Tim-4 Inhibits NO Generation by Murine Macrophages

Objective T cell immunoglobulin-and mucin-domain-containing molecule-4 (Tim-4) receives much attention as a potentially negative regulator of immune responses. However, its modulation on macrophages has not been fully elucidated so far. This study aimed to identify the role of Tim-4 in nitric oxide (NO) modulation. Methods Macrophages were stimulated with 100 ng/ml LPS or 100 U/ml IFN-gamma. RT-PCR was performed to detect TIM-4 mRNA expression. Tim-4 blocking antibody and NF-kappa B inhibitory ligand were involved in the study. NO levels were assayed by Griess reaction. Phosphorylation of NF-kappa B, Jak2 or Stat1 was verified by western blot. Results Tim-4 was up-regulated in murine macrophages after interferon-gamma (IFN-gamma) stimulation. Tim-4 over-expression decreased NO production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS) or IFN-gamma-stimulated macrophages. Consistently, Tim-4 blockade promoted LPS or IFN-gamma-induced NO secretion and iNOS expression. Tim-4 over-expression decreased LPS-induced nuclear factor kappa B (NF-kappa B) p65 phosphorylation in macrophages, which was abrogated by NF-kappa B inhibitory ligand. On the contrary, Tim-4 blocking increased LPS-induced NF-kappa B signaling, which was also abrogated by NF-kappa B inhibition. In addition, Tim-4 blockade promoted Jak2 and Stat1 phosphorylation in IFN-gamma stimulated macrophages. Conclusion These results indicate that Tim-4 is involved in negative regulation of NO production in macrophages, suggesting the critical role of Tim-4 in immune related diseases.