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H+ exchange
Immunobiol. 207, 141 ± 148 (2003) ¹ Urban & Fischer Verlag http: // www.urbanfischer.de/journals/immunobiol
Intracellular pH regulation in U937 human monocytes: roles of V-ATPase and Na/H exchange Thomas A. Heming, Akhil Bidani Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas, USA Received: March 27, 2002 ¥ Accepted: October 8, 2002
Abstract The role of plasmalemmal V-type H translocating ATPase (V-ATPase) in regulation of intracellular pH (pHi) is unclear in monocytes. This study examined the plasmalemmal VATPase and Na/H exchanger (NHE) in U937 human monocytes. The fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein was used to monitor baseline pHi and the kinetics of pHi recovery from cytosolic acid-loads (NH4Cl prepulse). Bafilomycin A1 and 5(N-ethyl-N-isopropyl)amiloride (EIPA) were used to delineate the activities of the H-pump and NHE, respectively. Baseline pHi was 7.13 at an extracellular pH (pHo) of 7.4 and fell progressively at lower pHo values. EIPA had no effect on baseline pHi at pHo 7.4, but caused a sustained decrement in pHi at pHo 6.0 ± 7.0. Bafilomycin A1 had biphasic effects on baseline pHi at pHo 6.5 ± 7.4: pHi declined 0.1 units over the course of several minutes and then recovered. At pHo 6.0, bafilomycin A1 caused a sustained decrement in baseline pHi. Recovery from the bafilomycin-induced acidosis at pHo 6.5 ± 7.4 was prevented by EIPA. Similarly, pHi recovery from NH4Cl prepulse acid-loads (pHo 7.4) was sensitive to both EIPA and bafilomycin A1. During this recovery process, Na/H exchange (EIPA-sensitive component of apparent H efflux) was the predominant mechanism for H extrusion at acid-loaded pHi values < 7.0. At acid-loaded pHi values 7.0, the V-ATPase (bafilomycinsensitive component) and NHE contributed almost equally to H extrusion. The data provide the first evidence that plasmalemmal V-ATPase participates in pHi regulation in U937 cells. The H-pump and NHE interacted to set baseline pHi and for pHi recovery following cytosolic acid-loading of the monocytes. Abbreviations: BCECF 2',7'-biscarboxyethyl-5,6-carboxyfluorescein; b cytosolic nonbicarbonate buffering capacity; dpHi/dt rate of pHi recovery; EH equilibrium H potential; EIPA 5-(N-ethyl-N-isopropyl)amiloride; JH, apparent H flux; NHE Na/ H exchanger; pHi intracellular pH; pHo extracellular pH; PSS physiologic salt solution; SVR surface area-to-volume ratio; V-ATPase V-type H translocating ATPase.
Introduction Phagocytes, namely monocytes, macrophages, and neutrophils, are fundamental components of innate
immunity (Medzhitov & Janeway, 2000; Hawiger, 2001). Resident tissue macrophages serve as sentinel cells. They respond to inflammatory/immune insults by up-regulating (activating) their host defense
Correspondence author: Thomas A. Heming, Ph.D., Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Texas Health Science Center, Houston, TX 77225-0708, USA. Phone: 1-713-500-6835; Fax: 1-713-500-6829; E-mail: [email protected]
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functions, including phagocytosis, generation of reactive oxygen species, and cytokine production. Activated resident macrophages release factors that attract and activate additional leukocytes. Neutrophils are among the first leukocytes to infiltrate sites of inflammation. Monocytes are also recruited and infiltrate the inflammatory sites where, over the course of several days, they differentiate into exudate (elicited) macrophages. Inflammatory loci tend to be acidic. For example, the pH of the interstitial fluids of tumors and abscesses can be 6.5 (Bessman et al., 1990; Griffiths, 2001). Further, phagocyte activation causes a substantial increase in cell metabolic rate (Auger & Ross, 1992) and, concurrently, the rate of metabolic H production (Heming & Bidani, 1995a). Thus, during normal host defense, phagocytes are confronted with both exogenous and endogenous challenges to their acid-base homeostasis. The consequences of these challenges for intracellular pH (pHi) are an important determinant of phagocyte effector functions (Swallow et al., 1990; Lardner, 2001). The host defense functions of phagocytes are selectively influenced by cell acidbase status, including the extracellular pH (pHo). For example, acidosis (induced either by cell exposure to low pHo or by inhibition of plasma membrane H transport) suppresses some phagocyte functions (e.g., phagocytosis, generation of superoxide anions, and phorbol ester-induced cytotoxicity) (Geffner et al., 1993; Bidani & Heming, 1995; Bidani et al., 2000), but enhances other functions (e.g., nitric oxide production and immune complexinduced cytotoxicity) (Geffner et al., 1993; Bellocq et al., 1998). The pHi of phagocytes is controlled by the interplay of multiple plasmalemmal acid-base transporters. V-type H-translocating ATPase (V-ATPase), the Na/H exchanger (NHE), Na-dependent and independent Cl /HCO 3 exchangers, and voltagegated H channels have been described in the plasma membrane of various phagocytes (Swallow et al., 1990, 1991; DeCoursey & Cherney, 1996). The plasmalemmal V-ATPase and NHE normally function to extrude H from the cells, while the Nadependent Cl /HCO 3 exchanger transports HCO 3 into the cells. These transporters serve to protect the cells from cytosolic acid-loads. The Na-independent Cl /HCO 3 exchanger normally functions to extrude HCO 3 from the cells and, hence, serves to protect the cells from cytosolic alkaline-loads. H channels enable H flux down the prevailing electrochemical H gradient and, thus, facilitate H extrusion under conditions in which the plasma membrane potential is less negative than (i.e.,
depolarized with respect to) the equilibrium H potential (EH). Phagocytes exploit a number of strategies for regulating pHi, particularly as regards the relative contributions of Na/H exchange and plasmalemmal V-ATPase activity. Circulating (resting) neutrophils rely primarily on the NHE for basal H extrusion (Grinstein et al., 1988; Swallow et al., 1990). Activation of neutrophils is accompanied by up-regulation of Na/H exchange (Grinstein et al., 1988; Swallow et al., 1990) and the expression of plasmalemmal V-ATPase activity (Nanda et al., 1996). In contrast, resting resident macrophages rely primarily on plasmalemmal V-ATPase activity for pHi regulation (Bidani et al., 1989, 1994). Activation of resident macrophages is accompanied by a general up-regulation of Na/H exchange, but has stimulus-specific effects on the plasmalemmal H-pump (Heming & Bidani, 1995a; Bidani & Heming, 1998). In elicited macrophages, plasmalemmal V-ATPase and the NHE share responsibility for pHi regulation (Swallow et al., 1988). Previous studies have shown that Na/H exchange plays an important role in pHi regulation in resting and activated monocytes (Forsbeck et al., 1988; Ladoux et al., 1988; Swallow et al., 1990). However, the role of plasmalemmal V-ATPase in monocyte pHi regulation is unclear. The present study investigated the plasmalemmal V-ATPase and NHE in human histiocytic lymphoma U937 cells. This cell line has phenotypic properties of immature monocytic cells (Sundstrom & Nilssom, 1976) and can be induced to differentiate into macrophage-like cells. Baseline pHi and the kinetics of pHi recovery following a cytosolic acid-load (NH4Cl prepulse) were monitored. 5-(N-ethyl-Nisopropyl)amiloride (EIPA: a selective blocker of Na/H exchange) and bafilomycin A1 (a selective inhibitor of V-ATPase) were used to delineate the activities of the NHE and H-pump, respectively. The results showed that the H-pump and NHE interacted to set baseline pHi and for pHi recovery following cytosolic acid-loading of the monocyte cell line. The data demonstrate that plasmalemmal V-ATPase participates in pHi regulation in U937 cells.
