Nitrated Serum Proteins in the Aqueous Humor of Experimental Autoimmune Uveitis and Endotoxin Induced Uveitis

Abstract

Abstract: : Purpose: We have previously demonstrated in-vitro chemotaxis and superoxide production in neutrophils stimulated with nitrated bovine serum albumin generated by reaction with SIN-1, a peroxynitrite donor. Chemotactic factors from the aqueous humor in rabbits with endotoxin-induced uveitis (EIU) have been found in fractions that co-eluted with serum albumin. In this study we investigated the aqueous humor of experimental autoimmune uveitis (EAU) rats and aqueous humor of EIU rabbits for presence of nitrated proteins. Methods: EAU was induced in 9 male Lewis rats by footpad injection of 100 µg of human S-Ag peptide with complete Freund’s adjuvant and intravenous injection of 1µg pertussis toxin in 300 µl saline. Aqueous humor was collected on day 15 post immunization. Eight non-immunized Lewis rats were used as controls. Three female New Zeland white rabbits were injected with lipopolysaccharide (E.coli; serotype 055:B5) in 50 µl normal saline intravitreally in the right eye. Aqueous humor was collected at 24 hours post-injection and nitrated proteins were probed with anti-nitroTyr antibody in western blot and confirmed by liquid chromatography/mass spectometry. Results: A major band of nitrated protein at 68 kDa was seen corresponding to albumin. The EIU rabbit eyes demonstrated a major band of nitrated protein at 50 kDa corresponding to heavy chain IgG. Conclusions: This study demonstrates the presence of nitrated serum proteins in the aqueous humor of EAU as well as EIU. Such nitrated protein may contribute to the recruitment of leukocytes and amplification of the intraocular inflammation.

Keywords: inflammation • uveitis-clinical/animal model • nitric oxide