Download Cytosolic pH and the inflammatory

PHAGOCYTES
Cytosolic pH and the inflammatory microenvironment modulate cell
death in human neutrophils after phagocytosis
Raymond J. Coakley, Clifford Taggart, Noel G. McElvaney, and Shane J. O’Neill
Following phagocytosis in vivo, acidification of extracellular pH (pHo) and intracellular metabolic acid generation contribute to cytosolic proton loading in
neutrophils. Cytosolic pH (pHi) affects
neutrophil function, although its regulation is incompletely understood. Its effect
on mechanisms of neutrophil death is
also uncertain. Thus, we investigated pHi
regulation in Escherichia coli–exposed
neutrophils, at various pathogen-tophagocyte ratios (0:1-50:1), under conditions simulating the inflammatory milieu
in vivo and correlated pHi changes with
mechanisms of neutrophil death. Following phagocytosis, proton extrusion was
dominated early by passive proton conductance channels, and later by Naⴙ/Hⴙ
exchange (NHE). Hⴙ-translocating adenosine triphosphatase (V-ATPase) pHi regulation was evident primarily at lower bacterial densities. At physiologic pHo, lower
pathogen-to-phagocyte ratios alkalinized
pHi and inhibited apoptosis, whereas
higher ratios acidified pHi (despite proton
extrusive mechanisms) and promoted
apoptosis. Necrosis was induced by highdensity bacterial exposure at reduced pHo.
Following phagocytosis, targeted inhibition of NHEs, proton conductance channels, or V-ATPases (amiloride, ZnCl2, or
bafilomycin, respectively) moderately hyperacidified pHi and accelerated apoptosis. However, in combination they
profoundly acidified pHi and induced necrosis. Proinflammatory mediators in vivo
might affect both pHi regulation and cell
death, so we tested the effects of bronchoalveolar lavage (BAL) fluid from patients with cystic fibrosis (CF) and healthy
subjects. Only CF BAL fluid alkalinized
pHi and suppressed apoptosis at physiologic pHo, but failed to prevent necrosis
following phagocytosis at low pHo. Thus,
a precarious balance between cytosolic
proton loading and extrusion after phagocytosis dictates the mode of neutrophil
cell death. pHi/pHo might be therapeutically targeted to limit neutrophil necrosis
and protect host tissues during necrotizing
infections. (Blood. 2002;100:3383-3391)
© 2002 by The American Society of Hematology
Introduction
Neutrophil phagocytosis is an essential and generally effective host
response to microbial invasion. However, during necrotizing
infections and in abscesses, neutrophils are ineffective at killing
bacteria and instead contribute to host tissue destruction. Both
intracellular and extracellular factors might contribute to dysregulation of neutrophilic inflammation in such situations, although these
are not well characterized. pH may be important in this regard
because it has the capacity to modulate neutrophil function. Thus,
we investigated the mechanisms of intracellular pH (pHi) regulation following phagocytosis, under conditions simulating the
inflammatory microenvironment, and assessed how pHi might
contribute to the regulation or dysregulation of acute inflammation
by altering mechanisms of neutrophil death.
pHi homeostasis is uniquely challenged in phagocytosing
neutrophils because cellular activation generates sufficient acid
equivalents to lethally acidify the cell, and extracellular pH (pHo) at
inflammatory sites is often decreased.1,2 The balance between
cytosolic proton loading and extrusion is important because many
neutrophil functions, including microbicidal behavior, cell migration, intracellular oxidant generation, tumor cell cytotoxicity, and
azurophil granule exocytosis, are pH dependent.3-8 Mechanisms
potentially contributing to pHi regulation in neutrophils include
Na⫹/H⫹ exchangers (NHEs), proton-translocating adenosine
triphosphatases (V-ATPases), and reduced nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase–associated proton conductance channels.9-11 Although NHEs are activated following
phagocytosis in neutrophils,12 it is not known whether additional
mechanisms also contribute to proton extrusion, or indeed whether
they are collectively sufficient to preserve pHi close to physiologic
levels, under conditions of increased proton loading. Further, the
combined effect on pHi regulation of mediators in the inflammatory
milieu could also be important in vivo, although it is poorly
characterized. Although some soluble inflammatory mediators
activate proton extrusion in vitro,13-16 others, such as lipopolysaccharide
(LPS), may impair it.17 Thus, in the first series of experiments reported
here, we investigated which pumps and channels are involved in pHi
regulation after phagocytosis and tested how their function is affected by
proinflammatory mediators from the lungs of cystic fibrosis (CF)
patients and healthy subjects. CF epithelial lining fluid (ELF) is an
attractive model of an inflammatory microenvironment for such studies
because its pulmonary phenotype is characterized by intense, persistent
inflammation and neutrophil-mediated parenchymal destruction.
pHi and the inflammatory microenvironment may also affect
survival of phagocytosing neutrophils by modulating rates of
From the Division of Respiratory Research, Department of Medicine, Royal
College of Surgeons in Ireland Education and Research Center, Beaumont
Hospital, Dublin, Republic of Ireland.
