Mediation of inflammation by encephalitogenic cells: interferon gamma induction of nitric oxide synthase and cyclooxygenase 2.
Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory demyelinating disorder of the central nervous system (CNS) which serves as a prime animal model for the human disease multiple sclerosis. Previous studies from these laboratories demonstrated excess nitric oxide (NO) in the CNS of EAE-affected mice, and amelioration of EAE with a selective inhibitor of the inducible nitric oxide synthase (iNOS). Recent studies from other laboratories have indicated that prostaglandin PGE2 is increased in CNS tissues of EAE-affected rodents and that EAE is prevented by the inhibition of cyclooxygenase activity. The present study investigated the ability of encephalitogenic lymphoid cells to induce NOS and cyclooxygenase (COX-2) in the murine macrophage line, RAW 264.7. In order to mimic the extracellular milieu present in EAE lesions, conditioned medium (CM) of activated EAE-inducer cells was added to this macrophage line. CM caused a time-dependent increase in nitrite, indicating NO production. Reverse-transcriptase PCR demonstrated iNOS mRNA in RAW 264.7 cells, first detected at 3 h, and Western blots confirmed the induction in RAW cells of the 130-kDa iNOS protein. Production of nitrite by CM-exposed RAW 264.7 cells was blocked by inhibitors of NOS (L-N-methylarginine or aminoguanidine) or by antibodies to murine IFN-gamma or IL-1 beta. CM of activated encephalitogenic cells induced production of PGE2 by RAW 264.7 cells, as determined by ELISA, and Western blots identified the presence of the 70-80-kDa inducible COX (COX-2) protein. Induction of COX-2 could be inhibited by antibody to IFN-gamma. Thus, encephalitogenic cells are capable of inducing the expression of the inflammatory enzymes iNOS and COX-2 in a murine macrophage line via the T cell cytokine IFN-gamma, alone or in combination with IL-1 beta.
Misko
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Inflammatory Diseases Research Group, G.D. Searle R & D, St. Louis, MO 63167, USA.
Trotter
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JL
Cross
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AH
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000201:enzymology*; D004790:Enzyme Induction; D005260:Female; D007249:Inflammation / Q000503:physiopathology*; D007371:Interferon-gamma / Q000502:physiology*; D008262:Macrophage Activation; D008264:Macrophages / Q000502:physiology; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis*
D017931:DNA Primers; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D011451:Prostaglandin-Endoperoxide Synthases