Abstract
Abstract: :
Purpose: Recently it has been shown that a–, ß– and γ–crystallins are expressed in the retina. Functions of these crystallins in the retina remain largely unexplored. The α–crystallins (αA and αB), members of the small heat shock family of proteins have been shown to inhibit aggregation of denatured polypeptides (chaperone–like function); the ß/γ–crystallins share significant similarities with epidermis–specific differentiation proteins. Here we tested the hypothesis that crystallins may have role in the retina during the early phase of EAU (day 7) when the retina is under immune mediated oxidative stress. Methods: Three groups of twelve B10R111 mice were used. The first group was immunized with IRBP (interphotoreceptor retinol binding protein) and complete Freunds adjuvant; the second group was injected with the adjuvant only; and the third group served as non–experimental controls. The animals were sacrificed on day 7, the retinas were harvested for isolation of RNA, which was used to generate probes for microarray analyses (screening of 8800 genes, Affymetrix, Santa Clara, CA) and by real–time PCR for the confirmation of the changes in crystallin transcripts. Specific antibodies were used for immunolocalization by confocal microscopy and/or histology. Other tissues such as the brain, liver, and heart were similarly processed for comparative analyses. Results: The microarray analyses revealed a 10– to 15–fold upregulation of αA, ßA1, ßB2, and γS crystallin genes in the retinas of the animals immunized with IRBP. Such upregulation was not detected in the brain, liver or in the heart. Among the α–crystallins only αA showed increased expression while αB expression remained unchanged. γB and γC showed about eight and ten fold increase respectively; the rest of the γ–crystallins (γD, γ E and γF) varied between three – five fold increases. Histologic examination revealed that none of the animal eyes had photoreceptor damage. Conclusions: In the early phase of EAU, the selective upregulation of crystallins is observed in the retina. These changes in the gene expression of the crystallins are not seen in other tissues examined suggesting a retina–specific role for these genes in early manifestations of EAU. This data also points to independent regulation of the genes within a family of crystallin gene (e.g. αA vs αB). While the mechanism of the upregulation of these genes remains to be elucidated, a likely mediator may be the peroxynitrite that is generated in the EAU retinas.
Keywords: crystallins • gene microarray • uveitis-clinical/animal model