Immunopathology of vitreous and retinochoroidal biopsy in posterior uveitis.

The authors used immunopathologic techniques to study vitreous and/or retinochoroidal biopsies from 23 patients with posterior uveitis unresponsive to conventional therapy or who had developed significant complications despite therapy. Results indicated that during active uveitis from many causes, T-helper cells predominated in the vitreous and retinochoroidal biopsies. Monocytes were not prominent constituents except in several cases of granulomatous etiology (e.g., syphilis and acute retinal necrosis). Class II major histocompatibility complex (MHC) antigens were increased on the retinal vascular endothelium, implicating an important role for these cells in the local cellular immune response. These results may be of great importance in our understanding of uveitis, as well as being helpful in categorizing posterior uveitis, permitting appropriate therapy to be given.

Advances in immunology and uveitis.

*Subspecialty synopses are intended to present major clinical developments and innovations in diagnosis, treatment, surgical technique, and recent clinical research. Each synopsis, written by a recognized knowledgeable subspecialist, is co-sponsored by a major subspecialty society or organization. This subspecialty synopsis is co-sponsored by the Ocular Microbiology and Immunology Group.

Role of fibronectin and fibrinogen in healing of corneal epithelial scrape wounds.

A provisional fibronectin (Fn)-fibrinogen (Fg) matrix forms de novo on the bare basement membrane (BM) surface of superficial epithelial scrape wounds in rabbit and guinea pig (GP) corneas. We determined whether such a substrate is essential for epithelial cell adhesion and migration, using three different approaches. (1) Polyclonal and monoclonal IgG reactive against GP Fn were topically administered to inhibit Fn formation in a GP epithelial scrape wound model. Immunofluorescence studies showed that deposition of both Fn and Fg was inhibited in antibody-treated corneas. During the first 38 hr after wounding, the healing rates were 0.46 +/- 0.06 mm2/hr in control eyes, 0.43 +/- 0.05 mm2/hr in those treated with rabbit polyclonal IgG anti-GP Fn and 0.45 +/- 0.09 mm2/hr in those treated with murine monoclonal IgG anti-GP Fn (P greater than 0.4). (2) In a rabbit epithelial scrape wound model, ancrod was administered intravenously to induce systemic Fg depletion. Fg deposition was completely inhibited on the wound surface, but Fn deposition was not suppressed. The healing rate was 1.24 +/- 0.41 mm2/hr in ancrod-treated corneal wounds and 1.19 +/- 0.29 mm2/hr in control eyes during the first 48 hr after wounding (P greater than 0.5). These data from the antibody and ancrod inhibition indicate that Fg binds to Fn and that Fn binds to components other than Fg.(ABSTRACT TRUNCATED AT 250 WORDS)

Class II antigens on retinal vascular endothelium, pericytes, macrophages, and lymphocytes of the rat.

Class II histocompatibility complex antigens on the retinal vascular endothelium may allow these cells to function as antigen-presenting cells to circulating T cells. The present study investigated induction of class II antigens in vitro to characterize the response under controlled conditions. Retinal vascular endothelium from Lewis and Brown Norway rats (high versus low responders in experimental autoimmune uveitis) were exposed in vitro to recombinant rat gamma interferon, interleukin-1, interleukin-2, or Concanavalin-A spleen supernatant. Retinal pericytes, macrophages and lymphocytes were studied in comparison. A newly adapted ELISA technique was used to assay levels of antigen expression. Class II antigens (I-A OX6, I-E OX17, polymorphic I-A OX3), class I antigens (OX18), macrophage marker (OX42), macrophage and T helper cell marker (W3/25), and T suppressor/cytotoxic cell marker (OX8) were studied. Results showed that retinal vascular endothelium normally expresses very little class II antigen. However, high levels of I-A and I-E were induced by interferon or spleen supernatant. The levels of class II antigen approached that of the traditional antigen-presenting cell (macrophage) and were much higher than levels for pericytes and lymphocytes. The same doses of interferon showed larger increases in the Lewis rat compared to Brown Norway. No effect was seen with interleukin-1 or -2. Therefore, retinal vascular endothelium may be induced by gamma interferon to express class II antigens with degree of induction greater than or equal to the macrophage, and higher levels of induction were seen in the high responder strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinal vascular endothelium expresses fibronectin and class II histocompatibility complex antigens in experimental autoimmune uveitis.

To analyze the role of the retinal vascular endothelial cells in the development of experimental autoimmune uveitis (EAU), we studied the presence of Ia antigen and FN in retinal vessels of Lewis rats immunized with retinal S antigen. Immunopathologic studies were performed on frozen tissues obtained during various stages of the disease. Our results show that Ia antigen was not present in the normal rat retina, and there was very little FN present in a few retinal vessels. One to two days prior to the histologic and clinical onset of EAU, FN was found to be increased in the retinal vessels. Ia antigen was found to be present in the retinal vessels coincident with the first signs of cellular infiltration. During the stage of maximal cellular infiltration, FN was present diffusely throughout the retina, as well as in the subretinal space, and Ia antigen was found diffusely in the cellular infiltrate. Therefore, FN and Ia antigen reflect the immunomodulation of vascular endothelial cells in EAU, which may be very important in the pathogenesis of retinal S antigen-induced uveitis. Two possible mechanisms for the role of the activation of the retinal vascular endothelium in the development of retinal inflammation in uveitis are discussed.

Immunohistochemical analysis of experimental autoimmune uveoretinitis (EAU) induced by interphotoreceptor retinoid-binding protein (IRBP) in the rat.

Experimental autoimmune uveoretinitis (EAU) was induced in rats by immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) and studied by immunohistochemistry. In general, the IRBP induction of inflammatory cellular components and expression of immune-related antigens on various non-lymphoid cells resembled those provoked by S-antigen (S-Ag). However, differences were found between the two diseases, including: 1) The increase in T suppressor/cytotoxic cells occurred in IRBP EAU more rapidly than in S-Ag EAU. 2) Fewer numbers of non-lymphoid cells expressed major histocompatibility complex class II surface antigens in IRBP EAU than in S-Ag EAU. The immunopathogenic mechanism of EAU induced by these two retinal antigens are discussed.

Chemotactic activity in aqueous humor from patients with anterior uveitis.

Anterior uveitis, inflammation of the iris or ciliary body of the eye, may be associated with a variety of systemic diseases. Although a leukocytic infiltrate is characteristic of anterior uveitis, few studies have sought to detect factors in aqueous humor that could attract neutrophils or monocytes into the anterior chamber. Using modified Boyden chambers, we found that a 5% concentration of aqueous humor from patients with anterior uveal inflammation induced monocyte movement comparable to optimal or near-optimal concentrations of C (complement)5a or platelet-derived growth factor. Aqueous humor from patients with anterior uveitis induced significantly more monocyte migration than did aqueous humor from two sets of controls (either patients undergoing cataract extraction or patients with posterior uveitis). "Checkerboard" or gradient analysis indicated that a majority of inflammatory disease samples induced monocyte chemotaxis (directed migration) while the control aqueous humor consistently induced chemokinesis (stimulated random migration) (P less than 0.02). Despite their ability to induce monocyte migration, samples tended to induce minimal neutrophil migration with the exception of aqueous humor that was obtained from one patient with acute anterior disease. This sample induced marked chemokinesis. Identification of chemotactic activity may clarify the pathogenesis of uveitis and the characterization of leukocyte migration factors in aqueous humor may help define subsets of anterior uveal inflammation.

Presence of HTLV-III in tears and cells from the eyes of AIDS patients.

Tears, conjunctival epithelium, and corneoscleral tissue from AIDS patients were used for the isolation of HTLV-III and also for identifying cell types which support its replication. HTLV-III was isolated from tears of AIDS patients (66.6%) by cultivation of cells and fluid from patients' eyes with fresh human mononuclear cells. The cells from the conjunctival scrapings of these patients (33.3%) revealed HTLV-III antigens by indirect immunofluorescence (IF) using anti-P24 and P15 monoclonal antibodies. HTLV-III from the cell-free supernatant of the infected mononuclear cells from two patients' cocultures were further transmitted into fresh cells. The cells from right and left central cornea, as well as limbal cornea from an asymptomatic HTLV-III antibody-positive individual and one AIDS patient revealed HTLV-III upon cocultivation. HTLV-III P15 and P24 antigens were detected in cultured primary cornea epithelial cells. The tears and conjunctival cells from a control group were found to be free of HTLV-III. Although no documented cases of AIDS have been reported in corneal transplant recipients, serologic screening of donors prior to the use of the tissues for transplantation is advisable. Our data also raises important questions regarding possible transmission of virus during ophthalmologic examination by way of examiner's hands, through instruments and during contact lens fittings. Moreover, these findings indicate the need for testing various eye disinfectants for virus inactivation and/or inhibition.

