Involvement of superoxide generated by polymorphonuclear leukocytes in endotoxin-induced uveitis

Background: Although superoxide is thought to be involved in the development of endotoxin-induced uveitis (EIU), the role of superoxide generation by polymorphonuclear leukocytes (PMNs) has not been fully elucidated. The purpose of this study was to investigate the role of peripheral blood PMNs in the development of EIU. Methods: EIU was induced in Lewis rats by injection of lipopolysaccharide (LPS) in one hind footpad. Superoxide generation was assayed by measuring the reduction of ferricytochrome c (cyt c). EIU severity was assessed by histological examination, and the relationship between the injected dose of LPS in vivo and the intensity of superoxide generation by peripheral PMNs or intraocular PMNs was studied. Twenty-four hours after the injection of LPS (2, 20, or 200 µg/rat), peripheral blood PMNs were collected and stimulated with phorbol 12-myristate 13-acetate (PMA). The time course of superoxide generation by PMNs after LPS injection (3, 6, 12, 24, 48, and 72 h) was also investigated. To test the possible inhibition of superoxide generation by protein kinase C (PKC) inhibitors, H-7 and staurosporine were added for the incubation. In addition to the measurement of cyt c reduction, western blotting was used to detect PKC activity. The direct effect of LPS on PMNs was tested by priming naive PMNs with LPS in vitro. Results: The intensity of superoxide generation by PMNs and the severity of EIU were dependent on the dose of injected LPS. No apparent superoxide generation was detected from intraocular PMNs. The time course of superoxide generation was similar to that of EIU severity. H-7 or staurosporine inhibited superoxide generation dose dependently and suppressed phosphorylation of PKC. Priming with LPS in vitro prompted minimal superoxide generation by naive PMNs. Conclusion: Superoxide generation by peripheral blood PMNs but not by intraocular PMNs from rats with EIU was demonstrated, and it is suggested that superoxide generation by PKC cascade might be involved in the pathogenesis of EIU.