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The activation of human phagocytic leukocytes by immune complexes (IC) or opsonized microbes via their Fc and complement receptors has been well-described (for reviews, see refs. [1–3]). The mechanisms involved in this process are complex and depend on the receptors involved. The biochemical events that lead to the destruction of invading organisms in turn display varying degrees of interdependence, but the controlling elements that lead to the ultimate killing of ingested organisms within phagosomes by lysosomal enzymes and reactive oxygen intermediates are still not completely understood. We have addressed these mechanisms by following and correlating the kinetics of responses by individual cells, using multiparameter flow cytometry [3, 4]. Using nonopsonized IC as stimuli, we document here the presence of a novel Ca2+/H+ voltage-independent channel in human neutrophils, which helps to control their cytoplasmic pH.

Neutrophils phagocytize high-valency immune complexes (HIC) by an Fcγ receptor-mediated mechanism, activating an oxidative burst and initiating degranulation. In contrast, neutrophils exhibit chemotaxis to N-formylated peptides [e.g., N-formylmethionyl-leucyl-phenylalanine (fMLP)] and secrete far fewer oxidants or granule contents than neutrophils activated by HIC. However, if neutrophils are treated with cytochalasin B (CB) or permeabilized with streptolysin O, chemoattractant-induced neutrophil secretion is increased to a level beyond that observed in response to HIC. Because priming neutrophils with CB, or permeabilizing them, also augments activation of phospholipase D (PLD) in response to fMLP, we reasoned that, in intact (i.e., nonpermeabilized) unprimed neutrophils, PLD may participate in a signaling pathway specific to phagocytic stimuli such as HIC and hence may contribute to degranulation control. PLD activity in response to HIC and fMLP correlated closely with stimulus-induced azurophilic degranulation under a wide variety of experimental conditions, including compounds that abrogated or augmented stimulus-induced PLD action. PLD activation preceded, and appeared to be necessary for, azurophilic degranulation. These results suggest that PLD may play a central role in controlling azurophilic degranulation and provide signaling specificity between pathways activated by fMLP and HIC in intact neutrophils.

Two Fc gamma receptor (Fc gamma R) subclasses on human neutrophils, Fc gamma RII and Fc gamma RIII, activate different cellular functions. To examine the involvement of each receptor subtype in polymorphonuclear leukocyte activation, Fab and F(ab')2 fragments of subclass-specific monoclonal antibodies ([mAbs] mAb IV.3 against Fc gamma RII and mAb 3G8 against Fc gamma RIII, respectively) were used to block the binding of low valency immune complexes (LICs) and high valency immune complexes (HICs). Flow cytometry then permitted the simultaneous quantitation of antibody and ligand binding, the elicited intracellular Ca2+ concentration (delta[Ca2+]int), initiation of the oxidative burst, and/or the phospholipase A activation in the same cell. We have previously demonstrated that subsaturating dosages of HIC bind uniformly to all the cells but elicit an "all-or-none" (i.e., dose independent) maximal delta[Ca2+]int in a dose-dependent subpopulation of the cells. In contrast, both the proportion of cells responding and the magnitude of the delta[Ca2+]int transient depend on the subsaturating dose of LIC, even though it too binds uniformly to all the cells, nonresponding as well as responding. These earlier findings have here been extended by single cell flow cytometric analysis to demonstrate that F(ab')2 Fc gamma RIII is the major Fc gamma R involved in HIC binding (and [Ca2+]int mobilization), as well as in oxidative burst and phospholipase A activation. In contrast, both receptor subclasses must be available for LIC-elicited delta[Ca2+]int, as blockage by either of the mAb Fab or F(ab')2 fragments abrogates this response, even though LIC binding to the receptors is not decreased. Furthermore, LIC elicited little oxidative burst activity and failed to activate phospholipase A but cross-linking to achieve multivalency, previously shown to induce [Ca2+]int and oxidative burst responses, elicited phospholipase A activity via Fc gamma RIII. Fc gamma RII's role appears to be modulation of the small, late Ca2+ influx observed at > 1 min, whereas Fc gamma RIII modulates all the earlier larger events. Thus, simultaneous observation of receptor identity, receptor occupancy, and consequent activation parameters in the same cell by flow cytometry permits use to demonstrate that Fc gamma RII is necessary for the small signal transduction elicited by LIC; it plays a relatively small role in polymorphonuclear leukocyte stimulation by HIC. Fc gamma RIII is the main receptor responsible for immune complex-elicited polymorphonuclear leukocyte responses; its efficacy is greatly enhanced when the receptors are cross-linked, either by preequilibrated multivalent complexes or by in situ cross-linking of bound LIC with excess antibody.