Reprints: Raymond J. Coakley, University of North Carolina at Chapel Hill, The
CF/Pulmonary Research and Treatment Center, Room 7013 Thurston-Bowles
Building, CB #7248, Chapel Hill, NC 27516-7248; e-mail: ray_coakley@med.
unc.edu.
Submitted August 3, 2001; accepted June 21, 2002.
Supported by the Higher Education Authority of Ireland, the Royal College of
Surgeons in Ireland, the Health Research Board of Ireland, and the Irish Lung
Association.
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The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
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apoptotic and necrotic cell death, both of which may occur in
response to bacterial exposure in vitro.18-20 This distinction is
important because the former is a protective, orderly involutionary
process, whereas the latter likely contributes to disease by exposing
host tissues to destructive neutrophil contents.21 pHi and pHo
directly affect apoptosis in many cell types,22-30 whereas severely
acidic pHi might nonspecifically injure cells and thereby promote
necrosis.31 Because phagocytosing neutrophils exhibit marked
alterations in metabolic acid generation and are subject to variable
pHo, it is surprising that the relationship between pHi and cell death
in neutrophils has not been characterized more rigorously. The
literature in this regard is, indeed, conflicting, as different studies
have implicated acidic pHi in both stimulation and inhibition of
neutrophil or myeloid cell apoptosis.6,13,32,33 Thus, we addressed
the consequences of altered pHi and pHo on cell survival in
neutrophils exposed to increasing bacterial loads. We focused on
pHi changes over the first 90 minutes to probe early metabolic
factors implicated in triggering these processes. Further, because
soluble inflammatory mediators affect granulocyte apoptosis, we
also evaluated the effect of bronchoalveolar lavage (BAL) fluid
from CF patients and healthy subjects on neutrophil survival.
We show that pHi following phagocytosis is not preserved by
active pHi regulatory mechanisms when neutrophils are exposed to
a high-density bacterial challenge, or when neutrophils are cultured
at low pHo, although proton extrusion is elevated by mediators
present in the inflammatory milieu. Further, we demonstrate that
changes in pHi/pHo and the inflammatory microenvironment determine mechanisms of cell death in phagocytosing neutrophils. Our
results illustrate that a precarious balance exists in vivo between
cytosolic proton loading and extrusion, and that the resultant change in
pHi is a significant determinant of cell survival and mode of death.
Materials and methods
Isolation of neutrophils and measurement of pHi
The institution’s ethical review board approved the studies. Subjects gave
informed consent (14 healthy individuals). Neutrophils were isolated from
heparinized peripheral venous blood by density gradient centrifugation, as
previously described.34 The neutrophil population was at least 96% pure by
morphologic assessment (Cytospin preparations and Giemsa staining).
Viability was at least 99% by trypan blue dye exclusion (light microscopy)
and propidium iodide (PI) exclusion (flow cytometry). Cells were cultured
in RPMI-1640 containing 25 mM NaHCO3 (Sigma Cell Culture; Poole
Dorset) and freshly supplemented with L-glutamine 0.3 g/L (pH 7.35;
Sigma Cell Culture). In additional experiments, cells were cultured under
HCO⫺
3 -free conditions in a HEPES (N-2-hydroxyethylpiperazine-N⬘-2ethanesulfonic acid)-buffered medium with pH adjusted to 5.8 with HCl
(Sigma Cell Culture). pHi was measured by flow cytometry (FACScan;
Becton Dickinson, Mountain View, CA) using the pH-sensitive fluorescent
dye carboxy-SNARF, as previously described.34 Briefly, carboxy-SNARF–
loaded cells were stimulated with argon laser light (488 nm), and the ratio of
emitted fluorescence at 580 nm and 630 nm was measured. pH was
extrapolated following calibration based on the nigericin/high K⫹ method,
which was carried out on cells from all subjects. Calibration was repeated in
stimulated cells to ensure that activation did not affect accuracy of the pH
measurements. During sample acquisition, gating according to characteristic side and forward scatter characteristics confined the analysis to
neutrophils, excluding clusters and debris.
