Fas ligand (CD95 ligand) controls angiogenesis beneath the retina.

A principal cause of blindness is subretinal neovascularization associated with age-related macular degeneration. Excised neovascular membranes from patients with age-related macular degeneration demonstrated a pattern of Fas+ new vessels in the center of the vascular complex, surrounded by FasL+ retinal pigment epithelial cells. In a murine model, Fas (CD95)-deficient (Ipr) and FasL-defective (gld) mice had a significantly increased incidence of neovascularization compared with normal mice. Furthermore, in gld mice there is massive subretinal neovascularization with uncontrolled growth of vessels. We found that cultured choroidal endothelial cells were induced to undergo apoptosis by retinal pigment epithelial cells through a Fas-FasL interaction. In addition, antibody against Fas prevented vascular tube formation of choroidal endothelial cells derived from the eye in a three-dimensional in vitro assay. Thus, FasL expressed on retinal pigment epithelial cells may control the growth and development of new subretinal vessels that can damage vision.

A reconsideration of immunological privilege within the anterior chamber of the eye.

The immunologically privileged status of the anterior chamber of the eye has been confirmed on the basis of skin and thyroid allografts with use of inbred strains of rats. Skin grafts, transplanted across both major and minor histocompatibility barriers, enjoy prolonged survival within the anterior chamber. However, several factors have been found to restrict the privilege exhibited by this site: the magnitude of the immunogenetic disparity between donor and recipient, graft size, type of tissue grafted, and, at least in the case of thyroid grafts, the endocrine status of the host. Although a defect in the afferent limb of the immune response has been the popular explanation offered for immunological privilege in an alymphatic site, this was not the basis of the privilege encountered in the anterior chamber. Furthermore, because the efferent limb of the immunological reflex are within the anterior chamber has been demonstrated to be intact, it was suggested that the unique properties of the anterior chamber may depend upon the forced intravascular presentation of antigen and consequent aberrant central processing.

Anterior chamber-associated immune deviation induced by TNP-splenocytes (TNP-ACAID). II. Suppressor T-cell networks.

Injection of trinitrophenyl (TNP)-modified splenocytes (TNP-Sp) into the anterior chamber (AC) of the eye results in systemic tolerance to TNP, a phenomenon termed "AC-associated immune deviation" or "TNP-ACAID". The systemic tolerance of TNP-ACAID is mediated by splenic suppressor T cells (Ts) that comprise an immunoregulatory network made up of two functionally distinct subsets (Ts-I and Ts-II). Ts-I is antigen-specific and cyclophosphamide (Cy)-sensitive, and requires a Cy-sensitive auxiliary cell. Conversely, Ts-II is antigen-nonspecific and Cy-resistant, and requires a TNP-derived accessory macrophage. Suppression via Ts-II is demonstrated only when Ts-I cells are inactivated functionally by Cy. Ts-II cells are regulated by T cells isolated from splenocytes containing active Ts-I suppressors. This contrasuppressive activity is mediated by Ts-I, or perhaps by a third T-cell subpopulation. The relationship between the two suppressor pathways in TNP-ACAID is discussed.

Anterior chamber associated immune deviation induced by TNP-splenocytes (TNP-ACAID). I. Systemic tolerance mediated by suppressor T-cells.

The relationship between the anterior chamber (AC) of the eye and the immune system was studied after intracameral inoculation with the hapten TNP. Injection of syngeneic TNP-modified splenocytes (TNP-Sp) into the AC of Balb/c mice results in suppressed systemic cell-mediated immunity to TNP as determined by the ear swelling assay. This aberrant immune responsiveness following AC injection of TNP-Sp has been termed TNP anterior chamber-associated immune deviation or TNP-ACAID. The phenomenon of TNP-ACAID is mediated by antigen-specific efferent suppressor T cells (Ts). The generation of Ts in TNP-ACAID requires a period of interaction between the intact eye and spleen, ie, the splenocameral axis.

In situ immune complex formation within the uvea. Potential role of cationic antibody.

