Interferon-gamma regulates chemokine expression and release in the human mast cell line HMC1: role of nitric oxide.
Mast cells (MCs) are critical immune effector cells that release cytokines and chemokines involved in both homeostasis and disease. Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that regulates multiple cellular activities. IFN-gamma modulates rodent MC responsiveness via production of nitric oxide (NO), although the effects in human MC populations is unknown. We sought to investigate the effects of IFN-gamma on expression of the chemokines interleukin-8 (IL-8) and CCL1 (I-309) in a human mast cell line (HMC1) and to determine the underlying regulatory mechanism. Nitric oxide synthase (NOS), IL-8 and CCL1 expression was determined using real-time polymerase chain reaction (PCR). NOS protein expression was analysed using western blot. NOS activity was determined using the citrulline assay. IL-8 and CCL1 release was measured by specific enzyme-linked immunosorbent assay (ELISA). IFN-gamma inhibited phorbol 12-myristate 13-acetate (PMA)-induced release of IL-8 and CCL1 (by 47 and 38%). Real-time PCR analysis of IFN-gamma-treated HMC1 showed a significant (P < 0.05) time-dependent increase in NOS1 and NOS3 mRNA. NOS3 protein was significantly increased at 18 hr, which correlated with a significant (P < 0.05) increase in constitutive NOS (cNOS) activity. IFN-gamma-induced inhibition of chemokine expression and release was NO dependent, as treatment with the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) reduced the IFN-gamma inhibitory effect on IL-8 and CCL1 mRNA expression. NO donors mimicked the IFN-gamma effect. IFN-gamma inhibited PMA-induced cAMP response element binding protein (CREB) phosphorylation and DNA-binding activity. Our observations indicate for the first time that IFN-gamma enhances endogenous NO formation through NOS3 activity, and that NO regulates the transcription and release of IL-8 and CCL1 in a human MC line.
Glaxo-Heritage Asthma Research Laboratory, Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. mgilchrist@systemsbiology.org
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D002460:Cell Line; D054402:Chemokine CCL1 / Q000096:biosynthesis / Q000235:genetics; D018925:Chemokines / Q000378:metabolism*; D004306:Dose-Response Relationship, Immunologic; D005786:Gene Expression Regulation / Q000276:immunology; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology*; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics; D008407:Mast Cells / Q000276:immunology*; D009569:Nitric Oxide / Q000096:biosynthesis / Q000276:immunology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction / Q000276:immunology; D014157:Transcription Factors / Q000378:metabolism
C065971:CCL1 protein, human; D054402:Chemokine CCL1; D018925:Chemokines; D016209:Interleukin-8; D012333:RNA, Messenger; D014157:Transcription Factors; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase