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(Journal of Leukocyte Biology. 2002;72:1172-1179.)
© 2002 by Society for Leukocyte Biology

Immune complex stimulation of human neutrophils involves a novel Ca²⁺/H⁺ exchanger that participates in the regulation of cytoplasmic pH: flow cytometric analysis of Ca²⁺/pH responses by subpopulations

John Bernardo, Hilary Hartlaub, Xin Yu, Heidi Long and Elizabeth R. Simons

Department of Biochemistry, Boston University School of Medicine, Massachusetts

Correspondence: Elizabeth R. Simons, Ph.D., or John Bernardo, M.D., R-304, Boston University School of Medicine, 80 E. Concord Street, Boston, MA 02118. E-mails: esimons@bu.edu or jbernardo{at}lung.bumc.bu.edu

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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 2 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.

Key Words: calcium channels • Fc receptors • pH regulation • flow cytometry

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In human neutrophils, transient changes in cytoplasmic Ca²⁺ and H⁺ concentrations ([Ca²⁺]_i and pH_i) induced by immune complexes (IC) precede the generation of an oxidative burst and degranulation within phagovacuolar compartments. This activity is designed to destroy the ingested complex [^{1 2 3 4 5 6} ]. We have previously demonstrated, using multiparameter flow cytometric cell-by-cell analysis, that there is a subpopulation specificity to this response, with the magnitude of the Ca²⁺ transient depending on the valency of the IC and the type of Fc receptor engaged [⁵ , ^{7 8 9 10} ].

Furthermore, using similar techniques and stimuli, we have demonstrated that the microbicidal functions of the cell can proceed in the absence of the normal, large transient increase in [Ca²⁺]_i, suggesting that Ca²⁺_i itself is not serving a second messenger function in this system [¹¹ ]. Grinstein and his colleagues [¹⁰ ] have shown that changes in [Ca²⁺]_i also do not affect the Na⁺/H⁺ antiport responsible for maintaining intracellular pH. In contrast to the extensive studies of the role of Ca²⁺, it has not yet been shown whether the pH_i changes that accompany such Ca²⁺ transients play a role in the regulation of the phagocytes’ killing processes or rather, whether they represent a concurrent, nonregulatory phenomenon [⁵ , ⁶ , ¹¹ , ¹² ].

Several pH-regulating systems have been identified in phagocytes. These include an electroneutral, amiloride-inhibitable Na⁺/H⁺ antiport [¹⁰ , ^{13 14 15 16 17} ] of the NHE-1 isoform [¹⁸ ], two types of Cl^-/HCO₃^- exchangers [¹³ ], and an adenosine 5'-triphosphate-dependent proton pump that can extrude protons from the cell and acidify phagovacuolar compartments [¹⁹ ]. The anion and cation exchangers appear to be the major mechanisms by which cellular pH is maintained in acidic environments, such as inflammatory sites. In turn, regulation of pH and pH changes within the cytoplasm of activated cells may be critical to the generation of an optimal pH in phagosomes [¹ ].

Intracellular pH changes in human neutrophils following IC stimulation consist of an early, rapid acidification followed by a slower, cytoplasmic alkalinization [¹⁵ , ¹⁶ ]. The former is attributable to the generation of phosphatidic acid via the activation of phospholipase D [⁶ , ¹² ], and realkalinization has been attributed largely to the Na⁺/H⁺ antiport thought to be triggered by cytoplasmic acidification [¹⁵ , ¹⁶ ]. The relation, if any, of the pH_i response to IC and to changes in [Ca²⁺]_i remains undefined, although current evidence suggests that changes in pH play a significant role in degranulation [¹ , ⁶ , ¹² ].

Previous studies of stimulus-induced, cellular pH_i changes have used spectrofluorimetric techniques that record averaged values for selected activation parameters in suspended populations of cells [¹⁶ ]. In contrast, multiparameter flow cytometry permits the analysis of several intracellular events simultaneously in real-time following engagement of a phagocytic stimulus [⁵ , ^{7 8 9} ].

In the present study, we use flow cytometry to examine the relationships between the changes in [Ca²⁺]_i and cellular pH in immune complex-stimulated human neutrophils. We show here, using cell-by-cell analysis, that pH_i and [Ca²⁺]_i changes proceed simultaneously (or, as a result of the limitations of our measurements, within <4 s of each other) in the same responding neutrophil population following IC stimulation. Taken together with our earlier finding that a calcium transient is not required [¹¹ ], this implies that pH_i and not [Ca²⁺] may control subsequent neutrophil effector functions such as degranulation and oxidative burst. Furthermore, we demonstrate here a new, voltage-independent Ca²⁺/H⁺ exchanger that contributes to cellular realkalinization following cell stimulation by IC, acting together with a Na⁺-dependent H⁺ change within the cell’s cytoplasm.