HTLV-III in the tears of AIDS patients.

The human T-cell leukemia/lymphotropic virus type III (HTLV-III), the causative agent of the acquired immunodeficiency syndrome (AIDS), has been isolated from the tears of five AIDS patients. When combined with data from our previous study, 5 of 16 samples from patients with AIDS or AIDS-related complex (ARC) showed positive isolates for HTLV-III from the tears. Normal control tears were negative for HTLV-III. Based upon these findings, the Centers for Disease Control (CDC) has issued precautions to prevent any possible spread of the virus by this route. Although there is no evidence to suggest transmission of HTLV-III by contact with the tears, until more is known about the possible transmissibility and infectious dose of this virus, such precautions should be taken during ophthalmic examination.

Immunohistochemistry and electron microscopy of cyclitic membrane. Report of a case.

An immunohistochemical study using various antibodies directed against antigens of inflammatory cells, neurons, and Müller cells, interstitial collagens (types I and III), basement membrane collagens (types IV and V), and basement membrane glycoproteins (laminin and fibronectin) was performed to characterize the components of two cyclitic membranes (in an enucleated eye and a surgical specimen) from a 15-year-old patient with an eight-year history of chronic bilateral uveitis. When specimens were obtained, no inflammation was seen clinically. Correlative light and electron microscopic examinations were also performed on one specimen. The preponderant cells in the cyclitic membrane were glial cells. The preponderant extracellular tissue within the cyclitic membrane consisted of basement membrane components. These observations suggest that cyclitic membranes could be formed mainly by the extension of proliferative glial elements from the retina, with a minor component derived from fibroblasts.

HLA-DR antigens on retinal pigment epithelial cells from patients with uveitis.

The pathogenic mechanisms involved in immunologically associated ocular diseases are not clearly understood. To further evaluate these types of disorders, we examined the expression of HLA-DR antigen on eye sections from normal individuals and five patients (six eyes) with ocular inflammation (sympathetic ophthalmia and chronic uveitis). Using immunoperoxidase staining technique and the complement-mediated cytotoxicity assay, we detected the presence of HLA-DR antigens on retinal pigment epithelial (RPE) cells from uveitic eyes. In contrast, HLA-DR antigen was not detected on RPE cells from normal ocular tissue. Our study shows that during the course of human ocular inflammatory disorders, the RPE cell is activated to express HLA-DR antigens. This antigen expression may be important in the initiation and/or perpetuation of immune reactivity in the eye.

Sympathetic ophthalmia. Immunopathological findings.

Ocular tissue from six patients with a clinical diagnosis of sympathetic ophthalmia (SO) was examined using immunohistochemical techniques. All patients presented with a history of bilateral panuveitis after penetrating ocular injury or multiple intraocular surgeries and clinical features of SO. In four cases, classic histopathological features of SO were observed, including granulomatous uveal tract infiltration and subretinal pigment epithelium (RPE) collections of inflammatory cells (Dalen-Fuchs nodules). Bone marrow derived monocytes were the major cellular components in these granulomas. In two cases, histopathology failed to demonstrate typical Dalen-Fuchs nodules or granulomas in the choroid. However, the choroidal infiltrates were composed primarily of T-helper and B lymphocytes, without macrophages or epithelioid cells. The eyes examined in this report indicate that a varied spectrum of immunopathological and histopathological findings may occur in clinically diagnosed SO.

Immunohistochemical characterization of extracellular matrix in the developing human cornea.

