Inhibition of human polymorphonuclear leukocyte function by 2-cyclohexene-1-one. A role for glutathione in cell activation

2-cyclohexene-1-one and diethyl maleate specifically decrease reduced glutathione (GSH) levels in human polymorphonuclear leukocytes (PMN) by direct conjugation, and by interaction with the glutathione-s-transferase system. Using these two nontoxic reagents we have examined the effect of decreased GSH levels on five parameters of PMN activation: superoxide generation, release of the lysosomal enzymes lysozyme and β-glucuronidase, and increases in the influx of Na⁺ and Ca²⁺. When PMN pretreated with 2-cyclohexene-1-one or diethyl maleate were incubated with formyl-methionyl-leucyl-phenylalanine (FMLP) or the proteolytic fragment of the fifth component of complement, C5a, agents that interact with surface membrane receptors, increases in all five parameters were inhibited in a dose-dependent manner. For O^-2 generation and lysosomal enzyme release the ID₅₀ for 2-CHX-1 was 40-90 μM corresponding with a 30-50% decrease in intracellular GSH. In contrast stimulation of treated PMN by the divalent cation ionophore A23187 or 5-hydroxyeicosatetraenoic acid was much less sensitive to depressed GSH; the ID₅₀ for 2-cyclohexene-1-one was 1 mM or greater, corresponding with an 80-90% decrease in GSH. The effect of lowered GSH was not the result of decreased binding of FMLP to surface receptors because [³H]-FMLP binding studies demonstrated a two- to three-fold increase in the number of available binding sites. These data indicate that normal GSH levels are necessary for the transduction of the activation signal from the exterior to the interior of the PMN, but once initiated the activation sequence proceeds normally despite markedly lowered intracellular GSH.