3C,D). Two of the major chemokines known to attract eosinophils are CCL11 and CCL24, which bind to the C-C-chemokine receptor-3 (CCR3) expressed on the surface of mature eosinophils.12 Expression of CCL11 in liver homogenates from mice treated with halothane was induced 9-fold, 15-fold, and 7-fold at 12, 18, and 24 hours, respectively, relative to the hepatic mRNA of vehicle-treated animals (Fig. 4A). The rise in the hepatic mRNA of CCL24 following halothane-treatment relative to that of vehicle-treated mice occurred only at 12 hours posttreatment (Fig. 4A). We compared the mRNA expression of CCL11 and CCL24 in total liver homogenates with that of levels in isolated hepatic leukocytes at 18 hours after halothane
treatment. CCL11 mRNA was enriched in the liver greater than 500-fold in relation to the expression in the mRNA isolated from the hepatic leukocytes (Fig. Selleckchem SB525334 4B). CCL24 mRNA expression was enriched 4.5-fold in liver homogenates compared to the mRNA expression in isolated hepatic leukocytes (Fig. 4B). These findings were not unexpected, Dabrafenib manufacturer as CCL11 and CCL24 are expressed
in mouse hepatocytes and liver sinusoidal endothelium.18 Moreover, halothane treatment also increased serum levels of CCL11 3-fold and 5.4-fold and CCL24 1.8-fold and 3.2-fold relative to the vehicle controls at 18 and 24 hours posttreatment, respectively (Fig. 4C). However not significant, the mean value of serum CCL11 was elevated at 12 hours after halothane treatment in relation to vehicle controls. Serum levels of CCL11 and CCL24 remained unchanged at all times following vehicle treatment in mice. To determine whether eosinophils played a pathogenic role in HILI, we repeated our toxicity study in mice that were depleted of eosinophils. The first
approach involved partially depleting eosinophils by pretreating Balb/cJ mice with Siglec-F monoclonal antibody prior to halothane administration. This antibody, which binds directly to the Siglec-F receptor, was reported to induce apoptosis of eosinophils resulting in ∼50% depletion in models TCL of eosinophilic inflammation.27 Mice pretreated with Siglec-F antibody had a significant reduction in the number of CD11c− CD11b+ Gr-1low Siglec-F+ eosinophils to 51% of those found in isotype-pretreated animals 24 hours after halothane treatment (Fig. 5A,B). In contrast to eosinophil depletion, anti-Siglec-F pretreatment did not significantly reduce the total number of hepatic leukocytes isolated 24 hours after administration of halothane, which are mainly comprised of neutrophils (Fig. 5B). Accordingly, the number of infiltrating neutrophils in the liver after halothane treatment did not change significantly (P = 0.11) between Siglec-F antibody- and isotype-pretreated animals (Fig. 5A,B). Depletion of eosinophils by anti-Siglec-F pretreatment resulted in a 49% decrease in serum ALT levels 24 hours after halothane treatment (Fig. 5C).