Neutrophil Chemotaxis and Local Expression of Interleukin-10 in the Tolerance of Endotoxin-Induced Uveitis

Abstract

To study the mechanism of lipopolysaccharide (LPS) tolerance in a rat model of footpad injection endotoxin-induced uveitis (EIU). EIU was produced by footpad injection of 1 mg/kg LPS in male Sprague-Dawley rats. Four experiments were undertaken in this study. First, on days 3, 7, 14, 28, 56, and 84 after LPS injection, the iris-ciliary body (ICB) was isolated. LPS tolerance-associated gene expression in the ICB was determined by quantitative polymerase chain reaction. Second, the distribution of IL-10-producing cells in frozen sections of ocular tissues was analyzed by fluorescence and confocal microscopy. Third, peripheral blood neutrophil chemotaxis was determined using a fluorescent in vitro migration assay. Fourth, for in vivo neutrophil chemotaxis assay, neutrophils isolated from EIU-tolerant or control rats were transfused into green fluorescence protein (GFP) rats injected with LPS 18 hours earlier. Six hours after transfusion, the percentage of GFP-negative neutrophils in the aqueous humor was determined by flow cytometry. IL-10 gene expression in ICB was significantly upregulated for at least 1 month. Immunohistochemical examination indicated that dendritic cells in the ICB produced IL-10. Peripheral blood neutrophil chemotaxis in EIU-tolerant rats was inhibited significantly in vitro and in vivo. IL-10 enhanced the reduction of neutrophil chemotaxis in EIU-tolerant rats in vitro. The results suggest that continuous high expression of IL-10 in the eye and the reduction of peripheral blood neutrophil chemotaxis play significant roles in the mechanism of LPS tolerance in a rat model of footpad injection EIU.