20 times in all specimens. Biopsies were categorized as "inflamed" if a cumulative inflammatory score of cellular staining in the substantia propria with CD4, CD8, and OKM1 monoclonal antibodies was greater than that of control tissues. CD4+ lymphocytes predominated over CD8+ lymphocytes in 5 of 7 inflamed biopsies, whereas CD8+ lymphocytes predominated over CD4+ lymphocytes in 5 of 7 noninflamed biopsies. Lymphoid aggregates were present in five inflamed biopsies, but lacked germinal centers, centrally located B cells, or parafollicular T cells typical of the acute stage of trachoma. CD4+ and CD8+ lymphocytes also were observed in the epithelium and lumen of Meibomian glands. These observations indicate that the inflammatory infiltrate of the tarsoconjunctiva in the cicatricial stage of trachoma is comprised predominantly of T cells, and suggests that T cells may be involved in the genesis of tarsal thickening and conjunctival scarring seen in the later stages of trachoma.     

Malignant melanoma arising from unusual conjunctival blue nevus

Cellular blue nevus is an uncommon pigmented tumor in the conjunctiva, where it generally appears as a deep, circumscribed, pigmented conjunctival mass. We report a case of conjunctival blue nevus that clinically resembled primary acquired melanosis and gave rise to conjunctival melanoma. A 41-year-old man developed a diffuse pigmented mass in the inferior fornix of his left eye. Over a 20-year period, he noted slight progression of the pigment. Foci of epibulbar pigmentation were also present. The lesion resembled primary acquired melanosis. Excisional biopsy and adjuvant cryotherapy were performed. Histopathologic examination disclosed an intense infiltrate of heavily pigmented dendritic melanocytes with aggregates of less pigmented plump cells in the substantia propria. The conjunctival epithelium was normal. Malignant cellular features consistent with melanoma were observed in some foci. Cellular blue nevus of the conjunctiva can simulate primary acquired melanosis and can give rise to malignant melanoma. Arch Ophthalmol. 2000;118:1581-1584     

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.     

Structural and cellular architecture of conjunctival lymphoid follicles in the baboon (Papio anubis)

Conjunctival lymphoid follicles (CLFs), present in normal individuals, undergo hyperplasia upon conjunctival infection by a specific array of pathogens; infection-associated enlargement of draining preauricular lymph nodes suggests that conjunctival follicles participate in the afferent limb of acquired immune responses for the ocular surface. The present study was performed to delineate the structural and lymphoid anatomy of CLFs in the baboon (Papio anubis), a non-human primate conjunctival model with close similarity to the human. Conjunctiva from both eyes, along with mesenteric lymph node, spleen, tonsil, and ileum controls were harvested from ten baboons at necropsy, and studied by histochemical and immunohistochemical methods. Baboon conjunctival follicles were identified as dense oval collections of leukocytes in the substantia propria with infiltration into a thinned overlying conjunctival epithelium. Goblet cells were universally absent, the overlying mucin layer was attenuated, and the follicle-associated epithelium (FAE) demonstrated comparatively diminished alkaline phosphatase expression. The basement membrane overlying each follicle appeared discontinuous. CD4-positive T lymphocytes were distributed in parafollicular areas and to a lesser degree in follicle germinal centers. B lymphocytes formed the predominant cell in follicles, and also heavily infiltrated the FAE. B cell IgM expression was prominent in germinal centers, while IgD staining occurred in a horseshoe-shaped distribution in the follicle mantle zone. Although B cell IgA expression was noted in the non-follicular conjunctiva, IgA expression was inconspicuous within conjunctival follicles. S-100- and CD1a-positive dendritic cells were found in FAE, while fascin-positive mature dendritic cells appeared in the deeper areas of each follicle. CD68-positive macrophages were dispersed throughout the follicles. CD35-positive follicular dendritic cells were observed only in germinal centers. CLFs appear highly organized consistent with a role in the adaptive immune response to conjunctival pathogens.     

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.     

Immune cells in a case of postherpetic marginal trophic ulcer.