Materials and methods Cell culture U937 cells (ATCC CRL-1593.2) were obtained from the Tissue Culture Core Facility, Department of Microbiology and Immunology, University of Texas
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Medical Branch (Galveston, TX). In the laboratory, the cells were washed and then incubated overnight in RPMI-1640 medium (pHo 7.4) supplemented with 15% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ ml streptomycin at 37 8C in an humidified atmosphere of 5% CO2.
bafilomycin A1 for 10 min (pHo 6.5 ± 7.4) and then titrated with a known concentration of sodium propionate (equimolar replacement of NaCl in PSS). b was computed from the resultant change in pHi, as described previously (Heming & Bidani, 1995b).
Measurement of pHi
HEPES was purchased from Research Organics (Cleveland, OH). RPMI-1640 was purchased from Gibco (Grand Island, NY). BCECF was purchased from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma (St. Louis, MO).
The pHi was measured using 2',7'-biscarboxyethyl5,6-carboxyfluorescein (BCECF), as described previously (Bidani et al., 1989, 1994). The cells were loaded with BCECF by incubation with the acetoxymethyl ester form of the probe (BCECF-AM in RPMI-1640 medium) for 30 min at room temperature. BCECF-loaded monocytes were then pelleted and suspended (1 106 cells/ml) in a physiologic salt solution (PSS) of 138 mM NaCl, 7 mM KCl, 2 mM CaCl2, 10 mM d-glucose, and 10 mM HEPES (pH 6.0 ± 7.4, 37 8C). Fluorescence intensities of the cells were monitored at 37 8C using excitation wavelengths of 504 nm (peak fluorescence) and 435 nm (isosbestic fluorescence), and an emission wavelength of 527 nm (model F-2000, Hitachi Instruments Inc., San Jose, CA). The fluorescence intensity ratio (peak/isosbestic) was calibrated to pHi using the high K-nigericin technique (Bidani et al., 1989). The experiments were conducted using a nominally CO2-free solution to minimize the confounding effects of plasmalemmal CO2-HCO3 flux on the assessment of H transport. To quantify plasmalemmal H extrusion, monocytes were subjected to a cytosolic acid-load (NH4Cl prepulse method) and pHi recovery was monitored as the cellular acid-base status returned back to baseline. Cells were pretreated with 10 ± 25 mM NH4Cl (equimolar substitution for NaCl in PSS) for 10 min and then transferred to NH4Cl-free PSS for pHi measurements. In studies with EIPA, the blocker was present in the recovery medium (i.e., the NH4Clfree PSS). In studies with bafilomycin A1, the inhibitor was present in both the NH4Cl-loading and recovery media. The rate of pHi recovery (dpHi/ dt) at given pHi values was computed from the digitized data record, as described previously (Bidani & Heming, 1997). Measurement of cytosolic buffering capacity The non-bicarbonate buffering capacity (b) of BCECF-loaded U937 cells was determined by titrating intact cells with weak acid (sodium propionate) in the nominal absence of CO2-HCO3 . The cells were incubated with 100 mM EIPA plus 10 mM
Chemicals
Data presentation and statistical analysis The data are presented as arithmetic means SE. Statistical analysis of the data included Student's t-tests or regression analysis, as appropriate (SigmaStat, Jandel Scientific, San Rafael, CA). A probability (P) value 0.05 was considered significant.
Results Under the present experimental conditions (nominally CO2-free experimental medium, 37 8C), the baseline pHi of U937 monocytes was 7.126 0.050 (n 7) at pHo 7.4. This yields an EH of -17 3 mV. The baseline pHi fell progressively and EH depolarized at lower pHo values (Figs. 1A and 1B). Figures 2A and 2B give representative data traces showing the effects on baseline pHi of 100 mM EIPA and/or 10 mM bafilomycin A1 at pHo 7.4 and 6.0, respectively. EIPA had no effect on the baseline pHi at pHo 7.4, but caused a sustained cellular acidosis at lower pHo values (Fig. 2C). This suggests that EIPAsensitive H transport (presumably Na/H exchange) played a role in setting the baseline pHi, at least at pHo values 7.0. In contrast, bafilomycin A1 elicited biphasic changes in baseline pHi at pHo 6.5 ± 7.4 (Figs. 2A and 2C). Exposure to bafilomycin A1 caused the baseline pHi to fall by 0.1 units over the course of 1 ± 2 minutes. The pHi then recovered back to or slightly overshot the original baseline value. At pHo 6.0, no recovery was detected during continued cell exposure to the V-ATPase inhibitor (figures 2B and 2C). Recovery from the bafilomycin-induced acidosis at pHo 6.5 ± 7.4 was sensitive to EIPA. The combination of bafilomycin A1 and EIPA caused a sustained decrease in pHi (figures 2A and 2C). These findings indicate that baseline pHi was influenced by
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Fig. 1. Effects of extracellular pH (pHo) on the intracellular pH (pHi: panel A) and equilibrium H potential (EH: panel B) of U937 cells. Data are mean values SE (n 6 ± 7). *Significantly different from paired value at pHo 7.4 (P < 0.05).
