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G-S Wu, TD Lee, NA Rao; Stimulation Of Neutrophils By Nitrated Bovine Serum Albumin: Production Of Superoxide And Chemotaxis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3613.
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Purpose: We previously demonstrated that in EAU, peroxynitrite-mediated tyrosine (Tyr) nitration of retinal proteins were mainly localized in photoreceptors and in retinal blood vessels (IOVS 1997;38:1333-9). Although Tyr nitration was found to alter protein function, the function of nitrated proteins as signaling molecules in inflammation has not been elucidated. In this study, we investigated the role(s) of nitrated bovine serum albumin (nitroBSA) as an inflammatory mediator. Methods:BSA was incubated with peroxynitrite donor, 3-morpholinosydnonimine (SIN-1; 6 mM), for 5h at 37ºC and dialyzed to remove SIN-1. Nitration was assessed by absorption at 354 nm (pH 7) and 425 nm (pH 11, ϵmax 4400/M/cm), by Western blot probed with anti-nitroTyr antibody and by mass spectrometry to define the site of nitration. Superoxide (O2-) production in rabbit peritoneal PMNs was assayed by SOD-inhibitable cytochrome C reduction. Chemotaxis of PMNs was assessed by 48-well microchemotaxis chamber. Cells migrated through polycarbonate filter were stained and counted per high power field. Results:SIN-1 nitration produced 5.9 µM nitroBSA in the sample and displayed an intense nitroTyr band in the Western blot. Tyr179 and Tyr424 are the primary nitration sites. In 1h, 0.97 µM nitroBSA-activated PMNs produced 4.53 0.51 nmoles O2-, compared with 0.92 0.079 nmoles produced by the BSA itself. At the same time, 0.5 µM N-formyl-methionyl-leucyl-phenylalanine (fMLP) produced 12.64 1.38 nmoles O2-. NitroBSA (2.2 µM) was chemotactic to PMNs, with 233 27.8 migrated cells/field compared 78 6.8 cells with BSA (2.2 µM). Positive control, 0.1 µM fMLP gave 273 32.5 cells/field. Conclusion: Nitrated proteins formed in the inflamed retina are capable of recruiting PMNs to inflammatory sites and can further enhance O2-production. Peroxynitrite derived from O2- and NO combination is a potent biological oxidant. Therefore, the secondary functions of these nitrated proteins might represent a major mechanism by which further degeneration of retina occurs in EAU.
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