Interphotoreceptor retinoid-binding protein (IRBP) promotes the release of all-trans retinol from the isolated retina following rhodopsin bleaching illumination.

All-trans retinol generated in rod photoreceptors upon the bleaching of rhodopsin is known to move from the rods to the retinal pigment epithelium (RPE), where it is enzymatically converted to 11-cis retinal in the retinoid visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) contained in the extracellular compartment (interphotoreceptor matrix) that separates the retina and RPE has been hypothesized to facilitate this movement of all-trans retinol, but the precise role of IRBP in this process remains unclear. To examine the activity of IRBP in the release of all-trans retinol from the rods, initially dark-adapted isolated retinas obtained from toad (Bufo marinus) eyes were bleached and then incubated in darkness for defined periods (5-180 min) in physiological saline (Ringer solution) supplemented with IRBP (here termed 'IRBP I') at defined concentrations (2-90 microm). Retinoids present in the retina and extracellular medium were then determined by extraction and HPLC analysis. Preparations incubated with > or =10 microm IRBP I showed a pronounced release of all-trans retinol with increasing period of incubation. As determined with 25 microm IRBP I, the increase of all-trans retinol in the extracellular medium was accompanied by a significant decrease in the combined amount of all-trans retinal and all-trans retinol contained in the retina. This effect was not mimicked by unsupplemented Ringer solution or by Ringer solution containing 25 or 90 microm bovine serum albumin. However, incubation with 'IRBP II', a previously described variant of IRBP with altered lectin-binding properties, led to the appearance of substantial all-trans retinol in the extracellular medium. The results suggest that in vivo, IRBP plays a direct role in the release of all-trans retinol from the rods during operation of the visual cycle.

Interphotoreceptor retinoid-binding protein (IRBP)-deficient C57BL/6 mice have enhanced immunological and immunopathogenic responses to IRBP and an altered recognition of IRBP epitopes.

Experimental autoimmune uveitis (EAU) and pinealitis (EAP) can be induced in susceptible mice by immunization with immunologically privileged retinal antigens. In the present study, we analyzed the immunologic and immunopathologic responses of mice deficient in the retinal autoantigen interphotoreceptor retinoid-binding protein (IRBP). The consequences of IRBP deficiency on the T-cell repertoire were also investigated. IRBP+/+, IRBP+/- and IRBP-/- mice on the C57BL/6 background were immunized with IRBP or with a pathogenic epitope, IRBP(1-20) peptide in adjuvant, and were evaluated for disease severity and immunological responses. C57BL/6 IRBP-/- mice were completely resistant to EAU and EAP, and had enhanced immunological responses to IRBP and to its pathogenic peptide 1-20, as compared to their IRBP+/+ counterparts. IRBP-/- mice exhibited an altered IRBP epitope recognition. T cell epitope mapping revealed a response to IRBP peptide 271-290 in IRBP-/- mice, that was absent in the wild type. Primed T cells of IRBP-/- mice transferred an exacerbated form of EAU to nai;ve wild type recipients. A gene-dose effect was evident in that C57BL/6 IRBP+/- mice, exhibited intermediate immunological responses and lower disease scores compared to wild type. We conclude that expression of IRBP in target tissues is a necessary prerequisite for disease induction, excluding other retinoid-binding or vision-related proteins as surrogate targets. Furthermore, endogenous expression of IRBP is directly responsible for lowering the threshold of susceptibility to uveitic disease.

Chemokine gene expression in iris-ciliary body during experimental autoimmune uveoretinitis.

PURPOSE: To evaluate, through differential gene expression of chemokines in iris-ciliary body (I/CB), the extent to which leukocyte recruitment to the ocular anterior segment participates in the pathogenesis of experimental autoimmune uveoretinitis (EAU) in mice. METHODS: B10.A mice were immunized with 50 microg of interphotoreceptor retinoid binding protein (IRBP) mixed with complete Freund's adjuvant. Aqueous humor (AqH) was collected at 0, 11, 17, and 28 days and assayed for leukocyte content and protein levels. Enucleated eyes were subjected to histologic analysis. Chemokine gene expression in I/CB was determined at these same time points by a multiprobe ribonuclease protection assay (RPA) system. RESULTS: Inflammation was detected in the anterior chamber (AC) at 11 days and leukocyte recruitment continued thereafter. Polymorphonuclear neutrophils (PMN) were the predominant cell type in the AC, whereas macrophages/monocytes and lymphocytes were predominant in the retina/subretinal space at 17 days. Peak gene expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and MIP-2 and interferon-gamma-inducible protein of 10 kd (IP-10) was detected in I/CB on day 11, whereas peak expression of RANTES and eotaxin was observed at 17 days. CONCLUSIONS: Early I/CB peak expression of mRNA for MIP-2 followed by PMN recruitment into the AC, suggests that PMN may play an important role in EAU pathogenesis.