V-ATPase activity (in addition to the influence exerted by Na/H exchange). Further, with respect to the maintenance of baseline pHi, the NHE was able to compensate for inhibition of the H-pump at pHo values 6.5. Figure 3A shows representative time courses for monocyte pHi following cytosolic acid-loading by Fig. 2. Effects of EIPA and bafilomycin A1 (baf) on baseline pHi of U937 monocytes. Panel A: representative time courses at pHo 7.4. Panel B: representative time courses at pHo 6.0. Panel C: change in baseline pHi (DpHi) at pHo 6.0 ± 7.4. baf(nadir) DpHi at bafilomycin-induced pHi nadir. baf(final) DpHi after attainment of steadystate in the presence of bafilomycin A1. Data are mean values SE (n 4 ± 5). *Significantly different from zero (P < 0.05).
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Fig. 4. Non-bicarbonate buffering capacity (b) of U937 monocytes, as a function of pHi. Data are mean values SE (n 6 ± 12).
Fig. 3. Effects of EIPA and bafilomycin A1 (baf) on pHi recovery of U937 cells following a cytosolic acid-load (NH4Cl prepulse, pHo 7.4). Panel A: representative time courses. Panel B: rate of pHi recovery (dpHi/dt) as a function of pHi during the recovery process. Data are mean values SE (n 6 ± 10).
way of an NH4Cl prepulse. The cells were pretreated with 10 ± 25 mM NH4Cl (pHo 7.4) for 10 minutes and then transferred to stock PSS (i.e., NH4Cl-free saline, pH 7.4) for measurement of pHi. Control cells acid-loaded to a pHi nadir of 6.346 0.013 (n 10) and pHi then recovered back to the original baseline value with an exponential time course. The rate of pHi recovery (i.e., dpHi/dt) of control cells declined in a non-linear fashion during the recovery process, as pHi approached the baseline value (figure 3B). Recovery of pHi from a cytosolic acid-load (NH4Cl prepulse) was sensitive to both EIPA and bafilomcyin A1 (Figs. 3A and 3B). Recovery was slowed markedly in the presence of 100 mM EIPA.
Bafilomycin A1 (10 mM) also slowed pHi recovery but to a lesser degree than EIPA. The combination of EIPA and bafilomycin A1 had an additive effect in slowing the recovery process. Under the present experimental conditions, the dpHi/dt was expected to be a function of the H extrusion rate (kH, mol H/ min/cm2 of cell surface), the cytosolic non-bicarbonate buffering capacity (b, mmol H/L/pH unit), and the monocyte surface area-to-volume ratio (SVR, cm 1): dpHi/dt kH ¥ SVR / b. Ignoring the modest changes in cell SVR that can occur during pHi recovery from an ammonia prepulse [Dr. G. Altenburg, University of Texas Medical Branch, Galveston, TX: personal communication], we calculated an apparent H flux (JH, mM/min) as the product of dpHi/dt and b. The b of U937 monocytes varied as an exponential function of pHi (Fig. 4). Activity of the NHE was computed as the EIPA-sensitive component of the pHi recovery process, that is, the difference in JH between paired control and EIPAtreated monocytes at a given pHi value. Similarly, plasma membrane V-ATPase activity was computed as the bafilomycin-sensitive component of pHi recovery, i.e., the difference in JH between paired control and bafilomycin-treated cells at a given pHi value. Figure 5 shows the activities of the H-pump and NHE during pHi recovery from a cytosolic acid-load (NH4Cl prepulse). Both transporters were sensitive to pHi. Na/H exchange and V-ATPase activity increased progressively at lower pHi values, albeit the NHE was more sensitive to pHi than was the Hpump. Na/H exchange was the predominant mechanism for H extrusion at pHi values < 7.0: the NHE-mediated JH tended to be greater than the
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Fig. 5. Apparent H flux (JH) mediated by plasmalemmal VATPase activity and Na/H exchange, as a function of pHi during the recovery of U937 cells following a cytosolic acid-load (NH4Cl prepulse). Data are mean values SE (n 6 ± 10).
pump-mediated JH at a given pHi value < 7.0. At pHi values 7.0, the NHE and V-ATPase contributed almost equally to H extrusion. The pHi set-point of the NHE (i.e., pHi value below which the exchanger was allosterically activated) obviously was > 7.0 and, perhaps, was close to the normal baseline pHi value of U937 cells.