Treatment of human leukemic HL-60 cells with N,N-dimethylformamide (DMF) induces them to mature until they reach granulocytoid morphology 3–6 d later. We have reported a maturation-dependent ability of these cells to respond to phorbol myristate acetate (PMA), as evaluated by membrane depolarization and by oxidative burst product formation (Newburger et al.: J. Biol. Chem. 259, 3771, 1984). More recently we have attempted to develop techniques for simultaneous evaluation of these parameters during HL-60 cell maturation. Here, we compare the cytoplasmic [Ca+ +] and membrane potential changes elicited by the chemotactic peptide fMLP via simultaneous measurement of individual cells in a fluorescence-activated cell sorter (FACS), as done previously for mature granulocytes (Lazzari et al.: J. Biol. Chem. 261,9710, 1986). The stimulus-induced [Ca++]in changes are detected with the fluorescent probe lndo-1 and reproducibly increase in magnitude for a subpopulation of cells as the cells mature into granulocytes. Ca+ + responsiveness to formyl peptide is restricted to a subpopulation of HL-60 granulocytes which expresses receptors for chemotactic peptide and consistently increases in magnitude (in response to the same concentration of agonist) with maturation. In contrast, there is less consistency in the direction or magnitude of membrane potential changes elicited under the same circumstances from the same maturing HL-60 cells.

Neutrophils phagocytize high-valency immune complexes (HIC) by an Fc receptor-mediated mechanism. Engaging Fc receptors in this manner induces PMN to generate superoxide and release the contents of both their specific and azurophilic granules. Signaling events that precede and accompany PMN secretion include activation of phospholipase D (PLD), as well as changes in cytoplasmic [Ca2+] (δ[Ca2+]in) and pH (δpHin). Although the role of PLD and δ[Ca2+]in in mediating Fc receptor-mediated PMN secretion has been studied, whether pHin plays a regulatory role has not yet been defined. HIC-stimulated PMN undergo an intracellular acidification followed by a prolonged Na+/H+ antiport-mediated alkalinization. To investigate the role of the pH transient in controlling degranulation, the Na+/H+ antiport was inhibited either with 100 μM dimethylamiloride (DMA) or by substituting N-methyl-glucamine for extracellular sodium. Blocking the antiport with DMA led to hyper-acidified PMN, which exhibited an increase in degranulation, but did not affect generation of superoxide. DMA did not alter the ability of neutrophils to phagocytose and oxidize dichlorodihydrofluoresceinated HIC, suggesting the increase in degranulation was not the result of failed phagocytosis. Investigation into whether the observed increase in degranulation when the antiport was blocked was mediated by PLD or δ[Ca2+]in revealed that blocking the antiport increased HIC-induced PLD activity but had no effect on HIC-induced δ[Ca2+]in. Blocking the Na+/H+ antiport by ion substitution caused similar effects on PMN signaling and secretion as was seen with DMA. These results indicate that Na+/H+ antiport activity is not necessary for degranulation or superoxide release in HIC-stimulated PMN and that hyperacidification of the cytoplasm can modulate degranulation. Therefore, pHin, via its effect on PLD, may be a control point of degranulation and may represent one way that neutrophils achieve differential control of their antibacterial products. J. Leukoc. Biol. 64: 98–103; 1998.

Receptor-mediated activation of neutrophils (PMN) initiates possibly interdependent events, including a rapid transient increase in [Ca2+]i, implicated as a second messenger. To investigate whether this transient is required for eventual degranulation, PMN were incubated with an intracellular Ca2+ chelator (BAPTA), then exposed to chemotactic peptide [N-formyl-methionyl-leucyl-phenylalanine (fMLP)] with or without cytochalasin B (CB) or to high-valency immune complexes (HIC); δ[Ca2+]i, δpHi, oxidative burst, and elastase release were then evaluated (plus or minus EGTA 15 s before stimulation) after 2 and 15 min incubation in 0.9 mM Ca2+. With either fMLP plus CB or HIC stimulation, BAPTA-treated cells were unable to achieve a Ca2+ transient with a 2-min incubation, whereas a 15-min incubation allowed the BAPTA-treated cells to recover a portion of the δ[Ca2+]i. Even though BAPTA-treated cells were unable to mount a δ[Ca2+]i at 2 min, HIC-stimulated BAPTA-treated cells were able to elicit an oxidative burst (33% of control) and degranulation (67% of control). Therefore, we conclude that δ[Ca2+]i modulates but is not required for oxidative burst or degranulation.