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across a range of pathogen-to-phagocyte ratios from 0:1 to 50:1. After
addition of E coli, the cells were agitated at moderate speed to promote
adherence, and pHi was measured. Some pathogen-to-phagocyte ratios
induced both alkalinization and acidification in the same cells over time,
compared with pHi in “non–bacteria-exposed neutrophils.” To quantify
these complex pH changes, we used commercial software (GraphPad) to
calculate the additional area “above” (alkalinization) or “below” (acidification) the pH trace recorded in bacteria-free cells that was induced by
exposure to varying densities of E coli, and expressed this area in arbitrary
units. This is effectively a measure of the net change in pHi. Positive values
indicate net alkalinization, whereas negative values indicate net acidification.
To assess the roles of individual mechanisms of proton extrusion, we
exposed phagocytosing cells to specific inhibitors of pHi regulatory pumps
and channels, including proton-translocating ATPases (bafilomycin A1, 100
nM), Na⫹/H⫹ exchange (amiloride, 0.2 mM), and NADPH oxidase passive
proton conductance–associated channels (ZnCl2, 50 ␮M). Amiloride,
bafilomycin A1, and ZnCl2 were obtained from Sigma.
Determination of intracellular oxidant generation
Neutrophil suspensions were incubated with normal saline or human
serum–opsonized E coli (pathogen-to-phagocyte ratio 0:1-50:1) at 37°C in
the presence of the oxidant-sensitive probe 123-dihydrorhodamine (Orpegen Pharma),35 after which they were immediately placed on ice. Cells were
partially fixed for 20 minutes with a solution containing formaldehyde and
less than 50% diethylene glycol (FACS lysing solution; Becton Dickinson,
Cowley, Oxford, United Kingdom) before washing in phosphate-buffered
saline (PBS). Finally, the samples were exposed to a cytoplasmic membrane
permeabilizing solution containing PI (Permeabilizing Solution; Becton
Dickinson) to stain DNA. At least 10 000 cells in each sample were
analyzed by flow cytometry (FACScan; Becton Dickinson). Neutrophils
were identified by characteristic size and granularity, as indicated by
low-angle forward-scattering and right-angle side-scattering properties of
argon laser light (488 nm). Mean emitted fluorescence intensity (mean
channel fluorescence; MCF) at 520 nm, expressed on a 4-decade logarithmic scale with constant photomultiplier gain values, was quantified as a
measure of intracellular oxidant generation. Analysis of the emitted
fluorescence of PI at 520 nm differentiated viable cells from bacterial
clumps on the basis of their DNA content. Only cells with the DNA binding
characteristics of mammalian cells were included in the analysis. In all
cases, background emitted fluorescence at 520 nm in unstained cells was
subtracted from fluorescence in stained cells.
Quantification of phagocytosis in isolated neutrophils
Neutrophils were gently stirred while being incubated with fluorescein
isothiocyanate (FITC)–labeled human serum–opsonized E coli (Orpegen
Pharma; pathogen-to-phagocyte ratio 0:1-50:1) for 15 minutes at 37°C. The
cells were then placed on ice to terminate phagocytosis, and a quenching
solution containing N-ethyl maleimide (Becton Dickinson) was added.
After sedimentation (500g 10 minutes at 4°C), cells were washed in PBS
and sedimented again before fixation, as described above. Following
fixation, cells were treated with a permeabilizing solution containing PI to
stain DNA, and at least 10 000 neutrophils in each sample were identified as
described above and analyzed by flow cytometry. Cells were stimulated
with an argon laser at 488 nm. Mean emitted fluorescence intensity (MCF)
at 520 nm was expressed on a 4-decade logarithmic scale with constant
photomultiplier gain values and was quantified as a measure of bacterial
ingestion. Analysis of the emitted fluorescence of PI at 580 nm differentiated cells from bacterial clumps on the basis of their DNA content. Only
cells with the DNA binding characteristics of mammalian cells were
included in the analysis. In all cases, background emitted fluorescence at
520 nm in unstained cells was subtracted from fluorescence in stained cells.
Cellular activation and determination of relative roles
of proton extrusive mechanisms
Measurement of viability and cell death
in phagocytosing neutrophils
During experimentation, cells were exposed to heat-killed, human serum–
opsonized Escherichia coli (Orpegen Pharma, Heidelberg, Germany)
Assessment of cell membrane integrity. Cell membrane integrity is
preserved until the late phase of apoptosis. Thus, we exploited the
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intracellular staining with the vital dyes (trypan blue via light microscopy
and PI via flow cytometry) to identify necrotic cells. Aliquots of cell
suspension were centrifuged and resuspended in a solution containing
either trypan blue or PI. In the former case, 200 cells were examined under
light microscopy, and the percentage of dye-excluding cells was calculated.
In the latter case, the PI-stained solution was assessed by flow cytometry.
