©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-1 Increases Vacuolar-type H-ATPase Activity in Murine Peritoneal Macrophages (*)

(Received for publication, October 19, 1995)

Guy F. Brisseau (1)(§) Sergio Grinstein (2) (3)(¶) David J. Hackam (1) Tommy Nordström (1) (3) Morris F. Manolson (3)(**) Aye Aye Khine (1) Ori D. Rotstein (1)(§§)

From the  (1)Department of Surgery, The Toronto Hospital and University of Toronto, Toronto, Ontario M5G 1X8, the (2)Department of Biochemistry, University of Toronto, and the (3)Hospital for Sick Children Research Institute, Toronto, Ontario M5G 2C4, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Maintenance of cytoplasmic pH (pH) within a narrow physiological range is crucial to normal cellular function. This is of particular relevance to phagocytic cells within the acidic inflammatory microenvironment where the pH tends to be acid loaded. We have previously reported that a vacuolar-type H-ATPase (V-ATPase) situated in the plasma membrane of macrophages and poised to extrude protons from the cytoplasmic to the extracellular space is an important pH regulatory mechanism within the inflammatory milieu. Since this microenvironment is frequently characterized by the influx of cells known to release inflammatory cytokines, we performed studies to examine the effect of one such mediator molecule, interleukin-1 (IL-1), on pHregulation in peritoneal macrophages.

IL-1 caused a time- and dose-dependent increase in macrophage pH recovery from an acute acid load. This effect was specific to IL-1 and was due to enhanced plasmalemmal V-ATPase activity. The increased V-ATPase activity by IL-1 occurred following a lag period of several hours and required de novo protein and mRNA synthesis. However, Northern blot analysis revealed that IL-1 did not exert its effect via alterations in the levels of mRNA transcripts for the A or B subunits of the V-ATPase complex. Finally, stimulation of both cAMP-dependent protein kinase and protein kinase C was required for the stimulatory effect of IL-1 on V-ATPase activity.

Thus, cytokines present within the inflammatory milieu are able to modulate pH regulatory mechanisms. These data may represent a novel mechanism whereby cytokines may improve cellular function at inflammatory sites.


INTRODUCTION

Maintenance of the intracellular pH (pH) (^1)close to the physiological range is crucial to normal cell function due to the narrow pH optima of many cellular processes. Most mammalian cells have therefore evolved regulatory mechanisms to ensure the constancy of the pH. Inflammatory cells such as macrophages are particularly prone to intracellular acid accumulation. Metabolic activation following exposure to microbial products and proinflammatory mediators results in endogenous acid generation. In addition, the acidic nature of the inflammatory microenvironment leads to cytosolic acidification due to passive diffusion of protons into the cell. At least four major mechanisms poised to combat intracellular acid accumulation have been reported in cells of monocyte/macrophage lineage. These include 1) the Na/H exchanger(1) , 2) the Na-dependent HCO(3)/Cl exchanger(2) , 3) a H-conductive pathway(3) , and 4) the vacuolar-type H-ATPase (V-ATPase)(4, 5) . Under physiological conditions, all four mechanisms would be expected to contribute to the maintenance of pH homeostasis. On the other hand, under conditions of extracellular acidification, the first three mechanisms are rendered largely ineffective, as they are susceptible to inhibition by external protons, whereas the V-ATPase remains functional and is the predominant pH regulatory mechanism in macrophages. Recent studies from our laboratory suggest that normal function of this proton extrusion mechanism is required for optimal cell function in acidic environments(6) .

Sites of inflammation are characterized not only by their accumulation of cells but also by the presence of a large number of mediator molecules, which serve to modulate cell function. Interleukin-1 (IL-1) is one of several pluripotent cytokines detected at these sites(7) . It is produced mainly by macrophages but may be derived from other cell types. This cytokine has effects on a wide range of cell types, including autocrine/paracrine effects on macrophages. These cells possess receptors for IL-1 and in response to stimulation by this cytokine elaborate tumor necrosis factor(8) , prostaglandins(9) , procoagulants(10) , thromboxanes(11) , and additionally IL-1(12) .

Since optimal macrophage function in the inflammatory microenvironment is dependent on the maintenance of pH, we performed studies to evaluate the mechanisms whereby mediator molecules within the local inflammatory milieu might modulate macrophage pH regulation, in particular V-ATPase activity. The present studies evaluated the effect of IL-1 on V-ATPase activity in elicited murine peritoneal macrophages. IL-1 augmented V-ATPase-mediated pH recovery in these cells, a process requiring both protein and RNA synthesis. This effect was mediated through both a cyclic AMP-dependent pathway as well as a pathway involving protein kinase C. These studies are the first to describe modulatory effects of proinflammatory cytokines on plasmalemmal V-ATPases responsible for proton extrusion in mammalian cells.


EXPERIMENTAL PROCEDURES

Materials and Solutions

Powdered Brewer's thioglycolate was purchased from Difco Laboratories (Detroit, MI). Calcium- and magnesium-free Hanks' balanced salt solution (HBSS), fetal bovine serum (FCS), bicarbonate-free RPMI 1640, SDS, ethidium bromide, and nylon transfer membranes were obtained from Life Technologies, Inc. RPMI 1640, glutamine, prostaglandin E(2), 8-bromo-cAMP, cycloheximide, actinomycin D, guanidine thiocyanate, MES, and Tris(hydroxymethyl)aminomethane (Trizma base) were purchased from Sigma. Endotoxin-free human recombinant IL-1alpha was obtained from ICN Biochemicals. The acetoxymethyl ester of the pH-sensitive fluorescent probe 2`,7`-biscarboxyethyl-5-(6)-carboxyfluorescein (BCECF) was procured from Molecular Probes. KT5720, H-89, and herbimycin A were from Calbiochem. The DNA labeling T(7) quick prime kit was purchased from Pharmacia Biotech Inc. Bafilomycin A(1) was from Kamiya Biochemical Co. (Thousands Oaks, CA). The KCl medium used for pH(i) recovery studies contained 145 mM KCl, 10 mMD-glucose, 2 mM CaCl(2), and 10 mM HEPES. The KCl medium and bicarbonate-free RPMI were adjusted to pH 7.35 at 37 °C and osmolality was adjusted to 295 ± 5 mosM with the major salt. The thioglycolate was solubilized in H(2)O, autoclaved, and stored in the dark at room temperature until uniformly green and clear.

