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.

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.

Immunohistochemical studies on melanin associated antigen (MAA) induced experimental autoimmune anterior uveitis (EAAU).

Experimental autoimmune anterior uveitis (EAAU), a model of uveitis induced by sensitization to melanin associated antigen (MAA) derived from the iris and ciliary body, closely resembles human acute anterior uveitis. The immunopathogenesis of EAAU was studied by immunohistochemical detection of immune cells and the expression of Ia, ICAM-1 and LFA-1 antigens. Male Lewis rats were immunized with bovine MAA, mixed with CFA and pertussis toxin in the hind foot pad. Animals were examined daily by slit-lamp biomicroscopy and serially sacrificed up to 30 days. Immunohistology of the enucleated eyes was performed with monoclonal antibodies W3/25 (CD4), OX-8 (CD8), ED2 (macrophage), OX-33 (B cell), OX-6 (Ia), IA29 (ICAM-1) and WT.1 (LFA-1). During each stage of EAAU, CD4+ T cells predominated over both CD8+ T cells and macrophages in the uvea. Very few B cells were detected during each stage of EAAU. EAAU could not be induced by the adoptive transfer of sera obtained from immunized animals. Low levels of constitutive ICAM-1 and Ia were observed. An increase in ICAM-1 expression was first noted on the epithelial cells of the uveal tract and RPE on day 9 post immunization and preceded LFA-1 and Ia upregulation by approximately 2 days. The immunopathogenesis of EAAU appears to be linked to the presence of the CD4+ T cells.

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.