The cell suspension was excited with argon laser light, and emitted light at
520 nm was quantified in at least 10 000 cells. The percentage of cells with
PI staining of DNA (necrotic cells) was quantified. Both techniques were in
close agreement (r2 ⫽ 0.93). Data are thus reported interchangeably.
Assessment of cellular morphology. Neutrophils were cytocentrifuged
and stained with Diff-Quick. Slides were mounted and viewed under oil
immersion microscopy. At least 500 cells per slide were inspected by an
observer who was blinded to the experimental condition. Features that
distinguished normal from apoptotic morphology (nuclear pyknosis, chromatin condensation, and cytoplasmic vacuolation) were sought.
Assessment of neutrophil apoptosis, annexin-V binding, and DNA
fragmentation. An alternative and independent method for quantification
of neutrophil apoptosis was also employed: the detection of phosphatidylserine expression on the external surface of the cytoplasmic membrane.
Briefly, following washing in PBS, 1 ⫻ 106 neutrophils were gently
resuspended in a solution containing FITC-labeled recombinant human
annexin V in a high-Ca⫹⫹ binding buffer (Boerhinger-Mannheim, Europe).
Following incubation in the dark at room temperature for 15 minutes, the
binding buffer was diluted and the samples were immediately analyzed by
flow cytometry. At least 10 000 cells were excited with argon laser light
(488 nm), and emitted fluorescence at 520 nm minus background fluorescence was quantified. Because “annexin positivity” could be due to
apoptosis-induced externalization of phosphatidylserine or annexin-V binding to intracellular phosphatidylserine in necrotic cells, additional experiments were always performed to quantify rates of necrosis, and the
appropriate correction was made. Reproducibility of this method compared
favorably with apoptosis detection by morphologic assessment at 16 hours.
To further validate this approach for the detection of apoptosis, we
(favorably) compared our results with those of a commercially available
enzyme-linked immunosorbent assay of intracellular histone-associated
DNA fragmentation. The percentage apoptosis reported is a mean of the
results obtained by morphology and annexin-V binding.
Figure 1. pHi in phagocytosing neutrophils is determined by bacterial load. Isolated human peripheral
blood neutrophils were exposed to increasing densities
of heat-killed opsonized E coli while intracellular pH (pHi)
was monitored by flow cytometry using cytosolic pHsensitive dyes and, in separate experiments, phagocytosis of fluorescently labeled bacteria was measured by
flow cytometry. (A) The correlation between increasing
pathogen-to-phagocyte ratio and mean number of bacteria ingested (mean channel fluorescence) by neutrophils
(4 replicates each from 5 healthy donors, coefficient of
determination r2 ⫽ 0.96). (B) The effect of increasing
pathogen-to-phagocyte ratios and thus mean number of
bacteria ingested (0:1-50:1) on pHi over time in normal
neutrophils (4 replicates each from 10 healthy donors).
pHi responses at each pathogen-to-phagocyte ratio differed significantly from the others (by MANOVA, P ⬍ .01).
(C,D) The correlation between bacterial load and the
change in pHi after 5 minutes and the lowest pH recorded. All data mean ⫾ SEM.
pH AND CELL DEATH IN PHAGOCYTIC NEUTROPHILS
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Effect of CF ELF on pH regulation and cell death
in phagocytosing neutrophils
Normal and CF epithelial lining fluid (ELF) was derived from bronchoalveolar lavage performed on healthy volunteers or CF patients (exacerbation
free for 6 weeks). In brief, during fiberoptic bronchoscopy, 3 ⫻ 1 mL/kg
body weight aliquots of sterile saline at 37°C were injected into a division
of the right middle lobe of the lung and reaspirated immediately.36 BAL
fluid was filtered through sterile gauze and centrifuged at 1100 rpm for 10
minutes, and the supernatant was stored at ⫺80°C. The volume of ELF was
calculated according to the urea dilution method,37 and the lavage was
concentrated using Centricon filter devices (Millipore, Bedford, MA).
Similar volumes (40 ␮L/mL) of saline or concentrated normal ELF or CF
ELF were added to suspensions of freshly isolated normal neutrophils. We
assessed the immediate effects on pHi, as well as the effects on pHi in E
coli–exposed neutrophils after preincubation (1 hour).
Data presentation and statistical analysis
All data are mean ⫾ standard error (SE) of the mean. Statistical analysis
was performed using GraphPad Prism software or Statistica software.
Linear regression analysis was used to test linearity of the correlation
between variables. Differences over time within individual groups were
analyzed using repeated-measures analysis of variance (ANOVA) and
Bonferroni post hoc correction. Differences over time between groups were
compared using either multivariate analysis of variance (MANOVA) or
2-way ANOVA and Bonferroni post hoc correction as appropriate.