Cell Harvest and Incubation Conditions

Peritoneal cells were obtained from 6-week-old female Swiss Webster mice (Taconic Farms Inc., Germantown, NY). The mice received an intraperitoneal injection with 2 ml each of thioglycolate 5 days prior to harvest. Cells were harvested by peritoneal lavage with ice-cold HBSS. Cells were then centrifuged at 200 times g for 10 min, washed twice in HBSS, and counted with a hemocytometer. The cells were diluted to 2 times 10^6/ml in RPMI 1640 supplemented with 10% FCS and 2 mM glutamine and incubated with or without treatment in a CO(2)-controlled environment (5% CO(2) and 95% room air) at 37 °C for the indicated time period.

The peritoneal exudate cells consisted of a population containing more than 85% macrophages as assessed by Wright's staining, nonspecific esterase, and electron microscopy. Viability exceeded 95% both before and at the end of the treatment periods as determined by trypan blue exclusion. These cells are considered to be at a heightened level of activation compared to resident macrophages(13, 14) . They are, however, more representative of cells migrating to sites of inflammation and are appropriate for investigation of the ability of inflammatory mediators to modulate cell function.

pH(i) Measurement and Characterization

Spectrofluorimetry was used to continuously monitor pH(i) in cells loaded with the pH-sensitive fluorescent probe BCECF. Briefly, cells (2 times 10^6) were removed from the incubator at the indicated time point, sedimented, and resuspended in bicarbonate-free RPMI 1640 without FCS containing 0.1 µg/ml of the acetoxymethyl ester form of BCECF. Cytoplasmic acid loading was performed simultaneously by incubation with 50 mM NH(4)Cl. After 20 min at 37 °C, the cells were sedimented and resuspended in 2 ml of NH(4)-free KCl medium in a fluorescence spectrometer cuvette maintained at 37 °C. The rationale for the NH(4)Cl ``prepulse'' technique is discussed elsewhere(15, 16) . This procedure resulted in consistent acid loading of cells (initial pH(i) approx 6.4-6.5), which did not differ between control groups and the various treatment groups being compared.

Changes in fluorescence were monitored using a Perkin Elmer LS-50 fluorescence spectrometer with excitation wavelength of 495 nm and emission wavelength of 525 nm using 5- and 9-nm slit widths, respectively. Calibration of the fluorescence signal versus pH(i) was performed using the K/H ionophore, nigericin. Cells were equilibrated in K medium of varying pH (adjusted by the addition of MES) in the presence of 5 M nigericin, and calibration curves were constructed by plotting extracellular pH (which is assumed to be identical to pH(i); see (17) ) against the corresponding fluorescence signal. The pH(i) recovery rate was defined as the slope of the pH change during the first 0.1 pH units of recovery and expressed as pH/min. The use of a Na- and HCO(3)-free KCl medium during recovery measurements ensured that the majority of the pH(i) recovery detected was attributed to the activity of the previously described V-ATPase in these cells rather than the Na/H antiport or the Na-dependent HCO(3)/Cl exchanger, which are rendered inoperative under these conditions. This was confirmed by the ability of the specific V-ATPase inhibitor, bafilomycin A1, to effect almost complete inhibition of pH(i) recovery under these conditions (see below).

Measurement of Proton Extrusion

Acid extrusion from the macrophages was quantitated by continuously measuring the extracellular pH (pH(o)) with a conventional pH electrode in a magnetically stirred, water-jacketed chamber at 37 °C filled with 2 ml of lightly buffered KCl medium containing 0.2 mM HEPES. Four million cells were added to the medium, and the pH was adjusted to 7.4 with potassium hydroxide (KOH). Changes in pH(o) were monitored continuously, and calibration of the rate of extracellular acidification was performed by addition of known amounts of KOH. Data are expressed as nmol of H/million cells/min.

Measurement of Buffering Capacity

Buffering capacity of macrophages was determined after 4 h of treatment with and without IL-1. Briefly, following the incubation period, cells were loaded with BCECF and then resuspended in a cuvette with KCl medium. Following stabilization of pH(i), ammonium chloride (5 mM, final concentration) was added to the medium, and the new stable pH(i) was determined. The buffering capacity of the cells was calculated as detailed elsewhere and expressed as mmol/pH unit/liter (17, 18) .

RNA Isolation and Northern Blotting

The levels of mRNA transcripts for the A and B subunits of the V-ATPase were assessed by Northern blot analysis. Briefly, 10 times 10^6 cells were pelleted, and total RNA was extracted using the method of Chomczynski and Sacchi(19) . After electrophoresis, RNA was transferred to Immobilon and hybridized with the P random-labeled cDNA probe for the A and B subunits of the V-ATPase complex. The cDNA probes for the A subunit were derived from bovine brain (PKP40a, donated by Dr. Michael Forgac (Tufts University, Boston, MA); see (20) ) and from human osteoclasts (F880, donated by Dr. Benoit van Hille (CIBA-Giegy, Basel, Switzerland); see (21) ). The cDNA probes for the B subunit were derived from bovine brain and bovine kidney (PKP7a-1 and PQL12b, respectively, donated by Dr. Michael Forgac; see (22) ). Comparable RNA loading between lanes was confirmed assured by probing with a cDNA probe for rat alpha-tubulin (23) .