Discussion The pHi of U937 monocytes was controlled by the interplay of plasmalemmal V-ATPase and the NHE over the experimental pHo range (6.0 ± 7.4). The baseline pHi was 7.13 at pHo 7.4, in reasonable agreement with values found in other phagocytes under similar experimental conditions (Grinstein et al., 1988; Swallow et al., 1988; Bidani et al., 1989). An early investigation reported the apparent baseline pHi of U937 cells as 6.61, however, the authors acknowledged problems with the calibration of their pH probe (Forsbeck et al., 1988). In the present study, baseline pHi declined progressively with decrements in pHo (see figure 1A). Both the plasmalemmal H-pump and NHE were involved in setting the baseline pHi. Inhibition of the NHE with EIPA caused a significant cell acidosis at pHo values 7.0 (see figure 2C). Similar results have been published previously for U937 monocytes, albeit NHE inhibition was found to cause an acidosis at pHo 7.4 (Forsbeck et al., 1988; Ladoux et al., 1988). Inhibition of V-ATPase with bafilomycin A1 caused a
biphasic change in baseline pHi at pHo 6.5 ± 7.4 (pHi declined and then recovered) and a sustained acidosis at pHo 6.0 (see Figure 2). The acidic shift in baseline pHi probably reflected effects on plasmalemmal V-ATPase. It occurred too rapidly to be explained by a bafilomycin-induced collapse of organelle (endomembrane) pH gradients (Lukacs et al., 1990). Recovery from the bafilomycin-induced acidosis at pHo 6.5 ± 7.4 was sensitive to EIPA and, thus, presumably involved a compensatory increase in Na/H exchange. These findings indicate that the plasmalemmal H-pump and NHE shared responsibility for basal H extrusion in U937 monocytes. Moreover, with respect to maintaining baseline pHi, the two transporters were capable of compensatory changes in activity one-for-another. Control cells recovered their pHi following a cytosolic acid-load (NH4Cl prepulse) with an exponential time course (see Fig. 3A). The measured dpHi/dt and b values are in general agreement with data reported for other leukocytes under the same experimental conditions (Swallow et al., 1988; Bidani et al., 1989; Heming & Bidani, 1995b; McKinney & Moran, 1995). Plasmalemmal V-ATPase and the NHE both contributed to the pHi recovery process (see Fig. 5). However, the relative contribution of the transporters varied with pHi during the recovery process. At pHi values 7.0, the H-pump and NHE contributed almost equally to pHi recovery. At pHi values < 7.0, Na/H exchange dominated the recovery process. This finding reflected differences in the sensitivity of the transporters to pHi: the NHE was more sensitive to pHi than was the H-pump. The difference in pHisensitivity of the transporters in U937 cells is similar to that reported previously for primary cultures of resident alveolar macrophages (Heming & Bidani, 2002). The present data provide the first evidence that plasmalemmal V-ATPase activity is involved in pHi regulation in U937 monocytes. V-ATPase shared responsibility with the NHE for maintaining baseline pHi and for pHi recovery following cytosolic acid-loads. The present studies also confirm the importance of Na/H exchange for pHi regulation in monocytes (Forsbeck et al., 1988; Ladoux et al., 1988; Swallow et al., 1991). In addition to the Hpump and NHE, monocytes express plasmalemmal Cl /HCO 3 exchangers (Swallow et al., 1990, 1991) and H channels (DeCoursey & Cherny, 1996) that probably play a role in pHi regulation in vivo. However, it is doubtful that these transporters influenced the present results. In our studies, Cl / HCO 3 exchange was minimized by the nominal absence of CO2/HCO 3 in the experimental medium.
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H channel activity may have contributed to the slow change in pHi following an NH4Cl prepulse that was observed in U937 cells treated with EIPA plus bafilomycin A1 (see figure 3A). Nonetheless, this would not have influenced our determinations of Na/H exchange (i.e., EIPA-sensitive JH) or VATPase activity (i.e., bafilomycin-sensitive JH). The available data are insufficient to ascertain the effects of cell differentiation on the overall pHi regulatory strategy and prowess of mononuclear phagocytes. Previous studies have shown that differentiation of U937 cells into macrophage-like cells is associated with an up-regulation of Na/H exchange (Ladoux et al., 1988; Swallow et al., 1990). However, U937 cells may not be entirely representative of human monocytes or pre-monocytic cells. Lee et al. (1995) found that the total cellular content of immunoprecipitable V-ATPase increases markedly during the differentiation of human peripheral blood monocytes and human monocytic leukemia THP-1 cells. However, their studies did not distinguish between plasmalemmal and endomembrane V-ATPase. As well, the kinetics of plasmalemmal H channel gating are altered during the differentiation of THP-1 cells into macrophage-like cells (DeCoursey & Cherny, 1996), with potential effects on pHi regulation. Nonetheless, it is noteworthy that the shared role of plasmalemmal V-ATPase and the NHE in pHi regulation by U937 cells is similar to that reported for thioglycolate-elicited (exudate) murine peritoneal macrophages (Swallow et al., 1988). The present data also are in general agreement with findings for J774 cells (McKinney & Moran, 1995), a murine monocyte-macrophage cell line. The present findings for U937 cells differ markedly from those found in resident alveolar or peritoneal macrophages (Bidani et al., 1989, 1994; Tapper & Sundler, 1992). The difference appears to be related, in large part, to differences in the pHi set-point of the NHE in resident macrophages versus U937 cells. In resting resident macrophages, plasmalemmal V-ATPase activity sets the baseline pHi and is solely responsible for pHi recovery from moderate acid-loads (i.e., from acid-loaded pHi values 6.8 at pHo 7.4). The NHE in resting resident macrophages is functionally silent (or masked by V-ATPase activity) at pHi values 6.8. In other words, the pHi set-point of the exchanger is 6.8 (pHo 7.4) or about 0.3 ± 0.4 pH units below the baseline pHi of resident macrophages. In contrast, the exchanger's set-point in U937 monocytes appears to be close to the normal baseline pHi value (see figure 5). This is similar to the situation in many cell types, including neutrophils and elicited macrophages (Grinstein et al., 1988;
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Swallow et al., 1988, 1990). The factors determining the pHi set-point for Na/H exchange under steady-state conditions are not yet fully understood. Phagocyte acid-base status probably has broad and complex effects on inflammatory/immune responses. There is a sizable body of evidence indicating that the host defense functions of phagocytes are selectively modulated by cellular acid-base status. Generally-speaking, cell acidification suppresses chemotaxis and random cell migration, superoxide anion generation, phagocytosis and intraphagosomal killing of bacteria, and the release of proinflammatory cytokines (e.g., tumor necrosis factor-a) (Swallow et al., 1990; Bidani et al., 2000; Lardner, 2001). At the same time, cell acidification promotes phagocyte spreading and adherence (including plasmalemmal expression of CD-18, an adhesion molecule), and the production of NO (Bellocq et al., 1998; Trevani et al., 1999; Bidani et al., 2000; Lardner, 2001). Moreover, monocyte acid-base status can influence the cell differentiation process itself and, hence, may modulate the phenotype of monocyte-derived cells (Eljaafari et al., 1998). The effects of cellular and whole body acid-base status on immune functions are worthy of further investigation. Acknowledgements. Supported by NIH HL51421 and Constance Marsili Schafer Research Fund.