Collagen, fibronectin and laminin are important components of the extracellular matrix of the human cornea. We used the immunofluorescence technique with polyclonal antibodies directed against these proteins and to bullous pemphigoid antigen (BPA), in order to study their distribution in human corneas from 8 weeks of gestation to term and in adult corneas. Immunoreactivity was observed with antibodies to type I collagen in the limbus and the corneal stroma at 8 weeks of gestation. At 11 weeks of gestation it was found in epithelial basement membrane (EBM) and Descemet's membrane (DM) and continued thus throughout fetal and adult life. Type II collagen was not detected in fetal or adult cornea. Type III collagen was detected during 8-20th weeks of gestation in the EBM, DM and stroma. After 27th weeks of gestation, type III collagen could no longer be detected in the central cornea. Type IV collagen was detected in the EBM as early as 8 weeks of gestation and remained positive throughout fetal and adult life. Descemet's membrane was negative for type IV collagen at 8 weeks of gestation and became positive thereafter. Immunostaining for fibronectin in DM was negative at 8 weeks of gestation, followed by patchy staining of corneal stroma and EBM up to the age of 37 weeks of gestation. Staining in the EBM was negative or variable up to 70 years of age, and then became positive again in a 77 year old individual. Staining for LN was positive in the EBM after 8 weeks of gestation. Staining was negative in DM at that age, but became positive after 9 weeks of gestation. Staining for BPA was negative at 8-9 weeks of gestation, then gradually became positive.

Treatment of cytomegalovirus retinitis with dihydroxy propoxymethyl guanine.

Eight patients with cytomegalovirus retinitis in one or both eyes were treated with 9-(1,3 dihydroxy 2-propoxymethyl) guanine. Of the 14 eyes with retinitis, eight demonstrated more than 90% resolution, four had partial improvement, one failed to respond, and one could not be evaluated. Two of the eight patients had chemotherapy-induced immunosuppression and had ocular remissions for at least several months. The remaining six patients had AIDS or probable AIDS and relapsed in five weeks or less after discontinuation of therapy. Dihydroxy propoxymethyl guanine appears to be effective biologically in treating human cytomegalovirus retinitis without the development of unacceptable side effects but a single course of therapy is not capable of eradicating the virus.

Conjunctival transplantation. Autologous and homologous grafts.

Autologous conjunctival transplants have been used successfully for restoration of damaged ocular surfaces. Homologous (allogeneic) conjunctival grafts have been explored less systematically. We developed a nonhuman primate model for comparison of autologous and homologous conjunctival transplantation in order to assess the clinical viability and immunopathologic characteristics of these grafts. Autologous or homologous grafts were performed in nine adult rhesus monkeys. Both autologous and homologous grafts were compared for clinical viability and immunopathologic change. Clinical results suggest that, although homologous grafts incited a greater inflammatory and scarring response, there was minimal graft shrinkage and a normal surface epithelium. Immunopathologic studies using laminin, bullous pemphigoid antigen, and fibronectin indicate that, despite the increased inflammatory response seen in homografts, the epithelial surface is normal. With our increasing ability to modulate the immune response, conjunctival homografts may play a role in restoration of the ocular surface.

Local cyclosporine therapy for experimental autoimmune uveitis in rats.

The use of locally applied cyclosporine was investigated in the retinal S-antigen-induced experimental autoimmune uveitis (EAU) model in Lewis rats. A 2% cyclosporine solution applied topically four times a day for 14 days effectively prevented the expression of EAU. This treatment, however, produced circulating cyclosporine levels in the therapeutic range. Lower concentrations of cyclosporine applied topically did not produce therapeutic levels and were not capable of reliably preventing disease. Intraocular levels of cyclosporine, measured by radioimmunoassay, were extremely low and outside the accepted therapeutic range. Intravitreal cyclosporine therapy appeared to protect eyes from EAU, without producing significant circulating cyclosporine levels. These findings show that, in its present form, cyclosporine in oil is not an efficacious topical therapy. Therefore, a local cyclosporine preparation with enhanced penetration into the globe may be a practical approach to therapy in the future.

Human T-cell leukemia/lymphotropic virus type III in the conjunctival epithelium of a patient with AIDS.

The human T-cell leukemia/lymphotropic virus type III (HTLV-III), the causative agent of the acquired immunodeficiency syndrome (AIDS), has been isolated from the conjunctival epithelium of a 33-year-old woman with AIDS, suggesting that an important reservoir of the virus may be the ocular surface epithelial cells. The tears and conjunctival epithelium from normal controls were negative for HTLV-III. The finding of HTLV-III in the tears and conjunctival epithelium indicated that HTLV-III may be ubiquitous in bodily cells and fluids. Repeated contact with the tears and ocular surface epithelium of patients with AIDS may possibly facilitate transmission of HTLV-III, and precautions are advisable during routine ophthalmologic procedures such as glaucoma testing and contact-lens fitting.