Western Blot Analysis

After 4 hours of incubation in the presence or absence of IL-1, cells were solubilized in Laemmli's buffer(24) , resolved by SDS-polyacrylamide gel electrophoresis using the Protean II minigel system (Bio-Rad) and then transferred onto nitrocellulose membranes. Membranes were then incubated in blocking buffer (0.25% gelatin, 10% ethanolamine, 1 M TrisbulletCl, pH 9.0), exposed to primary antibody solution overnight at 4 °C (dilution 1:1000), and washed three times for 10 min in antibody buffer (50 mM TrisbulletHCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20, 0.04% Nonidet P-40). Membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, washed, and developed using the enhanced chemiluminescence detection system (Amersham Corp.)(25) . The anti-39-kDa antibody was kindly provided by Dr. Nathan Nelson(26) . The antibody directed against the B subunit was generated using a fusion protein as follows.

A BglII fragment of the B subunit (generously provided by Dr. Gary Dean, University of Cincinnati Medical Center, Cincinnati, OH) encoding amino acid residues 206-378 was subcloned into BamHI-cut pGEX-3X (Pharmacia Biotech, Inc.) generating the plasmid pGEX-DHB. DH5alpha cells were then transformed with this plasmid by incubation for 45 s at 37 °C. Transformation efficiency was >90%. These cells were then grown to A = 0.4, induced by the addition of 0.4 mM isopropyl-beta-D-thiogalactopyranoside, and harvested after 3 h. Cells were pelleted in a JA-10 rotor, resuspended in 15 ml of ice-cold phosphate-buffered saline, and then sonicated with a 5-mm-diameter probe for 3 times 30 s on ice. Triton X-100 (1%) was added to the lysate and mixed for 5 min at 4 °C, and the suspension was centrifuged for 5 min in a Beckman JA-20 rotor at 9500 rpm. The supernatant was mixed with 1 ml of a 50% slurry of glutathione-Sepharose 4B (Pharmacia Biotech Inc.) for 30 min at 4 °C and then washed with 50 ml of ice-cold phosphate-buffered saline for 3 times 10 min. The fusion protein was eluted by the addition of 1 ml of 50 mM TrisbulletCl (pH 8.0)/5 mM reduced glutathione for 2 min followed by centrifugation at 500 times g for 30 s. Eluted fractions were resolved by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie Blue staining. The entire glutathione S-transferase fusion protein was injected into rabbits, and antibodies were generated.

Endotoxin Contamination

RPMI 1640, HBSS, and FCS were tested for endotoxin contamination using the standard Limulus amoebocyte lysate assay (Association of Cape Cod, Woods Hole, MA) and were found to contain <0.1 ng of endotoxin per ml, which constituted the lower limit of the test.

Statistics

All results are expressed as means ± S.E. for n experiments. Statistical significance was determined by one-way analysis of variance followed by post hoc Newman-Keuls testing. Significance was considered for p < 0.05.


RESULTS

IL-1 Increases V-ATPase-mediated pH(i) Recovery

The ammonium chloride ``prepulse'' technique induced a similar level of cytoplasmic acidification in control and IL-1-treated macrophages (Fig. 1A). Soon after the nadir was reached, pH(i) recovery began. Fig. 1A shows a representative tracing of pH(i) recovery in Na- and HCO(3)-free K-medium. These conditions permitted evaluation of V-ATPase-mediated pH(i) recovery in a setting where other major pH(i) regulatory mechanisms were inactive. Macrophages treated with IL-1 for 6 h demonstrated an increased rate of pH(i) recovery following an induced acid load compared to control cells. The dose response of IL-1 on pH(i) recovery is shown in Fig. 1B. IL-1 induces a dose-dependent increase in the initial rate of pH(i) recovery from an imposed acid load, reaching a maximum at 290 pM. This increase was not due to an alteration in the buffering capacity of the cell, since this did not differ between treatment groups (control cells 45.1 ± 3.7 mmol/pH unit/liter versus IL-1-treated cells 47.0 ± 4.0 mmol/pH unit/liter, n = 3 per group). As noted above, the conditions for pH(i) recovery were established to favor a V-ATPase-mediated recovery. To ensure that the observed increase in the recovery rate after treatment with IL-1 was due to stimulation of the V-ATPase and not some other previously undescribed mechanism, pH(i) recovery from acid-loaded macrophages following stimulation with the IL-1 was determined in the presence and absence of bafilomycin A1, a potent and selective inhibitor of V-ATPases (Fig. 1C). In both control and IL-1-treated cells, bafilomycin A1 reduced the pH(i) recovery to low and equivalent rates (control 0.191 ± 0.038 pH/min versus IL-1 0.194 ± 0.006 pH/min, n = 3). When the effect of IL-1 on V-ATPase activity is expressed as a change in bafilomycin-sensitive pH(i) recovery, the cytokine causes a 107% increase in activity compared to control cells. To confirm that the IL-1-induced increase in the rate of pH(i) recovery was due to augmented translocation of protons across the plasma membrane, a finding consistent with an effect on the plasmalemmal V-ATPase, the rate of proton extrusion into the extracellular medium was measured. As shown in Fig. 1D, proton extrusion rates were significantly increased in IL-1-treated cells compared to control. The increase was inhibited by the addition of bafilomycin A1, indicating an effect on the plasmalemmal V-ATPase. Finally, neutralizing anti-IL-1 antibody, but not an isotype-matched control antibody, completely reversed the stimulatory effect of IL-1 on the rate of pH(i) recovery (Fig. 1E), indicating the specificity of the effect of IL-1. When considered together, the data are consistent with the notion that IL-1 stimulates V-ATPase-mediated translocation of protons across the plasma membrane into the extracellular space.