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Materials
The fluorescent probes indo-1-acetoxymethyl ester, BCECF-acetoxymethyl ester, and the chelator 1,2-bis(O-aminophenyl-ethane-ethane)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester were obtained from Molecular Probes, Inc. (Eugene, OR), as was dimethylamiloride. The Ca²⁺ channel inhibitors verapamil and nifedipene as well as bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). The rabbit anti-BSA antibody was purchased from ICN (Costa Mesa, CA). All other chemicals were of reagent grade, purchased from Sigma Chemical Co. or from Fisher Scientific (Pittsburgh, PA).

Inhibitors of H⁺ transport
Bafilomycin A₁ stock (25 µM) was prepared in dry dimethylsulfoxide, and 10 µl was added to 1 ml BCECF and indo-1-loaded polymorphonuclear neutrophil (PMN; 10⁶/ml) at 37°C and was kept stirring in Krebs-Ringer phosphate (KRP) for 2 min before addition of 120 µg/ml IC. A similar procedure was followed with concanamycin A, whose stock was prepared at 10 µM. We found that ZnCl₂ at concentrations higher than 2 µM caused the PMN to clump, and therefore we used that final concentration prepared from 200 µM stock in phosphate-buffered saline (PBS).

Neutrophil isolation
Human PMN obtained from healthy volunteers were purified as previously described [²⁰ ] and were kept in PBS, pH 7.4 (125 mM NaCl, 2 mM NaH₂PO₄, 8 mM Na₂HPO₄, 5 mM KCl, 5 mM glucose), on a rocking platform at 4°C until needed.

Stimulus preparation
Multivalent IC were prepared as the insoluble portion of a fourfold molar excess of rabbit anti-BSA: BSA incubated and then isolated as previously described [⁵ ].

[Ca²⁺]_i measurements
[Ca²⁺]_i concentration was measured as previously described [⁸ ] using indo-1, the stimulus being injected after a 2-min incubation at 37°C in KRP buffer (PBS plus 0.9 mM CaCl₂ and 1.5 mM MgCl₂), which has been shown to allow stimulation of the neutrophils without permitting replenishment of their intracellular Ca²⁺ stores [¹¹ ].

pH_i measurements
pH_i was measured using BCECF-loaded neutrophils, as previously described [¹¹ ] and was measured on a Becton Dickinson FACS 440 flow cytometer. Although, in principle, BCECF can be used as a ratiometric pH probe, the FACS 440 did not permit us an excitation at 450 nm (the pH-independent excitation wavelength). We were only able to excite at the pH-dependent 488 nm wavelength (emission at 530 nm) and recorded data as % of maximal emission at t = 0, i.e., normalizing to allow for any differences in BCECF concentration; our pH_i calibrations [¹¹ ] have shown the pH_i of neutrophils at t = 0 to be 7.0.

Stimulus responses
Neutrophil stimulus responses were measured on the FACS 440 flow cytometer equipped with thermostating, stirring, and injection devices [⁵ , ^{7 8 9} ]. Data were collected at t = 0 and every 4000 cells (7 s) thereafter. Verification of these responses and of the calibration curve obtained previously [¹¹ ] was also obtained on a Hitachi 4500 fluorimeter.

Depolarization of neutrophils
Depolarization of neutrophils was accomplished as previously described [²⁰ ] using PRK, an isotonic buffer in which Na⁺ was largely replaced by K⁺ (25 mM NaCl, 120 mM KCl, 0.9 mM CaCl₂, 1.5 mM MgCl₂). Depolarization was verified by using the membrane potential cyanine probe diSC₃ [⁵ ], as described in that publication, on a Hitachi 4500 spectrofluorimeter. Instrumental limitations did not permit simultaneous measurement of membrane potentials and pH in the FACS 440.