Ligand-induced cross-linking of Fcγ receptors (FcγR) on neutrophils plays a significant role in their stimulation, shown here by contrasting the responses induced by low valency immune complexes (LICs) and high valency immune complexes (HICs) and by cross-linking LICs in situ (L/Ab) after their addition to the cells. Multiparameter flow cytometry was used to measure immune complex (IC)-elicited changes in cytoplasmic Ca2+ concentration and initiation of the oxidative burst simultaneously in the same cell and to correlate these with FcγR occupancy. We have previously shown that subpopulations of neutrophils respond maximally to subsaturating concentrations of HIC; saturating dosages stimulate the entire population. This discrepancy was not due to differences in receptor occupancy. The magnitude of the transient Ca2+ increase was independent of the dose of HIC but depended on the dose when an LIC was used. As shown here, L/Ab cross-linking elicited Ca2+ responses similar to those observed in HIC-stimulated cells. In contrast, LIC elicited only minimal intracellular ΔpH and no oxidative burst or membrane potential changes at all unless FcγR was cross-linked, accomplished by HIC or by L/Ab. However, azurophilic degranulation, as determined by elastase release, was not observed in cells stimulated by the in situ cross-linking method, whereas the HIC preparation triggered azurophilic degranulation. Thus, some FcγR-mediated neutrophil effector functions such as azurophilic degranulation and oxidative burst initiation have an absolute requirement for FcγR cross-linking, whereas signaling functions such as changes in membrane potential, intracellular pH, and intracellular Ca2+ concentration can occur, albeit more slowly and to a lesser extent, if single FcγR are occupied.

Stimulation of phagocytic leukocytes with chemotactic factors results in transient acidification, followed by alkalinization of the cytosol. Human monocytes are known to alter their functional responses to the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) in a complex fashion as they mature in vitro to macrophages. To examine the evolution of the cytoplasmic pH (pHi) response of monocytes to fMLP as they mature into macrophages, we incubated cells for 0, 24, 48, and 96 h (Medium-199 + 10% fetal bovine serum; 37°C) and examined pHi using the fluorescent probe 2′, 7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF; 1 μΜ) and a Perkin-Elmer 650/10 spectrofluorimeter (λem = 530 nm, λ_ex = 500, 450 nm) as previously described. The resting pH; of fresh (0 h) monocytes was 7.07 ± 0.16 (SD) and was unchanged after incubation for 24, 48, or 96 h (7.09, 7.11, 7.05, respectively). Cells exhibited an fMLP dose-dependent cytoplasmic acidification, with maximal δpHi occurring 30-60 s after exposure to 10−7 M fMLP. The response to fMLP did not change with the duration of incubation and, as with neutrophils, cytoplasmic realkalinization was blocked by dimethyl- amiloride (20 μΜ). Incubation with 2-deoxyglucose (10 min, 5 mM), sufficient to inhibit by more than 90% the formyl peptide-stimulated superoxide generation by monocytes, slowed fMLP-induced acidification and abrogated the alkalinization. In addition, monocytes isolated from the blood of a patient with X-linked chronic granulomatous disease (CGD) underwent fMLP-induced acidification that was unmasked further by coincubation with di- methylamiloride, in a manner quantitatively similar to that of normal monocytes, despite the inability of the CGD cells to produce superoxide. The chemotactic factor-induced cytoplasmic pH responses of mono- cytes/macrophages remained constant as the cells matured in vitro and exhibited a dimethylamiloride-independent acidification and dependent alkalinization, as did the response in neutrophils. The cytoplasmic acidification of these cells thus did not correlate with the cells’ production of superoxide and with the concomitant hexose monophosphate shunt activation, as has been suggested for other leukocyte types. J. Leukoc. Biol. 53: 673–678; 1993.

Soybean polypeptides and diisopropylfluorophosphate (DFP) have been reported to inhibit neutrophil functions such as the oxidative buret, 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-α-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.I 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.