3B). Eosinophils CX-4945 solubility dmso were abundant in areas juxtaposed to lesions in IL-10 KO animals; however, they were absent in IL-10 KO/PHIL mice, demonstrating that eosinophils were not critical in the development of hepatic necrosis. Results of ALT activity assays and hepatic leukocyte counts corroborated this interpretation (Fig. 3C,D). During infection, neutrophils were significantly increased in the livers of IL-10 KO animals in comparison with WT mice, with peak numbers (day 10) occurring just prior to the time of maximal lesion size (days 12-14; Fig. 4A). Numbers remained low in both WT and IL-4 KO mice, whereas those in IL-10/IL-4 KO animals initially rose but then fell, never achieving

the values observed in IL-10 KO mice. This was confirmed by flow cytometric analysis of hepatic leukocytes (data not shown). Additionally, the prevalence of neutrophils in the liver was suppressed by IL-10. For example, neutrophils represented 14% ± 1.7% of total leukocytes in IL-10 KO livers on day 12 versus 9% ± 1% in WT animals (P < 0.05). Loss of endogenous IL-4 decreased the prevalence to 2.7% ± 1.5% in IL-10/IL-4 KO mice. Activated neutrophils can release cytotoxic mediators, suggesting their potential participation in lesion development. We used expression

of CD11b and CD62L to determine if IL-10 and IL-4 influenced RG7204 supplier the neutrophil activation state in the liver. Infection resulted in a significant increase in the number of Ly6-G+F4/80− cells (markers of neutrophils) with an activated CD11b+CD62Llo phenotype in WT and IL-10 KO mice in comparison with naive animals (Fig. 4B). Upon infection, only the number of activated neutrophils in IL-10 KO mice differed significantly from that in WT animals. Taken together, the data suggested that IL-10 and IL-4 may have differential effects on neutrophil trafficking and activation state. To determine whether neutrophils

played a role in initial hepatocyte injury and/or subsequent development of hepatic necrosis, see more we depleted mice of neutrophils with one of two monoclonal antibodies. Figure 5A shows the effect of antibody administration on peripheral neutrophils. Both depleting antibodies reduced the prevalence of neutrophils to less than 2%. Control antibody-treated and neutropenic IL-10 KO mice had greater ALT values and hepatic leukocyte numbers than WT mice after infection (Fig. 5C,D). However, only control antibody-treated IL-10 KO mice developed hepatic necrosis (Fig. 5B). The number of CD4+α4β7+ cells was significantly increased in both control and depleted IL-10 KO mice in comparison with WT mice, corresponding to the elevated serum ALT activity in these animals (Fig. 5C,D). Thus, in the absence of IL-10 and in the presence of IL-4, neutrophils were necessary for the development of hepatic necrosis but were not required for the initiation of hepatocyte injury.