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Time and IC dose dependence of [Ca²⁺]_i
As we had shown earlier [⁵ ], the overall [Ca²⁺]_i averaged for all cells, as would have been determined in a fluorimetric measurement of neutrophil suspensions or in an ungated flow cytometric summary (Fig. 1a ), showed an apparent dependence of the magnitude of [Ca²⁺]_i (expressed as % of maximal [Ca²⁺]_i at the saturating dose, 120 µg/ml) and the time at which that maximum was achieved for doses of 120, 90, 60, and 30 µg/ml IC. When the data were restricted to the responding subpopulation (Fig. 1b) , data for 120 down to 60 µg/ml IC became super-imposable, reconfirming our findings that a maximal response, found at the saturating concentration of 120 µg/ml, was also obtained in the responding subpopulation at lower doses. The time to reach this maximal response also became equal within <7 s after addition of the stimulus.

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Figure 1. Dependence of [Ca2+]i and pHi on stimulus concentration. (a and c) Ungated; (b and d) gated so that only responding cells are included. (a and b) % of Maximal [Ca2+]i observed for 120 µg/ml IC; (c and d) % of maximal pHi observed for 120 µg/ml IC. All studies were performed in KRP as described in Materials and Methods. Figure is representative of 3–10 separate experiments.

Time and IC dose dependence of pH_i
Simultaneous flow cytometric measurements of [Ca;2⁺]_i and pH_i in neutrophils have not previously been reported. As was true for changes in [Ca²⁺]_i, the dose dependence of the initial rapid acidification in response to doses from 120 to 60 µg/ml IC is not detectable on the overall neutrophil scans (Fig. 1c) nor when the data were "gated" to restrict them to the responding subpopulation (Fig. 1d) , confirming our previous report [¹¹ ] that [Ca²⁺]_i and pH_i are separate events and appear to be differently controlled in phagocytic cells. In contrast, the Na⁺/H⁺ antiport-controlled alkalinization does appear to be smaller with 30 µg/ml, a subsaturating IC dose (Fig. 1d) .

Role of Na⁺/H⁺ antiport in neutrophil [Ca²⁺]_i and pH_i response
Dimethylamiloride (DMA), a specific Na⁺/H⁺ antiport-blocking agent [¹⁴ ], did not alter the neutrophils’ Ca²⁺ response to 120 µg/ml IC in KRP (i.e., in the presence of 145 mM Na⁺) but did, at doses from 50 to 200 µM DMA, inhibit the realkalinization of the cells that normally occurs approximately 1 min after stimulation (Fig. 2a and 2b ). As the highest DMA dose of 200 µM appeared to perturb other cellular functions, we used 100 µM DMA for all subsequent studies.

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Figure 2. Effect of DMA, a Na+/H+ antiport-blocking agent on pHi. (a) Effect of DMA on [Ca2+]i; (b) effect of DMA on % of maximal pHi. All studies were performed in KRP as described in Materials and Methods. Figure is representative of 3–10 separate experiments.

Role of influx of extracellular Ca²⁺ in neutrophil [Ca²⁺]_i and pH_i responses
As described in Materials and Methods and in many publications by others as well as ourselves, neutrophils are routinely kept rocking at 4°C in PBS (or other non-Ca²⁺-containing buffer) to prevent preactivation and then are equilibrated at 37°C in the presence of Ca²⁺ and Mg²⁺ (e.g., in KRP) for 2 min to restore full functional responsiveness to stimuli such as formyl-Met-Leu-Phe (fMLP) or IC. Chelating this extracellular Ca²⁺ 15 s before stimulation failed to affect the high-transient [Ca²⁺]_i response mediated via the chemotactic receptor (Fig. 3 and ref. [¹¹ ]) but reduced and retarded the responses via the Fc receptor-mediated pathways by approximately 30%. These data support our previous findings [⁶ ] of specific mechanistic differences between the activation pathways mediated by these two classes of neutrophil receptors. The indication that both pathways lead to a final [Ca²⁺]_i 2.5 min after stimulation was initiated which is lower in the absence than in the presence of extracellular Ca²⁺, implies the presence of an influx into the stimulated cells from the extracellular milieu via one of the calcium channels.

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Figure 3. Effect of extracellular Ca2+ chelation with EGTA on [Ca2+]i and pHi. Neutrophils were stimulated with 100 nM fMLP (a and c) or 120 µg/ml IC (b and d) with or without addition of 1 or 5 mM EGTA 15 s before the stimulus. All studies were performed in KRP as described in Materials and Methods. Figure is representative of 3–10 separate experiments.