Figure 1: A, effect of IL-1 on the rate of pH recovery. Macrophages were treated for 6 h in the presence or absence of IL-1 (290 pM). Cells were then loaded with BCECF and incubated with NH(4)Cl (50 mM) for 20 min at 37 °C to induce an acute acid load as described under ``Experimental Procedures.'' Cells were then resuspended in KCl medium, and pH was allowed to recover. The figure illustrates a representative trace of pH recovery in control and IL-1-treated cells. B, dose response of the effect of IL-1 on pH recovery in acid-loaded macrophages. Macrophages were incubated for 6 h with varying concentrations of IL-1. pHrecovery rate following an acute acid load was then determined as described under ``Experimental Procedures.'' The data represent the mean ± S.E. of 5-11 experiments. *, p < 0.05 versus no IL-1. C, effect of bafilomycin A1 on IL-1-induced enhancement of pH recovery. Macrophages were incubated with and without IL-1 (290 pM) for 6 h. During the last 20 min of the incubation period, bafilomycin A(1) (solid bars) or vehicle (open bars) was added, and cells were subsequently studied for pH recovery rate. The data represent the mean ± S.E. of three experiments. *, p < 0.05 versus no IL-1;**, p < 0.05 versus no bafilomycin A1. D, effect of IL-1 on macrophage proton extrusion rate. Macrophages were exposed to IL-1 (290 pM) for 6 h followed by quantitation of proton extrusion into the extracellular medium as described under ``Experimental Procedures.'' Cells were acid-loaded using the NH(4)Cl technique for the 20 min prior to the measurement of proton extrusion. Studies were performed in the presence of bafilomycin A(1) (closed bars) or vehicle (open bars). The data are expressed as the mean ± S.E. of seven to eight experiments. *, p < 0.05 versus no IL-1;**, p < 0.05 versus no bafilomycin A(1). E, effect of neutralizing anti-IL-1 antibody on pH recovery in IL-1-treated cells. Macrophages were pretreated with anti-IL-1 antibody (10 g/ml) (solid bars) or vehicle control (open bars) during a 4-h incubation with IL-1 (290 pM). The rate of pH recovery from an acid-loaded was then determined. The data represent the mean ± S.E. of three experiments. *, p < 0.05 versus all other groups.



IL-1-induced V-ATPase Activity Requires Protein and RNA Synthesis

An increase in V-ATPase activity may be due to exocytic translocation of V-ATPases present in intracellular organelles, activation of quiescent pumps already present in the plasmalemma, or de novo synthesis of additional pumps. Fig. 2illustrates a time course of the effect of IL-1 on V-ATPase-mediated pH(i) recovery. The stimulatory effect of the cytokine became evident only after a lag period of approximately 4 h, suggesting a requirement for protein synthesis. To directly test this possibility, the effects of cycloheximide, an inhibitor of protein synthesis, and actinomycin D, an inhibitor of RNA synthesis, on the IL-1-mediated stimulation were examined. As shown in Fig. 3, A and B, respectively, cycloheximide and actinomycin D were able to completely abrogate the ability of IL-1 to augment V-ATPase activity. Trypan blue exclusion remained >95% at the end of the incubation period, suggesting that the effect was not due to cellular toxicity.


Figure 2: Time course of the effect of IL-1 on macrophage pH recovery. Macrophages were incubated with or without IL-1 (290 pM) for varying times followed by quantitation of pH recovery. The data are the mean ± S.E. of four experiments. *, p < 0.05 versus all other groups.




Figure 3: A, effect of inhibition of protein synthesis on IL-1-enhanced pH recovery. Macrophages were pretreated with cycloheximide (0.05 g/ml) (solid bars) or vehicle control (open bars) for 20 min. Cells were then incubated with IL-1 (290 pM) or vehicle for 4 h in the presence of the inhibitor followed by quantitation of pHrecovery. Data are expressed as the mean ± S.E. of three experiments. *, p < 0.05 versus all other groups. B, effect of inhibition of RNA synthesis on IL-1-enhanced pH recovery. Macrophages were pretreated with actinomycin D (0.1 g/ml) (solid bars) or vehicle control (open bars) for 20 min. Cells were then incubated with IL-1 (290 pM) or vehicle for 4 h in the presence of the inhibitor followed by quantitation of pH recovery. Data are expressed as the mean ± S.E. of four experiments. *, p < 0.05 versus all other groups.



V-ATPases are multisubunit complexes consisting of two domains, the V(1) domain facing the cytosolic side of the membrane, which is responsible for the catalytic activity of the complex and the V(0) domain embedded within the membrane, which functions as the proton translocating pore. The A and B subunits of the V(1) domain have been shown to be responsible for ATP hydrolysis by the pump complex. Given the requirement for de novo mRNA synthesis, we hypothesized that IL-1 might exert its effect by inducing increased mRNA for the A and/or B subunits. Northern blot analysis was used to study the effect of IL-1 on the level of mRNA transcripts for the A and B subunits following 3 h of stimulation (Fig. 4A). Control cells were shown to constitutively express transcripts for both subunits using two different probes for each subunit. However, treatment with IL-1 had no effect on the level of A or B subunit mRNAs. Since an early and transient increase in V-ATPase mRNA may have been missed after 3 h of treatment with IL-1, similar studies were performed at an earlier time point. Following 1 h of treatment, the level of mRNA did not differ between control and IL-1-treated cells for either the A or B subunit (data not shown). The absence of a change in V-ATPase mRNA levels does not preclude a change in the amount of protein. Western blot analysis was used to study the levels of protein for two subunits of the V-ATPase complex, the B subunit, and the 39-kDa subunit. While control cells constitutively express the protein for the V-ATPase subunits, treatment of cells for 4 h with IL-1 had no effect on the levels of protein for either subunit (Fig. 4B).