The corneal surface was examined by means of replica histology, and the excised limbic conjunctiva was examined by routine histological and immunohistochemical methods with monoclonal antibodies directed against major histocompatibility class II antigens, lymphocyte subsets, Langerhans cells (HLA-DR, OKT4-Leu3a, OKT8, BA1, B1, and OKT6) and immunoglobulins A, G, M, and D. The findings were compared with those found in normal conjunctiva. No inflammatory cells were present in the replica of the corneal surface. An inflammatory infiltrate composed of B lymphocytes and null cells, in addition to T lymphocytes, Langerhans cells, and polymorphs, was present in the epithelium as well as in the stroma of the limbic conjunctiva. The composition of the infiltrate points towards the involvement of cell mediated immunity as well as humoral immunity. No immunoglobulins were bound to the conjunctival epithelium.     

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.

     

Characterization of bone marrow derived cells in the substantia propria of the human conjunctiva

PURPOSE: To characterize the main population of bone marrow-derived cells (BMCs) in human normal subconjunctiva and make a comparison with BMCs in the corneal stroma and epithelium. METHODS: Normal human donor corneas with attached conjunctiva were examined by fluorescence microscopy after single and double staining for multiple markers. CD68(+) cells were separated from the conjunctival tissues by using magnetic beads, and the expression of toll-like receptor (TLR) 2 and TLR4 was examined. Surface markers of CD68(+) cells were compared with those of BMCs from the corneal stroma and epithelium. RESULTS: CD45(+) cells were detected in the substantia propria of the conjunctiva, and approximately 60% of these cells were CD68(+). All the CD68(+) cells expressed HLA-DR and CD14. CD68(+) cells isolated from conjunctival tissues expressed TLR2 and TLR4 on flow cytometry. BMCs in both the corneal stroma and the subconjunctiva expressed scavenger receptor CD163. Macrophage mannose receptor CD206 was expressed by BMCs in the substantia propria of the conjunctiva, but not by BMCs in the corneal stroma or epithelium. CONCLUSIONS: These findings demonstrated that the main population of BMCs in the substantia propria of normal human conjunctiva is CD68(+)CD14(+)HLA-DR(+) cells. These BMCs express scavenger receptor, macrophage mannose receptor, TLR2, and TLR4 and may play a role in adaptive and innate immune responses in the human ocular surface. These cells are phenotypically different from the CD68(-)CD206(-) monocyte- lineage cells found in the corneal stroma and the CD11c(+)CD68(-)CD163(-)CD206(-) dendritic cells residing in the corneal epithelium.     

Mooren ulcer: an immunopathologic study

PURPOSE: To determine the pattern and distribution of mononuclear cells, adhesion, and co-stimulatory molecules in the conjunctiva of patients with Mooren ulcer. METHODS: Conjunctival biopsy specimens were obtained from 6 patients with Mooren ulcer and 6 healthy individuals. Immunohistochemistry was performed on frozen sections of the cryopreserved human conjunctivas using monoclonal antibodies directed against CD1alpha, CD3, CD4, CD8, CD20, CD25, CD57, and CD68 cells; the adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), very late activation-4 (VLA-4), ICAM-1, and LFA-1; and the co-stimulatory molecules CD28, B7-1, B7-2, and CTLA-4. RESULTS: Differences in expression on the conjunctival epithelium from patients with Mooren ulcer and normal subjects were noted only for VCAM-1, VLA-4, ICAM-1, and LFA-1. The ratio of CD4+/CD8+ cells in Mooren ulcer specimens was significantly higher (3.5-fold). However, in the substantia propria, Mooren ulcer specimens revealed significantly increased numbers of CD1alpha+, CD3+, CD4+, CD20+, CD28+, B7-1+, B7-2+, and CD68+ cells. The ratios of CD4+/CD8+ cells and B7-2+/antigen-presenting cells in Mooren ulcer specimens were significantly higher (5-fold). All tested adhesion molecules showed significant up-regulation in the patients' conjunctivas. Mooren ulcer vascular endothelial cells prominently expressed E-selectin, VCAM-1, VLA-4, and ICAM-1 compared with normal conjunctiva. CONCLUSION: The simultaneous presence of multiple types of inflammatory cells, adhesion, and co-stimulatory molecules in Mooren ulcer conjunctiva suggests that their interaction may contribute to a sustained immune activation as at least part of the pathogenic mechanism of this disorder.     