Simultaneous measurement of pH_i showed a very pronounced and previously undocumented acidification in the absence of this Ca²⁺ influx, whether the stimulation proceeded via the fMLP (Fig. 3c) or the Fc receptor (Fig. 3d) . The effect was maximal at 5 mM EGTA, the concentration used in subsequent experiments. These findings implied that there was not only the previously known Ca²⁺ influx in response to neutrophil stimulation but also a previously undocumented Ca²⁺ influx-coupled H⁺ efflux, i.e., a Ca²⁺/H⁺ channel. It should be noted that these findings extend those reported by Grinstein’s laboratory [¹⁰ ] using 1 mM EGTA; we found the higher dose necessary to achieve saturation of the pH effects (Fig. 3d) .

Role of extracellular Na⁺ in neutrophil [Ca²⁺]_i and pH_i responses in the presence or absence of extracellular Ca²⁺
As shown in Figure 4 , when extracellular Na⁺ is totally replaced by choline (112 mM choline chloride, 2 mM KH₂PO₄, 8 mM K₂HPO₄, 55 mM sucrose, pH 7.4–pH adjusted with KOH), thereby altering all Na⁺-involving mechanisms without depolarizing the cells [²⁰ ], the initial acidification remains, but the subsequent alkalinization is suppressed and is followed, approximately 1 min after stimulation is initiated (a time corresponding to the start of degranulation; refs. [²¹ , ²² ]), by acidification of the cells’ cytoplasm. This acidification is already apparent after just 30 s if extracellular Ca²⁺ has been chelated, i.e., if loss of H⁺ via a Ca²⁺/H⁺ exchange has been prevented, but the phospholipase D-mediated acidification remains unperturbed [⁶ , ¹² ]. In the absence of extracellular Na⁺ and Ca²⁺, the acidification is significantly greater than in their presence (compare Fig. 4 , a and b), as neither the classic Na⁺/H⁺ antiport nor the novel Ca²⁺/H⁺ channel we describe here can function. In PRK, an isotonic buffer in which the extracellular K⁺ matches its intracellular concentration, and the membrane potential drops from -75 to approximately -30 mV [²⁰ ], the effect of EGTA is comparable with that in KRP (Fig. 5 ), implying little or no voltage-dependence of the Ca²⁺/H⁺ exchanger.

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Figure 4. Effect of extracellular Na+ replacement by choline on pHi. pHi Response to stimulation of PMN by 120 µg/ml IC in (a) PBS ([Na+]=145 mM) or (b) choline buffer ([Na+]=0 mM) in the presence of 5 mM EGTA and/or 100 mM DMA. Figure is representative of 3–10 separate experiments.

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Figure 5. Effect of depolarization on the novel Ca2+/H+ channel. (a) [Ca2+]i and (b) pHi in response to 120 µg/ml IC were measured for neutrophils incubated in KRP or PRK (a depolarizing buffer) with/without addition of 5 mM EGTA 15 s before the stimulus. Figure is representative of 3–10 separate experiments.

To probe for any direct interaction or partial control of the Ca²⁺/H⁺ channel by Na⁺, we repeated the above experiments in the presence of 100 µM DMA in KRP versus choline buffers. In the presence of extracellular Ca²⁺, the pH_i changes elicited in the two buffers were identical, as would be anticipated, as DMA successfully competes for the Na⁺ site on the antiport, and it is therefore immaterial whether Na⁺ is present. Unexpectedly and conversely, if Ca²⁺ and Na⁺ are absent (choline buffer+EGTA), and DMA has been added, the cytoplasmic acidification is present but significantly smaller (Fig. 4b) . This highly reproducible finding implies that H⁺ efflux or OH^- influx is possible, i.e., that DMA binds to some entity other than the classic Na⁺/H⁺ antiport under these conditions (absence of extracellular Na⁺ and Ca²⁺) and/or that it permits some leakage of H⁺ from the cell when it binds to the antiport, whether Na⁺ is present or not. To date, no such binding or function for DMA has been reported in the literature.

Is the novel Ca²⁺/H⁺ channel voltage-dependent?
To answer this question, we investigated the Ca²⁺ and H⁺ responses of neutrophils in the depolarizing buffer PRK (120 mM KCl, 25 mM NaCl, 8 mM Na₂HPO₄, pH 7.4) in comparison with KRP (125 mM NaCl, 8 mM Na₂HPO₄, 5 mM KCl, pH 7.4) [²⁰ ]. Comparing Figures 3 and 5 , it is clear that the [Ca²⁺]_i transient is considerably lower, and the Na⁺/H⁺-mediated alkalinization is abolished when the membrane potential is reduced from -75 to -30 mV [²⁰ ], and the [Na⁺] is reduced from 145 to 25 mM. The acidification effect of extracellular Ca²⁺ chelation per se is apparent after 15 s and is similar in the presence of 145 or 25 mM extracellular Na⁺. This implies that the Ca²⁺/H⁺ channel may be [Na⁺]-dependent.