Figure 4: A, effect of IL-1 on levels of mRNA transcripts for A and B subunits of the V-ATPase complex. Macrophages were treated in the absence or presence of IL-1 (290 pM) for 3 h, and Northern analysis was performed as described under ``Experimental Procedures'' using the cDNA probe for A and B subunits of the V-ATPase complex. Blots were also probed with the cDNA probe for rat alpha-tubulin to ensure equivalent RNA loading. The blot depicted the levels of B subunit transcripts using the probe PKP7a-1 and was done after stripping the membrane following hybridization with the A subunit probe F880. Therefore, the same alpha-tubulin blot was used for both. Each blot is representative of at least three independent studies. B, effect of IL-1 on levels of protein for V-ATPase subunits. Macrophages were treated in the absence or presence of IL-1 (290 pM) for 4 h, and Western blot analysis was performed as described under ``Experimental Procedures.'' Antibodies directed against the B subunit (alpha-62) or the 39-kDa subunit (alpha-39) of the V-ATPase complex were used. Each blot is representative of at least four independent studies.



Effect of IL-1 Involves cAMP-dependent Protein Kinase A and Protein Kinase C

Several studies have previously addressed the signaling pathways whereby IL-1 exerts its effects on various cell types, including lymphocytes and fibroblasts. Some investigators have invoked protein kinase C (PKC) (8, 27, 28, 29) as being a major contributor to the IL-1-induced signal, while others have documented a role for cAMP-dependent protein kinase (PKA)(28, 30, 31) . Since cAMP and phorbol esters have each been shown to modulate V-ATPase activity(32, 33, 34) , studies were performed to examine the contribution of these two signaling pathways to the IL-1-induced increase in V-ATPase activity. Two specific inhibitors of cAMP-dependent PKA, H-89 (35) and KT5720(36, 37) , markedly abrogated the increase in pH(i) recovery induced by IL-1 without affecting recovery rates in control cells (Fig. 5, A and B, respectively). This inhibitory effect occurred without significant cell toxicity, as demonstrated by trypan blue exclusion studies. Since IL-1 has been shown to exert its effects on PKA by increasing cAMP, the direct effects of cAMP agonists on V-ATPase-mediated pH(i) recovery was examined. Neither forskolin nor 8-bromo-cAMP over broad dose ranges was able to significantly augment pH(i) recovery (Fig. 6, A and B, respectively). Considered together, these data are consistent with the notion that activation of the PKA-dependent pathway is necessary but not sufficient to account for the IL-1-induced increase in V-ATPase activity.


Figure 5: Effect of protein kinase A inhibitors on pH recovery rate in IL-1-treated macrophages. A, macrophages were pretreated with H-89 (20 µM) (solid bars) or vehicle control (open bars) for 20 min. Cells were then incubated with IL-1 (290 pM) or vehicle for 4 h in the presence of the inhibitor and evaluated for pH recovery rate from an acid load. Data represent the mean ± S.E. of four experiments. *, p < 0.05 versus all other groups. B, macrophages were pretreated with KT5720 (100 nM) (solid bars) or vehicle control (open bars) for 20 min. Cells were then incubated with IL-1 (290 pM) or vehicle for 4 h in the presence of the inhibitor and evaluated for pH recovery rate from an acid load. Data represent the mean ± S.E. of four experiments. *, p < 0.05 versus all other groups.




Figure 6: Effect of protein kinase A agonists on macrophage pH recovery rate. A, macrophages were treated with IL-1 (closed bar), vehicle control (open bar), or varying concentrations of forskolin for 6 h. pHrecovery from an acute acid load was then quantitated. Data are expressed as the mean ± S.E. of three to four experiments. *, p < 0.05 versus all other groups. B, macrophages were treated with IL-1 (closed bar), vehicle control (open bar), or varying concentrations of 8-bromo-cAMP for 6 h. pH recovery from an acute acid load was then quantitated. Data are expressed as the mean ± S.E. for three to four experiments. *, p < 0.05 versus all other groups.



To define the role of PKC in the augmentation of V-ATPase activity by IL-1, cells were pretreated with a low concentration of staurosporine (10 nM) prior to IL-1 stimulation. This low concentration of staurosporine is close to the IC for PKC but is markedly lower than the IC for PKA(38) . As shown in Fig. 7A, staurosporine completely abrogates the effect of IL-1 on V-ATPase activity, suggesting that PKC activity is also necessary for the enhanced V-ATPase activity by IL-1. When cells were treated with the PKC agonist, phorbol 12-myristate 13-acetate (PMA) alone, there was a dose-dependent stimulatory effect on V-ATPase activity (Fig. 7B). At low concentrations (<1.0 nM), this agent had little effect, while at higher concentrations (1.0-10 nM), PMA clearly augmented V-ATPase activity. However, the combination of a nonstimulatory concentration of PMA (0.01 nM) with forskolin resulted in increased proton pump activity, comparable to levels attained with IL-1 stimulation (Fig. 7C). When considered in aggregate, these findings suggest that the effect of IL-1 on V-ATPase is mediated through activation of both the PKA- and PKC-dependent pathways.


