(Received for publication, October 19, 1995)
From the
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 pH
regulation 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.
Maintenance of the intracellular pH (pH) (
)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
/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.
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.
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 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
; see (17) )
against the corresponding fluorescence signal. The pH
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
-free KCl
medium during recovery measurements ensured that the majority of the
pH
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
/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
recovery under
these conditions (see below).
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. DH5 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-
-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
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
10 min. The fusion protein was eluted by the addition of
1 ml of 50 mM Tris
Cl (pH 8.0)/5 mM reduced
glutathione for 2 min followed by centrifugation at 500
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.
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
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. pH
recovery 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
(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
Cl technique for the 20 min prior to the measurement of
proton extrusion. Studies were performed in the presence of bafilomycin
A
(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
. 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.
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
pH
recovery. 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 domain facing the cytosolic side of
the membrane, which is responsible for the catalytic activity of the
complex and the V
domain embedded within the membrane,
which functions as the proton translocating pore. The A and B subunits
of the V
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 -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
-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 (
-62) or the 39-kDa subunit
(
-39) of the V-ATPase complex were used. Each blot is
representative of at least four independent
studies.
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. pH
recovery 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.
Previous studies have demonstrated the importance of the
plasmalemmal V-ATPase in pH 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
regulatory mechanisms such as the
Na
/H
antiport and the
HCO
/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
recovery
from an induced acid load in cells incubating in a Na
-
and HCO
-free medium without altering the
buffering capacity of the cell. Second, the specific V-ATPase
inhibitor, bafilomycin A
, 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
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
, demonstrate no effect on the
rate of pH
recovery (data not shown). Considered together,
these studies suggest a process whereby pH
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.