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Intracellular pH regulation in U937 human monocytes: roles of V-ATPase and Na/H exchange Thomas A. Heming, Akhil Bidani Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas, USA Received: March 27, 2002 ¥ Accepted: October 8, 2002
Abstract The role of plasmalemmal V-type H translocating ATPase (V-ATPase) in regulation of intracellular pH (pHi) is unclear in monocytes. This study examined the plasmalemmal VATPase and Na/H exchanger (NHE) in U937 human monocytes. The fluorescent probe 2',7'-biscarboxyethyl-5,6-carboxyfluorescein was used to monitor baseline pHi and the kinetics of pHi recovery from cytosolic acid-loads (NH4Cl prepulse). Bafilomycin A1 and 5(N-ethyl-N-isopropyl)amiloride (EIPA) were used to delineate the activities of the H-pump and NHE, respectively. Baseline pHi was 7.13 at an extracellular pH (pHo) of 7.4 and fell progressively at lower pHo values. EIPA had no effect on baseline pHi at pHo 7.4, but caused a sustained decrement in pHi at pHo 6.0 ± 7.0. Bafilomycin A1 had biphasic effects on baseline pHi at pHo 6.5 ± 7.4: pHi declined 0.1 units over the course of several minutes and then recovered. At pHo 6.0, bafilomycin A1 caused a sustained decrement in baseline pHi. Recovery from the bafilomycin-induced acidosis at pHo 6.5 ± 7.4 was prevented by EIPA. Similarly, pHi recovery from NH4Cl prepulse acid-loads (pHo 7.4) was sensitive to both EIPA and bafilomycin A1. During this recovery process, Na/H exchange (EIPA-sensitive component of apparent H efflux) was the predominant mechanism for H extrusion at acid-loaded pHi values < 7.0. At acid-loaded pHi values 7.0, the V-ATPase (bafilomycinsensitive component) and NHE contributed almost equally to H extrusion. The data provide the first evidence that plasmalemmal V-ATPase participates in pHi regulation in U937 cells. The H-pump and NHE interacted to set baseline pHi and for pHi recovery following cytosolic acid-loading of the monocytes. Abbreviations: BCECF 2',7'-biscarboxyethyl-5,6-carboxyfluorescein; b cytosolic nonbicarbonate buffering capacity; dpHi/dt rate of pHi recovery; EH equilibrium H potential; EIPA 5-(N-ethyl-N-isopropyl)amiloride; JH, apparent H flux; NHE Na/ H exchanger; pHi intracellular pH; pHo extracellular pH; PSS physiologic salt solution; SVR surface area-to-volume ratio; V-ATPase V-type H translocating ATPase.
Introduction Phagocytes, namely monocytes, macrophages, and neutrophils, are fundamental components of innate
immunity (Medzhitov & Janeway, 2000; Hawiger, 2001). Resident tissue macrophages serve as sentinel cells. They respond to inflammatory/immune insults by up-regulating (activating) their host defense
Correspondence author: Thomas A. Heming, Ph.D., Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Texas Health Science Center, Houston, TX 77225-0708, USA. Phone: 1-713-500-6835; Fax: 1-713-500-6829; E-mail: [email protected]
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functions, including phagocytosis, generation of reactive oxygen species, and cytokine production. Activated resident macrophages release factors that attract and activate additional leukocytes. Neutrophils are among the first leukocytes to infiltrate sites of inflammation. Monocytes are also recruited and infiltrate the inflammatory sites where, over the course of several days, they differentiate into exudate (elicited) macrophages. Inflammatory loci tend to be acidic. For example, the pH of the interstitial fluids of tumors and abscesses can be 6.5 (Bessman et al., 1990; Griffiths, 2001). Further, phagocyte activation causes a substantial increase in cell metabolic rate (Auger & Ross, 1992) and, concurrently, the rate of metabolic H production (Heming & Bidani, 1995a). Thus, during normal host defense, phagocytes are confronted with both exogenous and endogenous challenges to their acid-base homeostasis. The consequences of these challenges for intracellular pH (pHi) are an important determinant of phagocyte effector functions (Swallow et al., 1990; Lardner, 2001). The host defense functions of phagocytes are selectively influenced by cell acidbase status, including the extracellular pH (pHo). For example, acidosis (induced either by cell exposure to low pHo or by inhibition of plasma membrane H transport) suppresses some phagocyte functions (e.g., phagocytosis, generation of superoxide anions, and phorbol ester-induced cytotoxicity) (Geffner et al., 1993; Bidani & Heming, 1995; Bidani et al., 2000), but enhances other functions (e.g., nitric oxide production and immune complexinduced cytotoxicity) (Geffner et al., 1993; Bellocq et al., 1998). The pHi of phagocytes is controlled by the interplay of multiple plasmalemmal acid-base transporters. V-type H-translocating ATPase (V-ATPase), the Na/H exchanger (NHE), Na-dependent and independent Cl /HCO 3 exchangers, and voltagegated H channels have been described in the plasma membrane of various phagocytes (Swallow et al., 1990, 1991; DeCoursey & Cherney, 1996). The plasmalemmal V-ATPase and NHE normally function to extrude H from the cells, while the Nadependent Cl /HCO 3 exchanger transports HCO 3 into the cells. These transporters serve to protect the cells from cytosolic acid-loads. The Na-independent Cl /HCO 3 exchanger normally functions to extrude HCO 3 from the cells and, hence, serves to protect the cells from cytosolic alkaline-loads. H channels enable H flux down the prevailing electrochemical H gradient and, thus, facilitate H extrusion under conditions in which the plasma membrane potential is less negative than (i.e.,
depolarized with respect to) the equilibrium H potential (EH). Phagocytes exploit a number of strategies for regulating pHi, particularly as regards the relative contributions of Na/H exchange and plasmalemmal V-ATPase activity. Circulating (resting) neutrophils rely primarily on the NHE for basal H extrusion (Grinstein et al., 1988; Swallow et al., 1990). Activation of neutrophils is accompanied by up-regulation of Na/H exchange (Grinstein et al., 1988; Swallow et al., 1990) and the expression of plasmalemmal V-ATPase activity (Nanda et al., 1996). In contrast, resting resident macrophages rely primarily on plasmalemmal V-ATPase activity for pHi regulation (Bidani et al., 1989, 1994). Activation of resident macrophages is accompanied by a general up-regulation of Na/H exchange, but has stimulus-specific effects on the plasmalemmal H-pump (Heming & Bidani, 1995a; Bidani & Heming, 1998). In elicited macrophages, plasmalemmal V-ATPase and the NHE share responsibility for pHi regulation (Swallow et al., 1988). Previous studies have shown that Na/H exchange plays an important role in pHi regulation in resting and activated monocytes (Forsbeck et al., 1988; Ladoux et al., 1988; Swallow et al., 1990). However, the role of plasmalemmal V-ATPase in monocyte pHi regulation is unclear. The present study investigated the plasmalemmal V-ATPase and NHE in human histiocytic lymphoma U937 cells. This cell line has phenotypic properties of immature monocytic cells (Sundstrom & Nilssom, 1976) and can be induced to differentiate into macrophage-like cells. Baseline pHi and the kinetics of pHi recovery following a cytosolic acid-load (NH4Cl prepulse) were monitored. 5-(N-ethyl-Nisopropyl)amiloride (EIPA: a selective blocker of Na/H exchange) and bafilomycin A1 (a selective inhibitor of V-ATPase) were used to delineate the activities of the NHE and H-pump, respectively. The results showed that the H-pump and NHE interacted to set baseline pHi and for pHi recovery following cytosolic acid-loading of the monocyte cell line. The data demonstrate that plasmalemmal V-ATPase participates in pHi regulation in U937 cells.