Isolation of human T-lymphotropic virus type III from the tears of a patient with the acquired immunodeficiency syndrome.

Human T-lymphotropic virus type III (HTLV-III) was isolated from the tears of 1 out of 7 patients with the acquired immunodeficiency syndrome (AIDS) or the AIDS-related complex. The tears from 5 healthy individuals did not show HTLV-III. The recovery of HTLV-III from tears suggests that they may harbour free virus or contain virus-positive cells and is consistent with the recovery of HTLV-III from other body fluids. Although casual contact with such fluids has not been shown to cause AIDS, direct contact with the tears of AIDS patients, including contact during routine ophthalmologic procedures, should be minimised.

Fibronectin in developing rabbit cornea.

Fibronectin is believed to be important in tissue morphogenesis. We examined the distribution of fibronectin in developing rabbit cornea by immunohistofluorescence. Cryostat sections of cornea from 13, 15, and 20-day-old fetuses, 3-day neonates, and adults were incubated with affinity-purified fluoresceinated guinea pig anti-rabbit fibronectin antiserum (aFN). aFN bound to components within the presumptive stromal region and along the basal surfaces of corneal and lens epithelia during early stages of mesenchymal invasion. At 15 days of gestation, fluorescence was associated with the stromal extracellular matrix of the cornea, the subepithelial zone, and the lens capsule. In the 20-day fetus an intense aFN fluorescence was present along the inner corneal stromal border coincident with the formation of Descemet's membrane. Fluorescence within the corneal stroma appeared as fine lines, restricted to the collagen lamellae, remaining through birth and disappearing in the adult. Although stromal fluorescence disappeared in the adult, Descemet's membrane continued to fluoresce, albeit to a lesser extent. The results of our studies indicate the presence of fibronectin in developing rabbit cornea. Because fibronectin is important to cell adhesion in vitro, and because intercellular and cell-extracellular matrix interactions, including adhesion, are necessary for tissue morphogenesis, our observation suggests that fibronectin plays an important role in corneal morphogenesis.

Basement membrane components in healing rabbit corneal epithelial wounds: immunofluorescence and ultrastructural studies.

The nature of the substrate that supports epithelial migration in vivo is of interest, particularly with respect to mechanisms of wound healing. Immunofluorescence and electron microscopy were used to search for common substrate components in prototype rabbit corneal wounds: epithelial scrape wounds, in which the corneal or conjunctival epithelium migrated over the denuded lamina densa of the corneal basement membrane (CBM), and superficial keratectomy, in which the corneal epithelium migrated over a bare stroma without CBM. The corneal epithelium moved rapidly over the CBM or stroma to cover the defect within 2-3 d, whereas the conjunctival epithelium required 1-2 wk. In all wounds, fibronectin and fibrin/fibrinogen were deposited onto the bare surface within 8 h after wounding and persisted under the migrating epithelium until migration was complete. Bullous pemphigoid antigen (BPA), a normal component of the CBM, was removed with the epithelium upon scrape wounding and reappeared in the CBM after migration was completed. In contrast, the conjunctival epithelium had a continuous subepithelial band of BPA out to the migrating tip. Laminin, also a normal component of the CBM, was not removed in the scrape wounds, indicating that the region of least resistance to shear stress was between the BPA and laminin layers. Laminin was removed by superficial keratectomy and was not detectable under the leading edge of the migrating cells. Laminin and BPA were restored in the CBM by 2-4 wk. Type IV collagen could not be detected in normal CBM, but was conspicuously present in conjunctival basement membrane and in blood vessels. Focal bands of type IV collagen did appear in the newly synthesized CBM 2-4 wk after keratectomy. These results argue that BPA, laminin, and type IV collagen are not essential for the migration of corneal epithelium during wound healing and support the hypothesis that fibronectin and fibrin/fibrinogen are the common, perhaps the essential, components of the provisional matrix that serves as a substrate until the permanent attachment components are regenerated.