A murine model has been developed to study the role of immunoglobulin charge in the regulation of the intraocular distribution of circulating IgG antibodies. Intravenously injected cationic antibodies to the tracer enzyme horseradish peroxidase bind within the ciliary body and choroid (CB/Ch). These cationic antibodies can selectively entrap and bind circulating antigens forming immune complexes (IC) within the uveal tissues. The structure of the uvea with its fenestrated CB/Ch capillaries and fixed anionic sites (within Bruch's membrane and the stroma of the CB and processes) may predispose the CB/Ch to in situ IC formation. Local IC formation mediated initially by deposition of cationic antibodies within the uvea may play an important role in the immunopathogenesis of some forms of uveitis.

The wavelength of light governing intraocular immune reactions.

Injection of antigen into the anterior chamber of the eye results in the induction of suppressed systemic cell-mediated responses as measured by delayed-type hypersensitivity or contact hypersensitivity (CHS). Previous studies from the author's laboratories have determined that this response is governed by exposure of the eye to visible light during the initial intraocular encounter between T cells and antigen. To more fully understand the role of light, as well as to begin to understand the molecular mediators involved, the authors chose to explore the properties of light governing the effect. Neutral density filter were used to demonstrate that the minimum amount of light required to induce suppression of CHS following anterior chamber injection of antigen is 1-2 lux (lumens/meter2). With narrow band filters, the wavelengths responsible for suppression were shown to be 500-510 nm. The results show that the effect of light extends beyond the hapten-derivatized spleen cell system to other antigens placed in the anterior chamber of the eye. Studies also show that the retina and the pineal gland, two light absorbing structures, may not be involved. The results in this report show that light of very restricted wavelengths controls intraocular immune reactions.

The distribution and ontogeny of MHC antigens in murine ocular tissue.

The distribution of H-2 (Class I) and Ia (Class II) antigens in the mouse eye was determined with the use of monoclonal antibodies and found to be different. H-2 antigen was observed in the corneal epithelium, conjunctival epithelium, choroid, and inner and outer nuclear layer of the retina--in order of decreased intensity of staining. In contrast, Ia was detected most strongly, in a patchy distribution, in the choroid, limbus epithelium, and peripheral corneal stroma, followed by the iris and ciliary body. Thus, distinctively different patterns of distribution of H-2 and Ia were observed in the normal mouse eye. Surprisingly, adult eyes stained more intensely for both H-2 and Ia antigens than neonatal eyes, implying that the expression of MHC antigens varies with ontogeny. Since unique and important immunologic functions have been ascribed to class I and II antigens, their different distribution within the eye may indicate that various ocular structures can play distinctive roles in the immune response.

Identification, quantitation, and purification of a 36 kDa circulating protein associated with active pars planitis.

PURPOSE: To establish a correlation between the presence of a 36 kDa protein in the blood of patients with pars planitis and to characterize and purify this protein. METHODS: Blood samples were obtained from patients with pars planitis and other types of uveitis and from various controls. Samples were treated with polyethelene glycol and protein A and were analyzed on 10% SDS-PAGE for the presence of a 36 kDa protein. Quantitative estimation of the level of this protein was determined by densitometric tracing of the stained gels. Polyclonal antibodies were raised by immunizing New Zealand White rabbits with a mixture of the gel fragment containing the 36 kDa protein (p-36) and complete Freund's adjuvant. These antibodies were used in the immunoaffinity purification of this protein. RESULTS: The levels of p-36 were sixfold to eightfold higher in 81% of the patients with active pars planitis than in controls (P < 0.05). Furthermore, the levels of this protein correlated with disease activity. A partial amino terminal sequence analysis revealed that p-36 may be a novel protein. It has been purified from the patient's blood using affinity chromatography. CONCLUSIONS: A 36 kDa protein (p-36) is found in elevated concentrations in the blood of many patients with active pars planitis. Its putative role in the etiopathogenesis of pars planitis is unknown.

Fate of human retinal pigment epithelial cells seeded onto layers of human Bruch's membrane.

