title: Tim-4 Inhibits NO Generation by Murine Macrophages.
abstract: 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-γ. RT-PCR was performed to detect TIM-4 mRNA expression. Tim-4 blocking antibody and NF-κB inhibitory ligand were involved in the study. NO levels were assayed by Griess reaction. Phosphorylation of NF-κB, Jak2 or Stat1 was verified by western blot.
  
  
  RESULTS
  Tim-4 was up-regulated in murine macrophages after interferon-gamma (IFN-γ) stimulation. Tim-4 over-expression decreased NO production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS) or IFN-γ-stimulated macrophages. Consistently, Tim-4 blockade promoted LPS or IFN-γ-induced NO secretion and iNOS expression. Tim-4 over-expression decreased LPS-induced nuclear factor kappa B (NF-κB) p65 phosphorylation in macrophages, which was abrogated by NF-κB inhibitory ligand. On the contrary, Tim-4 blocking increased LPS-induced NF-κB signaling, which was also abrogated by NF-κB inhibition. In addition, Tim-4 blockade promoted Jak2 and Stat1 phosphorylation in IFN-γ stimulated macrophages.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China; Cell and Molecular Biology Laboratory, Zhoushan Hospital, 739 Dingshen Road, Zhoushan, Zhejiang, 316000, P.R. China.
authors.forename: Li-yun
authors.initials: LY
authors.lastname: Xu
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China.
authors.forename: Jian-ni
authors.initials: JN
authors.lastname: Qi
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China.
authors.forename: Hong-xin
authors.initials: HX
authors.lastname: Ma
authors.affiliation: Department of bone surgery, Zhangqiu Hospital, 1920 Huiquan Road, Jinan, Shandong, 250200, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yuan
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China.
authors.forename: Pei-qing
authors.initials: PQ
authors.lastname: Zhao
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China.
authors.forename: Xiao-hong
authors.initials: XH
authors.lastname: Liang
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China.
authors.forename: Hong-xing
authors.initials: HX
authors.lastname: Wang
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China.
authors.forename: Xiao-yan
authors.initials: XY
authors.lastname: Xu
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China.
authors.forename: Chun-hong
authors.initials: CH
authors.lastname: Ma
authors.affiliation: Department of Immunology, Key Laboratory for Experimental Teratology of Ministry of Education, Shandong Provincial Key Laboratory of Infection & Immunology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, 250012, P.R. China.
authors.forename: Li-fen
authors.initials: LF
authors.lastname: Gao
mesh_terms: D000818:Animals; D002460:Cell Line; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D008070:Lipopolysaccharides; D008565:Membrane Proteins; D012333:RNA, Messenger; C500399:TIM-4 protein, mouse; D009569:Nitric Oxide; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II