Materials and methods Cell culture U937 cells (ATCC CRL-1593.2) were obtained from the Tissue Culture Core Facility, Department of Microbiology and Immunology, University of Texas
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Medical Branch (Galveston, TX). In the laboratory, the cells were washed and then incubated overnight in RPMI-1640 medium (pHo 7.4) supplemented with 15% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ ml streptomycin at 37 8C in an humidified atmosphere of 5% CO2.
bafilomycin A1 for 10 min (pHo 6.5 ± 7.4) and then titrated with a known concentration of sodium propionate (equimolar replacement of NaCl in PSS). b was computed from the resultant change in pHi, as described previously (Heming & Bidani, 1995b).
Measurement of pHi
HEPES was purchased from Research Organics (Cleveland, OH). RPMI-1640 was purchased from Gibco (Grand Island, NY). BCECF was purchased from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma (St. Louis, MO).
The pHi was measured using 2',7'-biscarboxyethyl5,6-carboxyfluorescein (BCECF), as described previously (Bidani et al., 1989, 1994). The cells were loaded with BCECF by incubation with the acetoxymethyl ester form of the probe (BCECF-AM in RPMI-1640 medium) for 30 min at room temperature. BCECF-loaded monocytes were then pelleted and suspended (1 106 cells/ml) in a physiologic salt solution (PSS) of 138 mM NaCl, 7 mM KCl, 2 mM CaCl2, 10 mM d-glucose, and 10 mM HEPES (pH 6.0 ± 7.4, 37 8C). Fluorescence intensities of the cells were monitored at 37 8C using excitation wavelengths of 504 nm (peak fluorescence) and 435 nm (isosbestic fluorescence), and an emission wavelength of 527 nm (model F-2000, Hitachi Instruments Inc., San Jose, CA). The fluorescence intensity ratio (peak/isosbestic) was calibrated to pHi using the high K-nigericin technique (Bidani et al., 1989). The experiments were conducted using a nominally CO2-free solution to minimize the confounding effects of plasmalemmal CO2-HCO3 flux on the assessment of H transport. To quantify plasmalemmal H extrusion, monocytes were subjected to a cytosolic acid-load (NH4Cl prepulse method) and pHi recovery was monitored as the cellular acid-base status returned back to baseline. Cells were pretreated with 10 ± 25 mM NH4Cl (equimolar substitution for NaCl in PSS) for 10 min and then transferred to NH4Cl-free PSS for pHi measurements. In studies with EIPA, the blocker was present in the recovery medium (i.e., the NH4Clfree PSS). In studies with bafilomycin A1, the inhibitor was present in both the NH4Cl-loading and recovery media. The rate of pHi recovery (dpHi/ dt) at given pHi values was computed from the digitized data record, as described previously (Bidani & Heming, 1997). Measurement of cytosolic buffering capacity The non-bicarbonate buffering capacity (b) of BCECF-loaded U937 cells was determined by titrating intact cells with weak acid (sodium propionate) in the nominal absence of CO2-HCO3 . The cells were incubated with 100 mM EIPA plus 10 mM
Chemicals
Data presentation and statistical analysis The data are presented as arithmetic means SE. Statistical analysis of the data included Student's t-tests or regression analysis, as appropriate (SigmaStat, Jandel Scientific, San Rafael, CA). A probability (P) value 0.05 was considered significant.
Results Under the present experimental conditions (nominally CO2-free experimental medium, 37 8C), the baseline pHi of U937 monocytes was 7.126 0.050 (n 7) at pHo 7.4. This yields an EH of -17 3 mV. The baseline pHi fell progressively and EH depolarized at lower pHo values (Figs. 1A and 1B). Figures 2A and 2B give representative data traces showing the effects on baseline pHi of 100 mM EIPA and/or 10 mM bafilomycin A1 at pHo 7.4 and 6.0, respectively. EIPA had no effect on the baseline pHi at pHo 7.4, but caused a sustained cellular acidosis at lower pHo values (Fig. 2C). This suggests that EIPAsensitive H transport (presumably Na/H exchange) played a role in setting the baseline pHi, at least at pHo values 7.0. In contrast, bafilomycin A1 elicited biphasic changes in baseline pHi at pHo 6.5 ± 7.4 (Figs. 2A and 2C). Exposure to bafilomycin A1 caused the baseline pHi to fall by 0.1 units over the course of 1 ± 2 minutes. The pHi then recovered back to or slightly overshot the original baseline value. At pHo 6.0, no recovery was detected during continued cell exposure to the V-ATPase inhibitor (figures 2B and 2C). Recovery from the bafilomycin-induced acidosis at pHo 6.5 ± 7.4 was sensitive to EIPA. The combination of bafilomycin A1 and EIPA caused a sustained decrease in pHi (figures 2A and 2C). These findings indicate that baseline pHi was influenced by
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Fig. 1. Effects of extracellular pH (pHo) on the intracellular pH (pHi: panel A) and equilibrium H potential (EH: panel B) of U937 cells. Data are mean values SE (n 6 ± 7). *Significantly different from paired value at pHo 7.4 (P < 0.05).