PURPOSE: To determine the fate of human retinal pigment epithelial (RPE) cells seeded onto different layers of human Bruch's membrane (BM). METHODS: Bruch's membrane explants were prepared from 16 human cadaver eyes (7 eyes age <50 years; 9 eyes >50 years) by removing native RPE cells with ammonium hydroxide to expose the RPE cell basal lamina (BL). The inner collagenous layer (ICL) and elastin layer (EL) were exposed by removing apical layers sequentially by mechanical and enzymatic means. Synchronized first passage human RPE cells (15,000 cells/(6-mm-diameter explant) were plated onto each layer of human BM. The RPE cell reattachment and apoptosis rates at 24 hours, proliferation rates and mitotic index 24 hours after growth stimulation, and the ability of RPE cells to repopulate the explant surface were determined on each layer. RESULTS: RPE cell reattachment was highest on BL but decreased on deeper layers of BM. The apoptosis rate of attached cells increased as deeper layers of BM were exposed. The proliferation rate and mitotic index of the grafted cells were higher on BL than on deeper layers. RPE cells plated onto BL repopulated the explant surface within 14 +/- 3 days, whereas cells plated onto the ICL and EL eventually died and never reached confluence. CONCLUSIONS: The fate of RPE cells seeded onto BM depends on the ultrastructural layer of BM available for reattachment. These findings suggest that the ability of transplanted RPE cells to repopulate bare BM will depend on the layer of BM available for RPE cell reattachment.

Differential expression of the complement regulatory proteins in the human eye.

PURPOSE: The presence of complement activation products in the human eye during infection or inflammation has been well described. During complement activation the host must be protected from attack against self tissue; this is achieved by three membrane-bound complement regulatory proteins: membrane cofactor protein (MCP, CD46), decay accelerating factor (DAF, CD55), and membrane attack complex inhibiting protein (CD59). This study was undertaken to analyze the expression of these proteins in the normal human eye. METHODS: Tissues were sectioned by cryostat and both polyclonal and monoclonal antibodies to MCP, DAF, and CD59 were used. Control stains were performed with nonrelevant antibodies of the same immunoglobulin subclass and normal rabbit serum as well as by omission of the primary and secondary antibodies. RESULTS: All three proteins were found to be differentially expressed in the human eye. With anti-MCP, strong staining of the corneal epithelium and weak staining of the corneal keratocytes in stroma and photoreceptor cells was observed. Staining with anti-DAF was very strong in the corneal epithelium and the ciliary body and moderate in the corneal stroma (keratocytes) and iris. In contrast, anti-CD59 stained very strongly in the corneal epithelium, corneal stroma (keratocytes), iris, choroid, and all layers of the retina, and moderately in the ciliary body. CONCLUSIONS: Identification of MCP, DAF, and CD59 in the human eye provides evidence that a regulatory system exists to protect these cells from destruction by complement-activating events. It remains to be determined if other more specialized functions exist for these proteins, especially in the case of CD59 because of its extensive expression in the retina.

Replication of HIV in human fetal retinal cultures and established pigment epithelial cell lines.

The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in cells derived from ocular tissue was studied. Primary retinal cultures (containing both glial and neuronal cells) were found to support the replication of HIV upon transfection with molecularly cloned proviral DNA. In addition, established retinal pigment epithelial (RPE) cell lines also produced HIV particles upon transfection. HIV released by these cell lines was able to infect and induce characteristic cytopathic effects in T4+ cells. An indicator plasmid containing the HIV long terminal repeat sequences (LTR) linked to the chloramphenicol acetyltransferase gene showed barely detectable activity in RPE cells and was transactivated by the addition of the HIV "tat" gene. Based on these observations, direct infection of ocular tissue derived cells such as RPE, fetal retinal cells, retinoblastoma cells (Y 79, WER1), choroidal endothelial cells (Chor 55) (mix culture) and corneal fibroblasts (K61) by HIV was attempted. HIV replication in these cells was not detected by reverse transcriptase, antigen and transactivation function assays.

Chronic low level complement activation within the eye is controlled by intraocular complement regulatory proteins.