Effect of inhibitors of the Na⁺/H⁺ antiport versus classic Ca²⁺ channels
In contrast to the effect of DMA (Fig. 2) , the classic Ca²⁺ channel inhibitors verapamil and nifedipene, at concentrations of 10 and 100 µM, had no effect on the [Ca²⁺]_i or pH_i responses (Fig. 6a and 6b ). These findings imply that the novel Ca²⁺/H⁺ channel does not respond to inhibition by nifedipene or verapamil, inhibitors of some but not all mammalian Ca²⁺ channels.

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Figure 6. Effect of Ca2+ channel-blocking agents on the Ca2+/H+ channel. (a) [Ca2+]i and (b) pHi were measured for neutrophils stimulated with 120 µg/ml IC in the absence or presence of 5 mM EGTA added 15 s before the stimulus or of 10 or 100 µM verapamil (ver) or nifedipene (nif) added 1 min before the stimulus. All studies were performed in KRP as described in Materials and Methods. Figure is representative of 3–10 separate experiments.

Effect of inhibitors of proton pumps and channels
To determine whether neutrophil proton channels, which can act as H⁺ extruders, might play a role in our observed Ca²⁺-dependent extrusion, we repeated our experiments, ±5 mM EGTA, with PMN incubated (at 37°C with stirring) for 2 min before addition of 120 µg/ml IC in 250 nM bafilomycin, 100 nM concanamycin, or 2 µM ZnCl₂. As shown in Figure 7a and 7b , none of these inhibitors had any effect on the acidification and lack or alkalinization observed in the presence of extracellular EGTA, nor did they affect the stimulus-induced Ca²⁺ changes (data not shown). Therefore, although these proton channels and pumps have been demonstrated to be present in neutrophils, they do not affect the function of the Ca²⁺/H⁺ channel we describe in the present study.

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Figure 7. Effect of inhibitors of H+ transport. pHi response of BCECF-loaded PMN to 120 g/ml IC in the presence of inhibitors of H+ transport and (a) absence or (b) presence of 5 mM EGTA added 15 s before the stimulus. Inhibitors are 250 nM bafilomycin A1 (BAF), 100 nM concanamycin A (CON), or 2 µM ZnCl2. Figure is representative of 3–10 separate experiments. DMSO, Dimethyl sulfoxide.

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The findings presented in the previous section provide proof that a previously postulated [¹⁰ , ¹⁶ , ¹⁷ , ²³ ] but undocumented channel capable of reversing the stimulus-induced intracellular acidification of neutrophils exists. Its role, like that of the well-known Na⁺/H⁺ antiport, appears to be the reversal of the [Ca²⁺]_i and pH_i stimulus responses by replenishing the cytoplasmic [Ca²⁺]_i stores at the same time as it deacidifies that cytoplasm.

In three separate studies of phagosomal pH changes and their role in phagocyte killing [^{24 25 26} ], we have shown that Cryptococcus neoformans survives in a very acidic phagosome but is destroyed by the phagocyte’s oxidative and lytic activities when the phagosome’s pH is raised. As an acidic cytoplasm leads, by H⁺ exchange across the plasma membrane, to a more acidic phagosome, the natural destructive oxidative and lytic capabilities, which are the phagocyte’s effector functions, will be enhanced when its cytoplasm is less acidic. The relative importance of oxidative versus lytic activities initiated by neutrophil granule contents is still subject to some controversy [¹ , ²¹ ], but it seems agreed that the phagosomal pH plays a critical role in the bactericidal and fungicidal functions of neutrophils [²² , ^{27 28 29} ].

Therefore, we conclude that not only the Na⁺/H⁺ antiport and the various proton channels and pumps but also the novel Ca²⁺/H⁺ channel we describe here perform this alkalinizing function. In addition, the channel permits replenishment of the phagocyte’s intracellular Ca²⁺, although the function of this replenishment is less obvious, as it does not appear to be necessary for the effector functions to proceed [⁶ , ^{10 11 12} ].

 
We gratefully acknowledge the support of NIH grant DK31056.

Received April 6, 2002; revised August 22, 2002; accepted August 29, 2002.

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