The role of monovalent cationic gradients in human polymorphonuclear leukocyte (PMNL) stimulation was investigated by monitoring immune complex-stimulated transmembrane depolarization and superoxide production, events which accompany--and have been used as indicators of --PMNL activation. Abolishing only the Na+ gradient by substitution of choline for extracellular Na+ did not affect the resting membrane potential but reduced the rate of stimulus-induced transmembrane depolarization to 50% of control. In contrast, collapsing both Na+ and K+ gradients by suspension in K+ buffer (high K-PRK) depolarized the cells and reduced the stimulus-induced rate of depolarization to 11% of control. Pretreatment of cells suspended in Na+ buffers with 5-(N,N-dimethyl)amiloride hydrochloride (DMA) or with valinomycin reduced by one-half the rate of immune complex induced membrane depolarization. Conversely, in the absence of either or of both Na+ or K+ gradients, or in the presence of valinomycin, immune complex elicited an enhanced rate of superoxide production. However, PMNL prepared via NH4Cl (NH4Cl-PMNL) instead of H2O (H2O-PMNL) lysis of residual red blood cells exhibited an absolute requirement for an intact Na+ gradient in cell stimulation. The present results thus demonstrate that: 1) both Na+ and K+ gradients participate equally in the membrane depolarization elicited by immune complex; 2) neither a Na+ or a K+ gradient is required for immune complex activation, or for activity of the respiratory burst; and 3) an artifactual requirement for an intact Na+ gradient occurs in neutrophils prepared by the NH4Cl lysis technique.

Soybean polypeptides and diisopropylfluorophosphate (DFP) have been reported to inhibit neutrophil functions such as the oxidative burst, chemotaxis, and/or phagocytosis in response to soluble stimuli; these observations would be compatible with the involvement of an active serine protease in neutrophil stimulation. We have investigated the possibility of such involvement when particulate stimuli such as immune complexes are utilized. The depolarization of the neutrophils' membrane potential, one of the earliest indicators of stimulation, and superoxide production, which is detectable 30-45 sec later, were our indicators of neutrophil response to immune complexes. The neutrophils were equilibrated with, and after 5 min washed free of, up to 60 mM DFP, a potent covalent serine protease inhibitor. At DFP concentrations below 24 mM, such treatment did not perturb neutrophil activation as measured by either of the above parameters, nor did F- alone under comparable conditions. Additionally, the immune complex induced responses of neutrophils preincubated for 3 min with N-alpha-p-tosyl-L-lysine chloromethylketone (TLCK), L-1-tosylamido-2-phenyl-ethyl-chloromethylketone (TPCK), or phenyl-methyl-sulfonyl-fluoride (PMSF), covalent serine protease inhibitors which have, however, been shown to function in other capacities, e.g., as superoxide dismutases; 1 mM PMSF or 0.5 mM TLCK consistently reduced the observed membrane depolarization, one of the earliest consequences of neutrophil activation, by 20-30%, while 0.1 mM TLCK and 0.01 mM TPCK had little or no effect. The inhibition of superoxide production, a slightly later stimulus response, by PMSF, TLCK, and TPCK was more profound (50-75%); these compounds have, however, been shown to have activities other than serine protease inhibitors--for example, as superoxide dismutases. Since DFP is purely a serine protease inhibitor, and since the three other compounds do not affect depolarization (the earlier and superoxide independent event), our results indicate that active serine proteases do not appear to be necessary for immune-complex-initiated neutrophil stimulation. Other stimuli, which are known to activate neutrophils by different pathways, were not investigated.