Results
pHi in neutrophils following phagocytosis as a
function of bacterial load
Using FITC-labeled E coli, we confirmed that increasing bacterial
density led to a linear increase in the mean number of bacteria
ingested over the range of pathogen-to-phagocyte ratios we studied
(Figure 1A). We then investigated how increasing the number of
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bacteria ingested affected pHi. Following exposure to E coli, pHi
exhibited striking dependence on the bacterial load (Figure 1B).
The lowest pathogen-to-phagocyte ratios predominantly increased
pHi over 90 minutes. Somewhat higher ratios induced transient
alkalinization and later acidification of pHi. In contrast, marked and
rapid declines of pHi were evident at the highest pathogen-tophagocyte ratios studied. At all concentrations of E coli, pHi
returned toward baseline by 90 minutes. Pathogen-to-phagocyte
ratios correlated strongly with pHi at 5 minutes and the lowest pHi
value recorded over the first 90 minutes (r2 ⫽ 0.97 and 0.928,
respectively, by linear regression analysis; Figure 1C,D). Viability
at 90 minutes (by vital dye exclusion) was preserved at all densities
of E coli exposure at physiologic pHo (data not shown).
Contribution of NHE, V-ATPases, and ZnCl2-sensitive proton
channels to pHi regulation in phagocytosing neutrophils
We examined the effect of specific inhibitors of proton extrusive
mechanisms on pHi following phagocytosis. As can be seen in
Figure 2, the presence of each inhibitor singly and in combination
for 20 minutes did not significantly affect pHi, which suggests that
the proton extrusive mechanisms that they inhibit are inactive
under basal conditions. We studied cells exposed to a pathogen-tophagocyte ratio of 20:1, having established that this caused
sustained cytoplasmic acidification and therefore was likely to have
stimulated proton extrusion. Inhibition of NADPH-associated
passive proton conductance channels (ZnCl2, 50 ␮M) enhanced
acidification during the first 20 minutes following phagocytosis
(Figure 2A). Amiloride (0.2 mM) strongly affected pHi after
phagocytosis, between 10 and 60 minutes (Figure 2B). Amiloride
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(but not ZnCl2) treatment reduced pHi at lower pathogen-tophagocyte ratios (data not shown). At a pathogen-to-phagocyte
ratio of 20:1, inhibition of V-ATPases with bafilomycin had a small
(but significant) effect on pHi (Figure 2C). A combination of ZnCl2,
amiloride, and bafilomycin led to more profound acidification,
suggesting that the effects of these 3 proton extrusive mechanisms
are additive (Figure 2D). We were surprised that bafilomycin did
not have a more pronounced effect on pHi regulation. Because
V-ATPases are trafficked to the cytoplasmic membrane on cellular
activation,11 we speculated that they may also be rapidly reinternalized at high pathogen-to-phagocyte ratios (due to infolding of the
plasma membrane during continuing phagocytosis). Therefore, we
investigated the effect of bafilomycin on pHi regulation at a lower
pathogen-to-phagocyte ratio of 3:1. Bafilomycin (100 nM) did
induce significant acidification of pHi under these conditions
(Figure 2E). As can be discerned in Figure 2, amiloride, bafilomycin, or ZnCl2, either alone or in combination, did not affect pHi
before E coli exposure. Further experiments demonstrated that pHi
was not altered in the presence of these inhibitors in the absence of
bacteria over 120 minutes (data not shown).
Effect of low extracellular pH on pHi following
ingestion of bacteria
We investigated pHi regulation in phagocytosing neutrophils at
reduced pHo (5.8). Compared with physiologic pHo, bacterial
ingestion at acidic pHo led to significantly more acidic pHi (Figure
3), even though lower numbers of bacteria were ingested at acidic
pHo (bacterial ingestion, MCF: pH 7.4, 33 ⫾ 4.3 versus pH 5.8,
22 ⫾ 6.1; P ⬍ .05). pHi failed to return to basal levels over
Figure 2. Mechanisms of pHi regulation in phagocytosing neutrophils. The individual and collective contributions of several putative mechanisms of proton extrusion were
investigated in isolated human peripheral blood neutrophils. Neutrophils (4 replicates each from 10 healthy donors) were exposed to opsonized heat-killed E coli at a
pathogen-to-phagocyte ratio of 20:1 in the presence or absence of inhibitors. (A) The significant effect on pHi following phagocytosis of inhibition of passive proton conductance
channels (ZnCl2, 50 ␮M, P ⬍ .01 versus E coli alone by 2-way ANOVA). (B) The significant effect on pHi following phagocytosis of inhibition of Na⫹/H⫹ exchange (amiloride, 0.2
mM, P ⬍ .001 by 2-way ANOVA). (C) The small but significant effect on pHi following phagocytosis of inhibition of V-ATPases (bafilomycin, 100 nM, P ⬍ .05 by 2-way ANOVA).