V-ATPase activity (in addition to the influence exerted by Na/H exchange). Further, with respect to the maintenance of baseline pHi, the NHE was able to compensate for inhibition of the H-pump at pHo values 6.5. Figure 3A shows representative time courses for monocyte pHi following cytosolic acid-loading by Fig. 2. Effects of EIPA and bafilomycin A1 (baf) on baseline pHi of U937 monocytes. Panel A: representative time courses at pHo 7.4. Panel B: representative time courses at pHo 6.0. Panel C: change in baseline pHi (DpHi) at pHo 6.0 ± 7.4. baf(nadir) DpHi at bafilomycin-induced pHi nadir. baf(final) DpHi after attainment of steadystate in the presence of bafilomycin A1. Data are mean values SE (n 4 ± 5). *Significantly different from zero (P < 0.05).
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Fig. 4. Non-bicarbonate buffering capacity (b) of U937 monocytes, as a function of pHi. Data are mean values SE (n 6 ± 12).
Fig. 3. Effects of EIPA and bafilomycin A1 (baf) on pHi recovery of U937 cells following a cytosolic acid-load (NH4Cl prepulse, pHo 7.4). Panel A: representative time courses. Panel B: rate of pHi recovery (dpHi/dt) as a function of pHi during the recovery process. Data are mean values SE (n 6 ± 10).
way of an NH4Cl prepulse. The cells were pretreated with 10 ± 25 mM NH4Cl (pHo 7.4) for 10 minutes and then transferred to stock PSS (i.e., NH4Cl-free saline, pH 7.4) for measurement of pHi. Control cells acid-loaded to a pHi nadir of 6.346 0.013 (n 10) and pHi then recovered back to the original baseline value with an exponential time course. The rate of pHi recovery (i.e., dpHi/dt) of control cells declined in a non-linear fashion during the recovery process, as pHi approached the baseline value (figure 3B). Recovery of pHi from a cytosolic acid-load (NH4Cl prepulse) was sensitive to both EIPA and bafilomcyin A1 (Figs. 3A and 3B). Recovery was slowed markedly in the presence of 100 mM EIPA.
Bafilomycin A1 (10 mM) also slowed pHi recovery but to a lesser degree than EIPA. The combination of EIPA and bafilomycin A1 had an additive effect in slowing the recovery process. Under the present experimental conditions, the dpHi/dt was expected to be a function of the H extrusion rate (kH, mol H/ min/cm2 of cell surface), the cytosolic non-bicarbonate buffering capacity (b, mmol H/L/pH unit), and the monocyte surface area-to-volume ratio (SVR, cm 1): dpHi/dt kH ¥ SVR / b. Ignoring the modest changes in cell SVR that can occur during pHi recovery from an ammonia prepulse [Dr. G. Altenburg, University of Texas Medical Branch, Galveston, TX: personal communication], we calculated an apparent H flux (JH, mM/min) as the product of dpHi/dt and b. The b of U937 monocytes varied as an exponential function of pHi (Fig. 4). Activity of the NHE was computed as the EIPA-sensitive component of the pHi recovery process, that is, the difference in JH between paired control and EIPAtreated monocytes at a given pHi value. Similarly, plasma membrane V-ATPase activity was computed as the bafilomycin-sensitive component of pHi recovery, i.e., the difference in JH between paired control and bafilomycin-treated cells at a given pHi value. Figure 5 shows the activities of the H-pump and NHE during pHi recovery from a cytosolic acid-load (NH4Cl prepulse). Both transporters were sensitive to pHi. Na/H exchange and V-ATPase activity increased progressively at lower pHi values, albeit the NHE was more sensitive to pHi than was the Hpump. Na/H exchange was the predominant mechanism for H extrusion at pHi values < 7.0: the NHE-mediated JH tended to be greater than the
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Fig. 5. Apparent H flux (JH) mediated by plasmalemmal VATPase activity and Na/H exchange, as a function of pHi during the recovery of U937 cells following a cytosolic acid-load (NH4Cl prepulse). Data are mean values SE (n 6 ± 10).
pump-mediated JH at a given pHi value < 7.0. At pHi values 7.0, the NHE and V-ATPase contributed almost equally to H extrusion. The pHi set-point of the NHE (i.e., pHi value below which the exchanger was allosterically activated) obviously was > 7.0 and, perhaps, was close to the normal baseline pHi value of U937 cells.