T-Cell Subsets and Langerhans Cells in Normal and Diseased Conjunctiva

To identify the infiltrating mononuclear cells in conjunctival lesions, we stained frozen tissue sections of conjunctiva with a series of monoclonal antibodies against T cells and T-cell subsets with an immunoperoxidase method. In vernal conjunctivitis and ocular pemphigoid, most lymphocytes reacted with the anti-T4 antibodies that define helper/inducer T cells (T4₊); a minority reacted with the anti-T8 antibody that defines suppressor/cytotoxic T cells (T8₊). However, T8₊ cells predominated in a conjunctival specimen from a patient with chronic graft-vs-host disease. A few T8₊ cells and rare T4₊ cells were also found in normal conjunctiva. In some diseased conjunctivas, there was an increase in cells stained with anti-T6 antibody, which stains Langerhans cells in normal conjunctiva and skin. T lymphocyte subsets and Langerhans cells probably play an important role in the pathogenesis of inflammatory conjunctival lesions     

Unusual melanocytic nevi of the conjunctiva

In one patient, an epithelioid cell nevus of the conjunctiva contained numerous large, unpigmented, mononucleated, binucleated, and multinucleated benign-appearing nevus cells with abundant cytoplasm and frequent intranuclear vacuoles. Despite their overall size, the cells manifested a low nuclear-cytoplasmic ratio. After a partial excision of the lesion, the remainder spontaneously regressed during a two-year period. Another patient's lesion was dominated by a proliferation of spindle nevus cells developing in a long-standing epibulbar nevus. The spindle cells were moderately pigmented, frequently located within walls of epithelial inclusion cysts, and had benign cytologic features. Finally, in a third patient with the cutaneous B-K mole syndrome, a dysplastic conjunctival nevus developed that featured intraepithelial, atypical melanocytic proliferation with superficial colonization of the substantia propria. This portion coexisted with a deeper, preexistent lesion in the substantia propria that was comprised of orderly nests of unpigmented cuboidal nevus cells surrounded by pigmented, spindle-shaped blue nevus cells--a so-called "mixed nevus."     

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.     

The T-lymphocyte chemoattractant Mig is highly expressed in vernal keratoconjunctivitis

PURPOSE: To examine the expression of the three interferon-gamma-inducible CXCR3-binding chemokines, CXCL10/IP-10 (interferon-gamma-inducible protein of 10 KDa), CXCL9/Mig (monokine induced by interferon-gamma), and CXCL11/I-TAC (interferon-inducible T-cell alpha chemoattractant) in the conjunctiva of patients with vernal keratoconjunctivitis (VKC). These chemokines exhibit potent T-lymphocyte chemoattractant activity. DESIGN: Immunohistochemical study. METHODS: Conjunctival biopsy specimens from 16 patients with active VKC and nine control subjects were studied by immunohistochemical techniques using monoclonal antibodies directed against IP-10, Mig, and I-TAC. The phenotype of inflammatory cells expressing chemokines was examined by double immunohistochemistry. RESULTS: In the normal conjunctiva, very weak Mig immunoreactivity was observed on basal epithelial cells and on vascular endothelial cells in the upper substantia propria. There was no immunoreactivity for the other chemokines. In all VKC specimens, strong immunoreactivity for Mig was expressed by epithelial cells, vascular endothelial cells, and inflammatory mononuclear cells. Inflammatory mononuclear cells expressing IP-10 and I-TAC were noted in 10 and nine specimens, respectively. The numbers of Mig(+) inflammatory cells were significantly higher than the numbers of IP-10(+) and I-TAC(+) inflammatory cells (P <.001). Inflammatory cells expressing Mig were CD4(+) T-helper/inducer cells (71.6 +/- 3.2%), CD8(+) T-cytotoxic/suppressor cells (19.5 +/- 1.5%), and CD68(+) monocytes/macrophages (5.3 +/- 5%). All inflammatory cells expressing IP-10 and I-TAC were CD68(+) monocytes/macrophages. CONCLUSIONS: The CXC chemokine Mig is selectively and highly expressed in VKC suggesting a pathogenic role of the chemokine receptor CXCR3 and the ligand Mig in the recruitment of activated T lymphocytes.