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 i nflammatory demyelinating disorder of the central nervous system (CNS) which serves as a prime animal model for the human disease multiple s clerosis. Previous studies from these laboratories demonstrated excess nitric oxide (NO) in the CNS of EAE-affected mice, and amelioration o f EAE with a selective inhibitor of the inducible nitric oxide synthas e (iNOS). Recent studies from other laboratories have indicated that p rostaglandin PGE(2) is increased in CNS tissues of EAE-affected rodent s and that EAE is prevented by the inhibition of cyclooxygenase activi ty. The present study investigated the ability of encephalitogenic lym phoid cells to induce NOS and cyclooxygenase (COX-2) in the murine mac rophage line, RAW 264.7. In order to mimic the extracellular milieu pr esent in EAE lesions, conditioned medium (CM) of activated EAE-inducer cells was added to this macrophage line. CM caused a time-dependent i ncrease in nitrite, indicating NO production. Reverse-transcriptase PC R demonstrated iNOS mRNA in RAW 264.7 cells, first detected at 3 h, an d Western blots confirmed the induction in RAW cells of the 130-kDa iN OS protein. Production of nitrite by CM-exposed RAW 264.7 cells was bl ocked by inhibitors of NOS (L-N-methylarginine or aminoguanidine) or b y antibodies to murine IFN-gamma or IL-1 beta. CM of activated encepha litogenic cells induced production of PGE(2) by RAW 264.7 cells, as de termined by ELISA, and Western blots identified the presence of the 70 -80-kDa inducible COX (COX-2) protein. Induction of COX-2 could be inh ibited by antibody to IFN-gamma Thus, encephalitogenic cells are capab le of inducing the expression of the inflammatory enzymes iNOS and COX -2 in a murine macrophage line via the T cell cytokine IFN-gamma, alon e or in combination with IL-1 beta.