Investigation of the Pro–Inflammatory Function of Osteopontin in Experimental Autoimmune Uveitis

Abstract

Abstract: : Purpose: Osteopontin (OPN) is a multi–functional, matricellular protein with cytokine and chemoattractant activities and has been implicated in cell–mediated immune function. OPN has been localized to the retinal ganglion cell layer of embryonic mouse (Hikita et al., 2003) and adult rat retina (Ju et al., 2000). In Experimental Autoimmune Uveitis (EAU), OPN mRNA was significantly increased, although the retinal source of OPN was unidentified (Foxman et al., 2002). We have shown previously (ASCB, 2004, #B338) that OPN plays a pro–inflammatory role in EAU at 21 days post–IRBP immunization. OPN was expressed by both infiltrating leukocytes and activated microglia in EAU. OPN null mice (Rittling et al., 1998) had decreased numbers of vitreal infiltrates and retinal granulomas. They also showed an attenuated disease trend compared to wildtype mice. We confirm these results in a second OPN null strain (Liaw et al., 1998) and investigate the role of OPN in EAU at an earlier timepoint to assess whether absence of OPN delays disease onset. Methods: Two independently generated OPN null mice strains (Rittling et al., 1998, Liaw et al., 1998) and wildtype control mice were immunized with IRBP to assess differences in disease incidence, severity, and onset. Next, immunohistochemistry and histology experiments were performed to identify the cell type(s) expressing OPN protein in EAU and normal eyes. Vitreal infiltrates and granulomas were quantified. Finally, to determine whether OPN may induce an EAU–like condition, purified OPN proteins were injected intraocularly into wildtype mice and retinal sections were analyzed. Results: In both strains of OPN null mice, disease incidence was decreased and quantification of vitreal infiltrates and granulomas showed an attenuated disease trend. Disease onset did not appear to be affected by the absence of OPN. Infiltrating leukocytes and activated microglia expressed OPN. Intraocular injection of purified OPN proteins in wildtype mice induced retinal folding/detachment and leukocyte infiltration similar to EAU. Conclusions: We have demonstrated a pro–inflammatory function for OPN in EAU in two independently generated strains of OPN null mice. OPN is expressed in infiltrating leukocytes and activated microglia. Absence of OPN results in decreased vitreal infiltrates and granulomas. Exogenous OPN is sufficient to cause EAU – like disease. Our findings suggest that OPN may be involved in the recruitment and/or activation of infiltrating immune cells in EAU.

Keywords: inflammation • autoimmune disease • uveitis-clinical/animal model