PURPOSE: To explore the role of the complement system and complement regulatory proteins in an immune-privileged organ, the eye. METHODS: Eyes of normal Lewis rats were analyzed for the expression of complement regulatory proteins, membrane cofactor protein (MCP), decay-acceleration factor (DAF), membrane inhibitor of reactive lysis (MIRL, CD59), and cell surface regulator of complement (Crry), using immunohistochemistry, Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR). Zymosan, a known activator of the alternative pathway of complement system was injected into the anterior chamber of the eye of Lewis rats. Animals were also injected intracamerally with 5 microl (25 microg) of neutralizing monoclonal antibody (mAb) against rat Crry (5I2) or CD59 (6D1) in an attempt to develop antibody induced anterior uveitis; control animals received 5 microl of sterile phosphate-buffered saline (PBS), OX-18 (25 microg), G-16-510E3 (25 microg), or MOPC-21 (25 microg). The role of complement system in antibody-induced uveitis was explored by intraperitoneal injection of 35 U cobra venom factor (CVF), 24 hours before antibody injection. Immunohistochemical staining and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Western blot analysis were used to detect the presence of membrane attack complex (MAC) and C3 activation products, respectively, in normal and antibody-injected rat eyes. RESULTS: Complement activation product MAC was present in the normal rat eye, and intraocular injection of zymosan induced severe anterior uveitis. The complement regulatory proteins, MCP, DAF, CD59, and Crry, were identified in the normal rat eye. Soluble forms of Crry and CD59 were also detected in normal rat aqueous humor. Severe anterior uveitis developed in Lewis rats injected with a neutralizing mAb against Crry, with increased formation of C3 split products. Systemic complement depletion by CVF prevented the induction of anterior uveitis by anti-Crry mAb. Intracameral injection of anti-rat CD59 (6D1), anti-rat MHC class I antigen (OX-18), anti-rat Ig (G-16-510E3), or MOPC-21 caused no inflammatory reaction. CONCLUSIONS: The results suggest that the complement system is continuously active at a low level in the normal eye and is tightly regulated by intraocular complement regulatory proteins.

Complement regulatory activity of normal human intraocular fluid is mediated by MCP, DAF, and CD59.

PURPOSE: To identify the molecules in normal human intraocular fluid (aqueous humor and vitreous) that inhibit the functional activity of the complement system. METHODS: Aqueous humor and vitreous were obtained from patients with noninflammatory ocular disease at the time of surgery. Samples were incubated with normal human serum (NHS), and the mixture assayed for inhibition of the classical and alternative complement pathways using standard CH(50) and AH(50) hemolytic assays, respectively. Both aqueous humor and vitreous were fractionated by microconcentrators and size exclusion column chromatography. The inhibitory molecules were identified by immunoblotting as well as by studying the effect of depletion of membrane cofactor protein (MCP), decay-accelerating factor (DAF), and CD59 on inhibitory activity. RESULTS: Both aqueous humor and vitreous inhibited the activity of the classical pathway (CH(50)). Microcentrifugation revealed the major inhibitory activity resided in the fraction with an M(r) >/= 3 kDa. Chromatography on an S-100-HR column demonstrated that the most potent inhibition was associated with the high-molecular-weight fractions (>/=19.5 kDa). In contrast to unfractionated aqueous and vitreous, fractions with an M(r) >/= 3 kDa also had an inhibitory effect on the alternative pathway activity (AH(50)). The complement regulatory activity in normal human intraocular fluid was partially blocked by monoclonal antibodies against MCP, DAF, and CD59. Immunoblot analysis confirmed the presence of these three molecules in normal intraocular fluid. CONCLUSIONS: Our results demonstrate that normal human intraocular fluid (aqueous humor and vitreous) contains complement inhibitory factors. Furthermore, the high-molecular-weight factors appear to be the soluble forms of MCP, DAF, and CD59.

Induction of experimental autoimmune anterior uveitis by a self-antigen: melanin complex without adjuvant.

PURPOSE: Experimental autoimmune anterior uveitis (EAAU) is an organ-specific autoimmune disease induced by immunization with bovine melanin-associated antigen (MAA) and two adjuvants (complete Freund's adjuvant and purified pertussis toxin). This study was undertaken to explore whether an adjuvant is required in the induction of EAAU. METHODS: Insoluble MAA was extracted from the bovine iris and ciliary body. Soluble bovine MAA was derived by treatment of insoluble MAA with the proteolytic enzyme, V8 protease. Lewis rats were immunized with the insoluble or soluble antigen, with or without adjuvant (complete Freund's adjuvant and purified pertussis toxin). Adoptive transfer of CD4+ and CD8+ T cells was performed to investigate the pathogenesis of EAAU. RESULTS: Experimental autoimmune anterior uveitis can be induced in Lewis rats by immunization with 100 g insoluble bovine MAA alone without the use of adjuvants. The disease can be adoptively transferred to naive syngenic rats by primed CD4+ T cells. In contrast, soluble bovine MAA was not uveitogenic unless adjuvants were employed. CONCLUSIONS: The data suggest that EAAU can be induced in the Lewis rat without addition of an adjuvant. Future studies concerning the pathogenesis of EAAU can now be performed without the possible confounding effect of an adjuvant.