(D) The significant effect on pHi following phagocytosis of inhibition of passive proton conductance channels, Na⫹/H⫹ exchange, and V-ATPases (P ⬍ .0001 by 2-way ANOVA).
(E) At a lower pathogen-to-phagocyte ratio (3:1), inhibition of V-ATPases with bafilomycin (100 nM) has a more significant effect on pHi following phagocytosis (*P ⬍ .001 by
2-way ANOVA). All data mean ⫾ SEM.
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sis. Intracellular oxidant generation correlated less significantly
with rates of apoptosis (Figure 5C). Of note, at low pathogen-tophagocyte ratios, intracellular oxidant generation was modestly
increased, whereas apoptosis was suppressed rather than stimulated.
Effect of manipulation of intracellular pH and CF ELF on
apoptosis and necrosis in phagocytosing neutrophils
Figure 3. The effect of an acidic extracellular milieu on pHi in phagocytosing
neutrophils. Isolated human peripheral blood neutrophils (4 replicates from 8
healthy donors) were exposed to opsonized heat-killed E coli (pathogen-tophagocyte ratio 20:1) in standard HCO3⫺-buffered RPMI medium (pH 7.45) and a
bicarbonate-free HEPES-buffered medium (pH 5.8). pHi was monitored during
bacterial ingestion. Cytosolic acidification was significantly greater following phagocytosis in the acidic extracellular medium (P ⬍ .001 by 2-way ANOVA). All data
mean ⫾ SEM.
90 minutes. Under these conditions, a subpopulation of neutrophils
with pH similar to that of the extracellular milieu was noted after 30
minutes, potentially representing necrosing cells with impaired
membrane integrity (data not shown).
Effect of ELF from CF patients and healthy controls
on pHi in phagocytosing neutrophils
Neutrophils were incubated with saline, normal ELF, or CF ELF.
Normal ELF did not affect resting pHi compared with saline
vehicle, but CF ELF induced a sustained alkalinization (Figure
4A). CF ELF, but not normal ELF, also increased intracellular
oxidant generation in response to bacterial exposure (intracellular
oxidant generation, MCF: normal lavage, 400 ⫾ 24.5 versus CF
lavage, 550 ⫾ 36.7; P ⬍ .05) and efficiency of phagocytosis in
neutrophils (bacterial ingestion, MCF: normal lavage, 30 ⫾ 3.6
versus CF lavage, 44 ⫾ 4.2; P ⬍ .05). To test the effects of ELF on
pHi following E coli challenge, we incubated neutrophils for 1 hour
with equivalent volumes of saline, CF ELF, or normal ELF, and
then exposed them to bacteria. Upon E coli challenge, pHi in
normal ELF–treated cells was similar to that in cells treated with
vehicle, whereas pHi remained significantly higher in cells preexposed to CF but not normal ELF (Figure 4B), suggesting that
inflammatory mediators limit acidification in phagocytosing cells.
The correlation between pHi and apoptosis may be an epiphenomenon. To probe a direct link between pHi and apoptosis, we
manipulated pHi in phagocytosing neutrophils by inhibition of one
or more proton extrusive mechanisms and assessed the effect on the
mechanism of cell death. Either amiloride or ZnCl2 accelerated
apoptosis in neutrophils exposed to E coli at a pathogen-tophagocyte ratio of 20:1 (Figure 6A). Bafilomycin did not alter rates
of apoptosis at a pathogen-to-phagocyte ratio of 20:1, but at lower
pathogen-to-phagocyte ratios (3:1), at which we had established
that bafilomycin more clearly affected pHi, apoptosis was promoted
(apoptosis at 16 hours, mean ⫾ SE: E coli, 18.9% ⫾ 4.2% versus E
coli ⫹ bafilomycin, 31% ⫾ 4.8%; P ⬍ .05). At lower pathogen-tophagocyte ratios (3:1), amiloride and ZnCl2 acidified pHi and
accelerated apoptosis to a modest degree (apoptosis at 16 hours,
mean ⫾ SE: E coli (3:1), 18.9% ⫾ 4.2% versus E coli (3:1) ⫹
amiloride, 25.7% ⫾ 3.9%; P ⬍ .05). The presence of ZnCl2 at this
lower pathogen-to-phagocyte ratio, which had only a minor effect
on pHi, did not affect apoptotic rates significantly (apoptosis at 16
hours, mean ⫾ SE: E coli (3:1), 18.9% ⫾ 4.2% versus E coli
Effect of low- and high-level bacterial exposure on neutrophil
apoptosis at physiologic pHo, and its correlation with
pHi and intracellular oxidant production
At low pathogen-to-phagocyte ratios, E coli ingestion significantly
delayed apoptosis, whereas higher ratios accelerated it (Figure 5A).