Figure 7: A, effect of staurosporine on IL-1-enhanced pH recovery rate. Macrophages were pretreated with staurosporine (10 nM) (solid bars) or vehicle control (open bars) for 20 min. Cells were then incubated with IL-1 (290 pM) or vehicle for 4 h in the presence of the inhibitor. pH recovery rate was then determined following an acute acid load. Data are expressed as the mean ± S.E. of five experiments. *, p < 0.05 versus all other groups. B, effect of the PKC agonist, phorbol myristate acetate, on V-ATPase-mediated pH recovery. Macrophages were treated with IL-1 (closed bar) or vehicle control (open bar) or phorbol myristate acetate at varying concentrations for 4 h. pH recovery from an acute acid load was then quantitated. Data are expressed as the mean ± S.E. of three to five experiments. *, p < 0.05 versus no treatments. C, effect of forskolin and phorbol myristate acetate on pH recovery from an induced acid load. Macrophages were treated with IL-1 (closed bar) or vehicle control (open bar) or forskolin (1 µM) and phorbol myristate acetate (0.01 nM) alone or in combination for 4 h. pH recovery from an acute acid load was then quantitated. Data are expressed as the mean ± S.E. of four experiments. *, p < 0.05 versus no treatments.




DISCUSSION

Previous studies have demonstrated the importance of the plasmalemmal V-ATPase in pH(i) homeostasis in macrophages(6) . The requirement for pump activity was particularly evident under conditions of extracellular acidification, as might occur in the inflammatory milieu, where other pH(i) regulatory mechanisms such as the Na/H antiport and the HCO(3)/Cl exchanger are rendered ineffective. The present studies are the first to demonstrate that a cytokine, i.e. IL-1, known to be present at sites of inflammation(7) , is able to modulate plasmalemmal V-ATPase activity in macrophages. Several lines of evidence support this conclusion. First, IL-1 increased the rate of pH(i) recovery from an induced acid load in cells incubating in a Na- and HCO(3)-free medium without altering the buffering capacity of the cell. Second, the specific V-ATPase inhibitor, bafilomycin A(1), abrogated the effect of IL-1, implicating the V-ATPase as the major effector of the increase. Finally, the increase in V-ATPase-mediated pH(i) recovery occurred in parallel with a rise in the rate of bafilomycin-sensitive proton extrusion, indicating an effect on the plasmalemmal V-ATPase. Further, this effect also appeared to be specific for IL-1 since the effect was dose-dependent and was completely reversed by neutralizing anti-IL-1 antibody. Initial studies using another inflammatory cytokine, tumor necrosis factor alpha, demonstrate no effect on the rate of pH(i) recovery (data not shown). Considered together, these studies suggest a process whereby pH(i) regulatory mechanisms might be augmented within the inflammatory microenvironment to counteract the tendency for intracellular acid accumulation and resultant cellular dysfunction.

Several possible mechanisms may underlie the stimulatory effect of IL-1 on V-ATPase activity. These include exocytic translocation of pumps present in intracellular organelles, synthesis of new pumps with targeting to the plasmalemma, and activation of preexisting quiescent plasmalemmal pumps. The requirement for new protein and RNA synthesis and the 4-6-h lag phase for induction of increased pump activity lead us to consider the second possibility. In this regard, a recent study reported that V-ATPase activity might be modulated via transcriptional regulation of the B subunit(39) . We therefore examined the levels of mRNA transcripts for the A and B subunits of the V-ATPase complex in control and IL-1-treated cells. These studies showed that macrophages constitutively express mRNA transcripts for both subunits. However, IL-1 did not significantly alter their level over the time course of the study. Further, IL-1 failed to alter the level of protein for two subunits of the V-ATPase complex as assessed by Western blot analysis.

While other integral subunits might be involved, the present studies suggest the possibility that IL-1 may exert its effect by inducing proteins other than the pump subunits themselves. Recent studies have reported the existence of cytosolic proteins capable of modulating V-ATPase activity(40, 41, 42) . Theoretically, IL-1 might exert its effect through increased synthesis of an activator, resulting in increased activity of existing V-ATPase complexes.

While cells of monocyte/macrophage lineage are known to possess surface receptors for IL-1(43) , the subsequent signaling mechanisms responsible for the effects of IL-1 on macrophage activation have not been defined. In other cell types, both PKC- and PKA-dependent pathways have been reported to mediate the effects of IL-1. The present studies demonstrate that both pathways may be involved in the stimulatory effect of IL-1 on V-ATPase activity. Two inhibitors of PKA, H-89 and KT5720, reversed the ability of IL-1 to augment proton pump activity. Further, low dose staurosporine precluded the stimulatory effect of IL-1, implicating PKC in the signaling pathway. Neither PKA agonists nor low concentrations of the phorbol ester PMA were able to reproduce the effect of IL-1, while in combination they were able to do so. Considered together with the inhibitor data, these observations suggest that, while activation of either PKA- or PKC-dependent pathways alone is not sufficient to increase V-ATPase activity, combined activation of both pathways is both necessary and sufficient to mediate the stimulatory effect of IL-1.

Previous studies have demonstrated that activation of PKA augments V-ATPase activity in endocytic vesicles isolated from rabbit proximal tubule. This effect appears to be at least in part due to increased counterion conductance related to activation of the chloride channel. The failure of valinomycin to reproduce the effect of IL-1 in macrophages (data not shown), the chronic nature of the effect, and the requirement for new protein and RNA synthesis make increased counterion conductance an unlikely mechanism underlying the effect of IL-1.