Intraocular viral replication after systemic murine cytomegalovirus infection requires immunosuppression.

PURPOSE: Human cytomegalovirus retinitis is the most common blinding complication of acquired immune deficiency syndrome. However, the pathogenesis of the disease is poorly understood. The authors sought to characterize intraocular viral replication after systemic murine cytomegalovirus (MCMV) infection in the normal and immunosuppressed Balb/c mouse. METHODS: Normal or immunosuppressed mice (400 rads radiation plus antilymphocyte serum) were infected intravenously with a recombinant MCMV (RM408) that carries an MCMV IE1 promoter--LacZ insert. In vivo MCMV replication and its tissue distribution were monitored by beta-gal activity with x-gal staining on frozen tissue sections of multiple organs harvested from infected mice at different time points after inoculation. RESULTS: MCMV replication within the eye can be detected in the immunosuppressed Balb/c mouse but not in the normal host. Intraocular viral replication was noted first, and most frequently, in the ciliary body and was mainly restricted to the uveal tract. Intraocular viral replication coincided with the peak of systemic viral replication; however, the neurosensory retina was spared. In contrast, supraciliary inoculation of MCMV in the immunosuppressed Balb/c mouse resulted in massive viral replication and destruction of the neurosensory retina. CONCLUSIONS: This study demonstrated that intraocular MCMV replication after systemic infection requires systemic immunosuppression. Furthermore, the ciliary body is the portal of entry for the virus within the eye. MCMV can replicate in the epithelium of the uvea and retinal pigment epithelium, but it does not replicate within the neurosensory retina. The absence of MCMV replication within the neurosensory retina is not caused by either a defect in the recombinant virus or the inability of the host tissue to support viral replication.

Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein.

PURPOSE: Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation. The current studies were undertaken to determine the primary structure of this protein. METHODS: A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library. The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced. Southern blot analysis was used to study the genomic hybridization pattern. p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells. RESULTS: The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively. The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100. Southern blot analysis of human genomic DNA revealed a simple hybridization pattern. Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein. CONCLUSIONS: The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library. Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein. Thus, we refer to this protein as nup36.

Development of astrocytes and their relation to blood vessels in fetal monkey retina.

PURPOSE: To determine the development of astrocytes and their vascular relations in Macaca monkey retina. METHODS: Sections and wholemounts of retinas from fetal day (Fd) 65 to adult animals were analyzed immunohistochemically to detect glial fibrillary acidic protein (GFAP) and vimentin. RESULTS: Astrocytes appeared first near the optic disc, then subsequently further peripherally, but avoided the fovea. In the nerve fiber layer, round and ovoid cells extended processes parallel to ganglion cell axons. In the ganglion cell layer, ovoid and stellate cells exhibited anisotropic processes or a honeycomb network. The inner lamina of astrocytes developed ahead of the outer lamina, and both reached their final positions before birth. Astrocytes lay more peripherally than did developing blood vessels, and the growing edge of nerve fiber layer vessels lay between the two astrocytic layers. Spindle cells, which may be vascular precursor cells, often aligned along linear astrocytic processes. Occasional spindle-shaped cells containing GFAP or vimentin were identified as immature glia. Astrocytes and blood vessels coincided regionally during development, but astrocyte processes were typically not in register with the meshwork of growing blood vessels. Astrocyte-vessel associations increased during fetal life and postnatally. CONCLUSIONS: During development, astrocytes display the same bilaminar pattern and morphologies present in adult retina. Astrocytes and blood vessels exhibit a similar regional distribution, but develop in distinct spatial patterns. Vessel investment by astrocytic processes increases during fetal life but is variable at all ages.

Requirement of B7-mediated costimulation in the induction of experimental autoimmune anterior uveitis.

PURPOSE: To study the role of costimulatory signaling through the CD28-B7 interaction in experimental autoimmune anterior uveitis (EAAU). METHODS: Naive Lewis rats were immunized with insoluble melanin-associated antigen (MAA) derived from bovine iris and ciliary body. CTLA4-Fc, a recombinant protein comprised of the extracellular domain of human CTLA4 bound to mouse IgG2a Fc, was used to block the CD28-B7 interaction. A mutant version (CTLA4-Fc-mutant) was used as a control. The effect of CTLA4-Fc on the in vivo induction of disease with MAA was studied. Subsequently, the mechanism by which CTLA4-Fc blocked the interaction of CD28 and B7 was investigated in vivo, using the adoptive transfer of T cells derived from CTLA4-Fc-treated rats, and in vitro, using the proliferative response and cytokine production of MAA-T cells in the presence of CTLA4-Fc. RESULTS: CTLA4-Fc markedly reduced the incidence and severity of EAAU in Lewis rats after sensitization with MAA. The adoptive transfer of sensitized T cells from CTLA4-Fc-treated donors did not induce EAAU in naive recipients. CTLA4-Fc inhibited the expansion of antigen-specific MAA-T cells and the production of TNF-alpha. CONCLUSIONS: The costimulatory signal delivered through CD28-B7 is required for the induction and pathogenesis of EAAU. In the absence of this signal, antigen-specific expansion of MAA reactive T cells as well as production of TNF-alpha is inhibited. Abrogation of this costimulatory signal may be an important therapeutic option for EAAU.

Endotoxin-induced uveitis in the rat is attenuated by inhibition of nitric oxide production.

PURPOSE: These experiments were undertaken to assess the role of increased nitric oxide production in the pathogenesis of vascular dysfunction associated with endotoxin-induced uveitis. METHODS: Lipopolysaccharides (LPS) (100 micrograms of Salmonella minnesota) was injected into foot-pads of Lewis rats randomly assigned to an untreated group or to a group treated with subcutaneous injections of aminoguanidine, a selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS). Controls included untreated and aminoguanidine-treated rats. Twenty to 24 hours later, blood flow and vascular 125I-albumin permeation were quantified in ocular tissues. Eyes were graded histologically for leukocyte infiltration into the anterior uvea and anterior chamber, and leukocyte counts were performed on aqueous fluid. Plasma nitrate levels were measured fluorometrically after enzymatic reduction to nitrite. RESULTS: Lipopolysaccharides markedly increased plasma nitrate levels and 125I-albumin permeation in aqueous fluid, retina, anterior uvea, and choroid-sclera. Blood flow was increased only in the anterior uvea. Aminoguanidine normalized plasma nitrate levels and prevented or significantly ameliorated the 125I-albumin permeation and blood flow changes in ocular tissues. The increased aqueous fluid content of lymphocytes and neutrophils in LPS-treated rats, as well as the increased histologic score of iritis, were significantly reduced by aminoguanidine. CONCLUSIONS: These results suggest that the hemodynamic and vascular permeability changes associated with endotoxin-induced uveitis are mediated in large part by increased production of nitric oxide.

Experimental autoimmune anterior uveitis. Induction with melanin-associated antigen from the iris and ciliary body.

PURPOSE: This study was designed to investigate an animal model of uveitis that resembles anterior uveitis in humans after immunization with iris-ciliary body antigen. METHODS: Male Lewis rats 6 to 8 weeks of age were immunized with the buffer- and detergent-insoluble bovine iris-ciliary body antigen mixed with complete Freund's adjuvant and pertussis toxin. Antigen was digested with various proteolytic enzymes and tested in different rodent strains for a uveitogenic response. RESULTS: Acute iridocyclitis developed in both eyes of the Lewis rat during the second week after immunization, and the pattern of inflammation was similar to acute anterior uveitis in humans, with sudden onset, localization to the anterior uvea, and spontaneous resolution. Among the strains tested, F344 rats were susceptible to experimental autoimmune anterior uveitis but Long-Evans rats were not. Experimental autoimmune anterior uveitis did not develop in any of the mice studied, nor was it induced by immunization with synthetic melanin, amelanotic bovine tissues, pigmented bovine skin, or pigmented rat and rabbit iris-ciliary body. A soluble fraction derived from bovine melanin-associated antigen (BMAA) after digestion with the proteolytic enzyme V8 protease resulted in a disease similar to that observed with intact BMAA. CONCLUSIONS: A model of anterior uveitis has been induced in the Lewis rat after immunization with bovine uveal antigen, and it resembles the acute iridocyclitis observed in humans. These results suggest that the pathogenic antigen is a melanin-associated protein(s) present within the iris-ciliary body.