It is puzzling that a quantitatively dissimilar but qualitatively
similar signal produced opposite effects on apoptosis. We speculated that this might be the result of differential effects on pHi, and
thus correlated apoptosis with pHi changes and also with intracellular oxidant generation following phagocytosis, because the latter
has previously been implicated in neutrophil apoptosis. Apoptosis
correlated significantly with the lowest recorded pHi following E
coli exposure (r2 ⫽ 0.872) and with pHi 5 minutes after exposure to
E coli (r2 ⫽ 0.902). Net change in pHi (calculated by measuring the
curve area above and below pHi in non–bacteria-exposed neutrophils; see “Materials and methods”) over 90 minutes correlated
even more closely with rates of apoptosis (r2 ⫽ 0.95; Figure 5B).
Low pathogen-to-phagocyte ratios alkalinized pHi and inhibited
apoptosis, whereas higher ratios acidified pHi and activated apopto-
Figure 4. The effect of the inflammatory milieu from the lungs of CF patients on
baseline pHi and pHi in phagocytosing neutrophils. Isolated human peripheral
blood neutrophils were exposed to CF ELF (concentrated from BAL fluid from stable
CF patients or healthy controls), and pHi was measured. (A) The acute alkalinizing
effect on pHi of the addition of CF ELF (40 ␮L/mL) to neutrophils in culture. (B) The
effect of preincubation with CF ELF (30 minutes, 30 ␮L/mL) on pHi after exposure to
opsonized heat-killed E coli (EC; pathogen-to-phagocyte ratio 20:1) (*P ⬍ .02, CF
versus normal ELF by 2-way ANOVA). All data mean ⫾ SEM.
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COAKLEY et al
Figure 5. Correlation of phagocytosis-induced neutrophil apoptosis with changes in pHi and intracellular oxidant generation. Isolated peripheral blood neutrophils
were exposed to varying pathogen-to-phagocyte ratios, and early changes in pHi, intracellular oxidant production, and apoptosis were measured after 16 hours. (A) Neutrophil
apoptosis is inhibited at lower pathogen-to-phagocyte ratios (*P ⬍ .01 by 2-way ANOVA), but is activated at higher ratios (**P ⬍ .005 by 2-way ANOVA). (B) Linear correlation
between ⌬pHi over 90 minutes and ⌬% apoptosis at varying pathogen-to-phagocyte ratios. Net ⌬pHi is based on a calculation of the phagocytosis-induced change in net pHi
curve area above (net alkalinization) and below (net acidification) baseline pHi (baseline pHi curve is that of non–bacteria-exposed neutrophils) over time. (C) Linear correlation
between intracellular oxidant generation over 90 minutes and ⌬% apoptosis at varying pathogen-to-phagocyte ratios. All data mean ⫾ SEM; r2 indicates coefficient of
determination.
(3:1) ⫹ ZnCl2, 21.7% ⫾ 4.8%; P ⫽ .17). Exposure of neutrophils
to these inhibitors in the absence of E coli did not affect rates of
apoptosis, suggesting that they do not exert significant pHiindependent effects on the cell’s apoptotic machinery (apoptosis,
mean ⫾ SE: control, 38.5% ⫾ 4.3%, amiloride, 37.7% ⫾ 6.3%,
bafilomycin, 40.5% ⫾ 4.0%, ZnCl2, 43.5% ⫾ 6.1%; P ⫽ .3
by ANOVA).
Of note, all 3 inhibitors together at high bacterial density did not
increase apoptosis, but instead promoted necrosis (Figure 6B). In
the absence of inhibitors and at physiologic pHo, little necrosis was
induced following high-density bacterial exposure (Figure 6B),
suggesting efficient function of apoptotic machinery even when
bacteria are numerous. However, high-density bacterial exposure
when pHo was low (5.8) induced significant necrosis (Figure 6B),
suggesting that excessive declines in pHi interfere with the
inception or perpetuation of apoptosis.
Because the inflammatory milieu may contain many proapoptotic and antiapoptotic substances (eg, proapoptotic inflammatory
mediators soluble FAS ligand (CD95) and tumor necrosis factor–␣;
antiapoptotic inflammatory mediators LPS, N-formyl-methionylleucyl-phenylalanine [FMLP], and interleukin-8 [IL-8]), we monitored apoptosis following preincubation with CF ELF and normal
ELF. Normal ELF did not affect apoptosis (data not shown).