Recent studies have suggested that activated PKC may in part exert its signaling effect by stimulating tyrosine kinase activity(44, 45) . Further, staurosporine, at concentrations higher than those used in the present studies, has been shown to have inhibitory effects on tyrosine kinases(46, 47) . To discern the contribution of tyrosine phosphorylation to the IL-1-induced increase in V-ATPase activity, the effect of herbimycin, a tyrosine kinase inhibitor, was studied. Herbimycin was unable to inhibit the stimulatory effect of IL-1 (data not shown), demonstrating that IL-1 was exerting its effect through a tyrosine kinase-independent pathway and that staurosporine was unlikely to have caused inhibition via an effect on tyrosine kinases. This observation is also consistent with the finding that IL-1 did not induce phosphotyrosine accumulation in peritoneal macrophages (data not shown).

In summary, the present studies are the first to demonstrate that cytokines are able to modulate the activity of plasmalemmal V-ATPases. Elevated local concentrations of IL-1 have been measured at sites of inflammation in experimental models as well as in the clinical setting (7, 48) . Studies are required to discern whether IL-1 is able to exert this effect in vivo, particularly in view of the fact that IL-1 inhibitors are known to be present in extracellular fluid(49) . Studies by Ford et al.(7) , however, suggest that IL-1 biological activity in wound fluid persists despite this possible antagonism by inhibitor molecules. While we have focused on the role of plasmalemmal V-ATPases as effectors of cytosolic pH regulation, extracellular acidification mediated by this proton extrusion mechanism may also have functional consequences in these and other cells. Enhanced acidification of the extracellular space by stimulated macrophages at sites of infection may serve to augment microbicidal activity both by a direct effect on microbial viability as well as by enhancing the activity of lysosomal acid hydrolases. Osteoclasts, which are derived from the same hematopoietic cells as macrophages, cause bone resorption by pumping H into tightly sealed pericellular resorption compartments, a process mediated by V-ATPases localized to the plasma membrane lying in apposition to the bone surface. Since IL-1 is a potent inducer of osteoclastic activity both in vitro and in vivo, the data presented suggest the possibility that IL-1 may act, in part, by augmenting plasmalemmal V-ATPase activity in these cells.


FOOTNOTES

*
This work was supported by the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of an MRC-Industry fellowship.

Howard Hughes International Scholar.

**
Scholar of the Medical Research Council of Canada.

§§
To whom correspondence should be addressed: Toronto Hospital, EN 9-236, 200 Elizabeth St., Toronto, Ontario M5G 2C4, Canada. Tel.: 416-340-4988; Fax: 416-595-9486.

(^1)
The abbreviations used are: pH, intracellular pH; pH, extracellular pH; PMA, phorbol 12-myristate 13-acetate; BCECF, 2`,7`-biscarboxyethyl-5-(6)-carboxyfluorescein; HBSS, Hanks' balanced salt solution; FCS, fetal calf serum; PKC, protein kinase C; PKA, protein kinase A; IL-1, interleukin-1; V-ATPase, vacuolar-type H-ATPase; MES, 2-[N-morpholino]ethanesulfonic acid.


