ROLE OF GLUTATHIONE IN MACROPHAGE ACTIVATION - EFFECT OF CELLULAR GLUTATHIONE DEPLETION ON NITRITE PRODUCTION AND LEISHMANICIDAL ACTIVITY

We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gam ma + LPS, as measured by nitrite production and leishmanicidal activit y. Diethylmaleate (DEM), which depletes intracellular GSH by conjugati on via a reaction catalyzed by the GSH-S-transferase, strongly inhibit ed nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progre ssively lost when addition of DEM was delayed up to 10 hr. A close cor relation was observed between levels of intracellular soluble GSH duri ng activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfo ximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetas e, also inhibited macrophage activation, although to a lesser extent t han DEM despite a more pronounced soluble GSH depletion. This inhibiti on was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O-2(-) production by activat ed macrophages, suggesting that the inhibitory effects observed on nit rite secretion and leishmanicidal activity were not related to a gener al impairment of macrophage function. DEM and BSO treatment reduced iN OS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect, Although commo nly used as a GSH-depleting agent, DEM may have additional effects bec ause it can also act as a sulfhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-b ound GSH, Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-a ctivated macrophages. (C) 1995 Academic Press, Inc.