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Interferon‐γ regulates chemokine expression and release in the human mast cell line HMC1: role of nitric oxide
Author(s) -
Gilchrist M.,
Befus A. D.
Publication year - 2008
Publication title -
immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.297
H-Index - 133
eISSN - 1365-2567
pISSN - 0019-2805
DOI - 10.1111/j.1365-2567.2007.02688.x
Subject(s) - nitric oxide synthase , chemokine , nitric oxide , interferon gamma , western blot , mast cell , cytokine , chemistry , microbiology and biotechnology , omega n methylarginine , immune system , biology , immunology , endocrinology , biochemistry , gene
Summary Mast cells (MCs) are critical immune effector cells that release cytokines and chemokines involved in both homeostasis and disease. Interferon‐γ (IFN‐γ) is a pleiotropic cytokine that regulates multiple cellular activities. IFN‐γ 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‐γ 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‐γ inhibited phorbol 12‐myristate 13‐acetate (PMA)‐induced release of IL‐8 and CCL1 (by 47 and 38%). Real‐time PCR analysis of IFN‐γ‐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‐γ‐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‐γ inhibitory effect on IL‐8 and CCL1 mRNA expression. NO donors mimicked the IFN‐γ effect. IFN‐γ inhibited PMA‐induced cAMP response element binding protein (CREB) phosphorylation and DNA‐binding activity. Our observations indicate for the first time that IFN‐γ 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.