REFERENCES

  1. Swallow, C. J., Grinstein, S., Sudsbury, R. A., and Rotstein, O. D. (1993) J. Cell. Physiol. 157, 453-460 [Medline] [Order article via Infotrieve]
  2. Ladoux, A., Miglierina, R., Krawice, I., Cragoe, E. J., Jr., Abita, J. P., and Frelin, C. (1988) Eur. J. Biochem. 175, 455-460 [Abstract]
  3. Kapus, A., Romanek, R., Qu, A. Y., Rotstein, O. D., and Grinstein, S. (1993) J. Gen. Physiol. 102, 729-760 [Abstract]
  4. Swallow, C. J., Grinstein, S., and Rotstein, O. D. (1988) J. Biol. Chem. 263, 19558-19563 [Abstract/Free Full Text]
  5. Swallow, C. J., Grinstein, S., and Rotstein, O. D. (1990) J. Biol. Chem. 265, 7645-7654 [Abstract/Free Full Text]
  6. Swallow, C. J., Grinstein, S., Sudsbury, R. A., and Rotstein, O. D. (1990) Surgery 108, 363-369 [Medline] [Order article via Infotrieve]
  7. Ford, H. R., Hoffman, R. A., Wing, E. J., Magee, D. M., McIntyre, L., and Simmons, R. L. (1989) Arch. Surg. 124, 1422-1428 [Abstract]
  8. Bethea, J. R., Gillespie, G. Y., and Benveniste, E. N. (1992) J. Cell. Physiol. 152, 264-273 [Medline] [Order article via Infotrieve]
  9. Nakazato, Y., Simonson, M. S., Herman, W. H., Konieczkowski, M., and Sedor, J. R. (1991) J. Biol. Chem. 266, 14119-14127 [Abstract/Free Full Text]
  10. Carlsen, E., Flatmark, A., and Prydz, H. (1988) Transplantation 46, 575-580 [Medline] [Order article via Infotrieve]
  11. Conti, P., Cifone, M. G., Alesse, E., Fieschi, C., and Angeletti, P. U. (1985) Agents Actions 17, 390-391
  12. Dinarello, C. A., Ikejima, T., Warner, S. J. C., Orencole, S. F., Lonnemann, G., Cannom, J. G., and Libby, P. (1987) J. Immunol. 139, 1902-1910 [Abstract/Free Full Text]
  13. Karnovsky, M. L., and Lazdins, J. K. (1978) J. Immunol. 121, 809-813 [Medline] [Order article via Infotrieve]
  14. Cohn, Z. A. (1978) J. Immunol. 121, 813-816 [Medline] [Order article via Infotrieve]
  15. Roos, A., and Boron, W. F. (1981) Physiol. Rev. 61, 296-434 [Free Full Text]
  16. Swallow, C. J., Grinstein, S., and Rotstein, O. D. (1990) Biochim. Biophys. Acta 1022, 203-210 [Medline] [Order article via Infotrieve]
  17. Grinstein, S., and Furuya, W. (1986) Am. J. Physiol. 250, C283-C291
  18. Grinstein, S., Cohen, S., and Rothstein, A. (1984) J. Gen. Physiol. 83, 341-369 [Abstract]
  19. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  20. Puopolo, K., Kumamoto, C., Adachi, I., and Forgac, M. (1991) J. Biol. Chem. 266, 24564-24572 [Abstract/Free Full Text]
  21. Van Hille, B., Richener, H., Evans, D. B., Green, J. R., and Bilbe, G. (1993) J. Biol. Chem. 268, 7075-7080 [Abstract/Free Full Text]
  22. Puopolo, K., Kumamoto, C., Adachi, I., Magner, R., and Forgac, M. (1992) J. Biol. Chem. 267, 3696-3706 [Abstract/Free Full Text]
  23. Lemischka, I., and Sharp, P. A. (1982) Nature 300, 330-335 [Medline] [Order article via Infotrieve]
  24. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  25. Brisseau, G. F., Dackiw, A. P. B., Cheung, P. Y. C., Christie, N., and Rotstein, O. D. (1995) Blood 85, 1025-1035 [Abstract/Free Full Text]
  26. Siebert, A., Lottspeich, F., Nelson, N., and Betz, H. (1994) J. Biol. Chem. 269, 28329-28334 [Abstract/Free Full Text]
  27. Conquer, J. A., Kandel, R. A., and Cruz, T. F. (1992) Biochim. Biophys. Acta 1134, 1-6 [Medline] [Order article via Infotrieve]
  28. Maier, J. A. M., and Ragnotti, G. (1993) Exp. Cell Res. 205, 52-58 [CrossRef][Medline] [Order article via Infotrieve]
  29. Kracht, M., Heiner, A., Resch, K., and Szamel, M. (1993) J. Biol. Chem. 268, 21066-21072 [Abstract/Free Full Text]
  30. Ray, K., Thompson, N., Kennard, N., Rollins, P., Grenfell, S., Witham, S., Smithers, N., and Solari, R. (1992) Biochem. J. 282, 59-67 [Medline] [Order article via Infotrieve]
  31. Shirakawa, F., Yamashita, U., Chedid, M., and Mizel, S. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8201-8205 [Abstract]
  32. Gurich, R. W., and DuBose, T. D., Jr. (1989) Am. J. Physiol. 257, F777-F784
  33. Bae, H.-R., and Verkman, A. S. (1990) Nature 348, 637-639 [CrossRef][Medline] [Order article via Infotrieve]
  34. Nanda, A., Gukovskaya, A., Tseng, J., and Grinstein, S. (1992) J. Biol. Chem. 267, 22740-22746 [Abstract/Free Full Text]
  35. Geilen, C. C., Wieprecht, M., Wieder, T., and Reutter, W. (1992) FEBS Lett. 309, 381-384 [CrossRef][Medline] [Order article via Infotrieve]
  36. Kase, H., Iwahashi, K., Nakanishi, S., Matsuda, Y., Yamada, K., Takahashi, M., Murakata, C., Sato, A., and Kaneko, M. (1987) Biochem. Biophys. Res. Commun. 142, 436-440 [Medline] [Order article via Infotrieve]
  37. Shuntoh, H., Taniyama, K., Fukuzaki, H., and Tanaka, C. (1988) J. Neurochem. 51, 1565-1572 [Medline] [Order article via Infotrieve]
  38. Davis, P. D., Hill, C. H., Keech, E., Lawton, G., Nixon, J. S., Sedgwick, A. D., Wadsworth, J., Westmacott, D., and Wilkinson, S. E. (1995) FEBS Lett. 259, 61-63 [CrossRef]
  39. Lee, B. S., Underhill, D. M., Crane, M. K., and Gluck, S. L. (1995) J. Biol. Chem. 270, 7320-7329 [Abstract/Free Full Text]
  40. Zhang, K., Wang, Z.-Q., and Gluck, S. (1992) J. Biol. Chem. 267, 9701-9705 [Abstract/Free Full Text]
  41. Zhang, K., Wang, Z.-Q., and Gluck, S. (1992) J. Biol. Chem. 267, 14539-14542 [Abstract/Free Full Text]
  42. Xie, X. S., Crider, B. P., and Stone, D. K. (1993) J. Biol. Chem. 268, 25063-25067 [Abstract/Free Full Text]
  43. Uhl, J., Newton, R. C., Giri, J. G., Sandlin, G., and Horuk, R. (1989) J. Immunol. 142, 1576-1581 [Abstract/Free Full Text]
  44. Zen, K., Masuda, J., Sasaguri, T., Kosaka, C., and Ogata, J. (1994) Exp. Cell Res. 215, 172-179 [CrossRef][Medline] [Order article via Infotrieve]
  45. Buscher, D., Hipskind, R. A., Krautwakd, S., Reimann, T., and Baccarini, M. (1995) Mol. Cell. Biol. 15, 466-475 [Abstract]
  46. Wallace, B. G. (1994) J. Cell Biol. 125, 661-668 [Abstract]
  47. Fallon, R. J., Danaher, M., Saylors, R. L., and Saxena, A. (1994) J. Biol. Chem. 269, 11011-11017 [Abstract/Free Full Text]
  48. Nicolle, L. E., Brunka, J., Orr, P., Wilkins, J., and Harding, G. K. M. (1993) J. Urol. 149, 1049-1053 [Medline] [Order article via Infotrieve]
  49. Kronborg, G., Hansen, M. B., Svenson, M., Fomsgaard, A., Hoiby, N., and Bendtzen, K. (1993) Pediatr. Pulmonology 15, 292-297 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.