Discussion The pHi of U937 monocytes was controlled by the interplay of plasmalemmal V-ATPase and the NHE over the experimental pHo range (6.0 ± 7.4). The baseline pHi was 7.13 at pHo 7.4, in reasonable agreement with values found in other phagocytes under similar experimental conditions (Grinstein et al., 1988; Swallow et al., 1988; Bidani et al., 1989). An early investigation reported the apparent baseline pHi of U937 cells as 6.61, however, the authors acknowledged problems with the calibration of their pH probe (Forsbeck et al., 1988). In the present study, baseline pHi declined progressively with decrements in pHo (see figure 1A). Both the plasmalemmal H-pump and NHE were involved in setting the baseline pHi. Inhibition of the NHE with EIPA caused a significant cell acidosis at pHo values 7.0 (see figure 2C). Similar results have been published previously for U937 monocytes, albeit NHE inhibition was found to cause an acidosis at pHo 7.4 (Forsbeck et al., 1988; Ladoux et al., 1988). Inhibition of V-ATPase with bafilomycin A1 caused a
biphasic change in baseline pHi at pHo 6.5 ± 7.4 (pHi declined and then recovered) and a sustained acidosis at pHo 6.0 (see Figure 2). The acidic shift in baseline pHi probably reflected effects on plasmalemmal V-ATPase. It occurred too rapidly to be explained by a bafilomycin-induced collapse of organelle (endomembrane) pH gradients (Lukacs et al., 1990). Recovery from the bafilomycin-induced acidosis at pHo 6.5 ± 7.4 was sensitive to EIPA and, thus, presumably involved a compensatory increase in Na/H exchange. These findings indicate that the plasmalemmal H-pump and NHE shared responsibility for basal H extrusion in U937 monocytes. Moreover, with respect to maintaining baseline pHi, the two transporters were capable of compensatory changes in activity one-for-another. Control cells recovered their pHi following a cytosolic acid-load (NH4Cl prepulse) with an exponential time course (see Fig. 3A). The measured dpHi/dt and b values are in general agreement with data reported for other leukocytes under the same experimental conditions (Swallow et al., 1988; Bidani et al., 1989; Heming & Bidani, 1995b; McKinney & Moran, 1995). Plasmalemmal V-ATPase and the NHE both contributed to the pHi recovery process (see Fig. 5). However, the relative contribution of the transporters varied with pHi during the recovery process. At pHi values 7.0, the H-pump and NHE contributed almost equally to pHi recovery. At pHi values < 7.0, Na/H exchange dominated the recovery process. This finding reflected differences in the sensitivity of the transporters to pHi: the NHE was more sensitive to pHi than was the H-pump. The difference in pHisensitivity of the transporters in U937 cells is similar to that reported previously for primary cultures of resident alveolar macrophages (Heming & Bidani, 2002). The present data provide the first evidence that plasmalemmal V-ATPase activity is involved in pHi regulation in U937 monocytes. V-ATPase shared responsibility with the NHE for maintaining baseline pHi and for pHi recovery following cytosolic acid-loads. The present studies also confirm the importance of Na/H exchange for pHi regulation in monocytes (Forsbeck et al., 1988; Ladoux et al., 1988; Swallow et al., 1991). In addition to the Hpump and NHE, monocytes express plasmalemmal Cl /HCO 3 exchangers (Swallow et al., 1990, 1991) and H channels (DeCoursey & Cherny, 1996) that probably play a role in pHi regulation in vivo. However, it is doubtful that these transporters influenced the present results. In our studies, Cl / HCO 3 exchange was minimized by the nominal absence of CO2/HCO 3 in the experimental medium.
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H channel activity may have contributed to the slow change in pHi following an NH4Cl prepulse that was observed in U937 cells treated with EIPA plus bafilomycin A1 (see figure 3A). Nonetheless, this would not have influenced our determinations of Na/H exchange (i.e., EIPA-sensitive JH) or VATPase activity (i.e., bafilomycin-sensitive JH). The available data are insufficient to ascertain the effects of cell differentiation on the overall pHi regulatory strategy and prowess of mononuclear phagocytes. Previous studies have shown that differentiation of U937 cells into macrophage-like cells is associated with an up-regulation of Na/H exchange (Ladoux et al., 1988; Swallow et al., 1990). However, U937 cells may not be entirely representative of human monocytes or pre-monocytic cells. Lee et al. (1995) found that the total cellular content of immunoprecipitable V-ATPase increases markedly during the differentiation of human peripheral blood monocytes and human monocytic leukemia THP-1 cells. However, their studies did not distinguish between plasmalemmal and endomembrane V-ATPase. As well, the kinetics of plasmalemmal H channel gating are altered during the differentiation of THP-1 cells into macrophage-like cells (DeCoursey & Cherny, 1996), with potential effects on pHi regulation. Nonetheless, it is noteworthy that the shared role of plasmalemmal V-ATPase and the NHE in pHi regulation by U937 cells is similar to that reported for thioglycolate-elicited (exudate) murine peritoneal macrophages (Swallow et al., 1988). The present data also are in general agreement with findings for J774 cells (McKinney & Moran, 1995), a murine monocyte-macrophage cell line. The present findings for U937 cells differ markedly from those found in resident alveolar or peritoneal macrophages (Bidani et al., 1989, 1994; Tapper & Sundler, 1992). The difference appears to be related, in large part, to differences in the pHi set-point of the NHE in resident macrophages versus U937 cells. In resting resident macrophages, plasmalemmal V-ATPase activity sets the baseline pHi and is solely responsible for pHi recovery from moderate acid-loads (i.e., from acid-loaded pHi values 6.8 at pHo 7.4). The NHE in resting resident macrophages is functionally silent (or masked by V-ATPase activity) at pHi values 6.8. In other words, the pHi set-point of the exchanger is 6.8 (pHo 7.4) or about 0.3 ± 0.4 pH units below the baseline pHi of resident macrophages. In contrast, the exchanger's set-point in U937 monocytes appears to be close to the normal baseline pHi value (see figure 5). This is similar to the situation in many cell types, including neutrophils and elicited macrophages (Grinstein et al., 1988;
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Swallow et al., 1988, 1990). The factors determining the pHi set-point for Na/H exchange under steady-state conditions are not yet fully understood. Phagocyte acid-base status probably has broad and complex effects on inflammatory/immune responses. There is a sizable body of evidence indicating that the host defense functions of phagocytes are selectively modulated by cellular acid-base status. Generally-speaking, cell acidification suppresses chemotaxis and random cell migration, superoxide anion generation, phagocytosis and intraphagosomal killing of bacteria, and the release of proinflammatory cytokines (e.g., tumor necrosis factor-a) (Swallow et al., 1990; Bidani et al., 2000; Lardner, 2001). At the same time, cell acidification promotes phagocyte spreading and adherence (including plasmalemmal expression of CD-18, an adhesion molecule), and the production of NO (Bellocq et al., 1998; Trevani et al., 1999; Bidani et al., 2000; Lardner, 2001). Moreover, monocyte acid-base status can influence the cell differentiation process itself and, hence, may modulate the phenotype of monocyte-derived cells (Eljaafari et al., 1998). The effects of cellular and whole body acid-base status on immune functions are worthy of further investigation. Acknowledgements. Supported by NIH HL51421 and Constance Marsili Schafer Research Fund.
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