Evidence that proteases are involved in superoxide production by human polymorphonuclear leukocytes and monocytes.

Abstract

A B S T R A C T The possible participation of proteases in superoxide (O°) production by human polymorphonuclear leukocytes (PMN) and monocytes was explored using various protease inhibitors and substrates. Protease inhibitors used included naturally occurring inhibitors of serine proteases and synthetic inhibitors that modify the active site of serine proteases. Substrates used were synthetic substrates of the chymotrypsin type as well as trypsin type ofprotease. All these inhibitors and substrates inhibited O2 production by human PMN and monocytes induced by cytochalasin E and concanavalin A, though PMN were more sensitive to these inhibitors and substrates than monocytes. Inhibition appeared rapidly even when the inhibitors were added at the same time as the stimulants, during the "induction time of O2 production" or at the time of maximum O2 production, whereas much greater inhibition was observed when the cells were preincubated with the inhibitors. These observations suggest that enzymatically active serine proteases are essential for these phagocytic cells to initiate and maintain the O2 production in response to the stimuli. The inhibitory effect of the inhibitor and substrate for chymotrypsin type protease was greater than that of those substances for trypsin-type protease. Macromolecular inhibitors also inhibited the O2 production.These findings suggest that the serine proteases involved in the O2 production by human PMN and monocytes are similar to chymotrypsin rather than trypsin, and are possibly located at the cell surface membrane.

Effect of the chemotactic peptide on the subsequent superoxide releasing response in human polymorphonuclear leukocytes

“…induced by FMPare required for maximum O;-release on contact with con A [9]. Similar findings were reported [ lo,1 11.…”

“…PMN were obtained from healthy adult donors by the dextran sedimentation and Conray-Ficoll method as in [9]. PMN preparations were suspended in Hepessaline, and contained >98% PMN.…”

“…Hitachi, Tokyo), equipped with thermostatted cuvette holder as in [9,12]. The cell suspension was added to a 1 ml cuvette containing 2 mM glucose and 66 I.IM ferricyto-chrome c to obtain a final volume of 0.985-0.99 ml.…”

“…Various stimulating agents (S-10 ~1) were added to the reaction mixture in cuvettes to obtain final volume of 1 ml and the desired concentrations of these agents, while the time-course of cytochrome c reduction (the absorbance change at 550-540 nm) was followed on the recorder. The final concentrations of stimuli were 20 PM FMP, 5 pg cyt B/ml, 100 pg con A/ml and 100 pg WGA/ml [9]. Where indicated, Ca2+ was added as chloride salt in a final concentration of 1 mM.…”

Serine protease inhibitors inhibit superoxide production by human polymorphonuclear leukocytes and monocytes stimulated by various surface active agents

“…PMN and mononuclear cell preparations were obtained from healthy adult donors by the dextran sedimentation and Conray-Ficoll method as in [ 1,2]. Both cell preparations were suspended in Hepessaline.…”