The role of transmembrane cationic gradients in immune complex stimulation of human polymorphonuclear leukocytes

Abstract

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.

Neutrophil degranulation and phospholipase D activation are enhanced if the Na+/H+ antiport is blocked

“…Human PMN were obtained from healthy donors by Dextran sedimentation followed by Ficoll-Hypaque centrifugation and brief hypotonic lysis of remaining red cells, as previously described [11]. Cells were resuspended in phosphate-buffered saline with glucose (PBS-glucose), pH 7.4, and kept rocking at 4°C until experiments were performed, always within Ͻ3 h.…”

“…␤-Glucuronidase was measured by a colorimetric method employing phenolphthalein glucuronic acid as a substrate [11]. Elastase was measured by spectrofluorimetry as previously described [1].…”

“…The alkalinization can be inhibited by amilorides [4][5][6][7][8], including dimethylamiloride, a relatively potent and specific inhibitor of the Na ϩ /H ϩ antiport [8], or by substitution of extracellular Na ϩ with a cation that cannot be transported via the antiport, such as N-methylglucamine [11]. Therefore, to investigate what role, if any, the pH transient plays in controlling neutrophil degranulation, we used these methods to block the HIC-induced cytoplasmic alkalinization, thereby enhancing the acidification and measuring any consequent effects on HIC-induced degranulation.…”

Sequential Chemotactic and Phagocytic Activation of Human Polymorphonuclear Neutrophils

“…The rabbit anti-bovine serum albumin (BSA) antibody was obtained from MP Biomedicals (Irvine, CA). All buffers were prepared with high-performance liquid chromatography-grade chemicals in our laboratory as described below, employing our in-house distilled water that was additionally processed in four stages: charcoal, ion exchange, pyrogard filters, and, thereafter, redistillation (21,28). All other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO) or from Fisher Scientific (Pittsburgh, PA).…”

“…Peripheral venous blood (30 ml) was collected by venipuncture and instantly mixed with 3 ml of 4.3% sodium citrate. PMN were purified by dextran sedimentation followed by Ficoll-Hypaque centrifugation and brief hypotonic lysis of the remaining red blood cells, as previously described (28). As noted therein and above, special precautions were taken to ensure that PMN remained naïve and unprimed; this includes the use of distilled, three-cartridge-purified, and then redistilled H 2 O in the preparation of sterile buffers, rinsed plastic ware, and hypotonic lysis.…”