However, CF ELF delayed apoptosis in phagocytosing neutrophils,
without inducing necrosis (Figure 6A,B). CF ELF also delayed
constitutive apoptosis (apoptosis: control, 39.4% ⫾ 4.2% versus
Figure 6. Perturbations of pHi and the inflammatory milieu alter mechanisms of
cell death in phagocytosing neutrophils. (A) The effects of amiloride (0.2 mM),
ZnCl2 (50 ␮M), bafilomycin (100 nM), and CF ELF (40 ␮L/mL) on rates of apoptosis at
16 hours in phagocytosing neutrophils (4 replicates from 5 healthy donors) exposed
to E coli at a pathogen-to-phagocyte ratio of 20:1. Inhibitors or ELF was added 10
minutes after bacteria. *P ⬍ .05; % apoptosis greater than E coli alone; **P ⬍ .05; %
apoptosis less than E coli. Note that bafilomycin did affect apoptosis in neutrophils
exposed to lower pathogen-to-phagocyte ratios (see text). (B) The effects of
amiloride, ZnCl2, CF ELF, a combination of amiloride plus ZnCl2 plus bafilomycin, and
lowering of the extracellular pH on rates of necrosis in the same cells (*P ⬍ .005
versus E coli alone).
CF ELF treated, 31.0% ⫾ 4.2%; P ⬍ .05). However, when neutrophils were exposed to high bacterial densities at low extracellular
pH (5.8), pretreatment with CF ELF did not prevent necrosis
(necrosis: control, 76.4% ⫾ 9.2% versus CF ELF, 67.8% ⫾ 10.2%;
P ⫽ .56).
Discussion
These studies were designed first to elucidate the capacity and
mechanisms of pHi regulation in phagocytosing neutrophils under
conditions simulating the inflammatory environment in vivo, and
second to establish whether pHi has a role in regulating neutrophil
death. Our results define the relative contributions of NHEs, proton
conductance channels, and V-ATPases in neutrophil pHi regulation
following bacterial ingestion and show that cytosolic pH is not
preserved when many bacteria are ingested or when extracellular
pH is reduced, although proton extrusive capacity may be augmented by components of the inflammatory milieu. Furthermore,
they illustrate that early changes in pHi dictate the fate of the
phagocytosing cell. Acidification of pHi following phagocytosis
accelerates apoptosis, whereas more marked decrements in pHi
induce necrosis.
Our results confirm previous observations that NHEs are
implicated in pHi regulation following phagocytosis12 and extend
those observations by demonstrating that Zn⫹⫹-sensitive passive
proton conductance channels also participate, being relatively more
important in combating the early phase of acidification after
phagocytosis. V-ATPases are implicated predominantly upon lowlevel bacterial exposure. These latter pumps are stored internally
and are trafficked to the plasma membrane upon cellular activation.11 They have been shown to be reinternalized and incorporated
into the phagocytic vacuoles as bacteria are ingested, where they
reduce phagosomal pH. Their proportionately lesser role in pHi
regulation when bacterial density is high (and the cell ingests more
bacteria) may reflect their rapid reinternalization under these
circumstances. It is important to note that all 3 proton extrusive
mechanisms acting in combination did not maintain pHi near
resting levels at high-density bacterial exposures. The simplest
explanation for this is that increasing bacterial loads trigger
metabolic acid production that simply exceeds the cell’s maximal
capacity to extrude protons. Alkalinizing and acidifying effects of
low and high bacterial density thus reflect a scenario where
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
phagocytosis triggers signal transduction events that maximally
activate proton extrusive mechanisms. It is, however, also conceivable that early acidification of pHi, induced by high bacterial
density, reflects active inhibition of proton extrusion as a prerequisite for neutrophil apoptosis. Consistent with this postulate, H2O2
and caspase-8 (which is activated upstream of mitochondrial
changes during apoptosis) induce cytosolic acidification by inhibiting proton extrusion in the course of apoptosis in HL60 cells and
MCF-7 cells, respectively.25,27 This is also consistent with our
unpublished observations that phorbol-12-myristate-13-acetate
(PMA) is more potent than bacterial ingestion as an activator of
NADPH oxidase (the major source of metabolically generated
protons in activated neutrophils38), but yet does not induce the
same rapid acidification of pHi (May 1998). This lack of a rapid fall
in pHi is also consistent with the observation that, although PMA
induces some features of apoptosis in neutrophils, it does not
appear to stimulate caspase-mediated DNA fragmentation,39 which
may require a decline in pHi for its promotion.
These studies implicate pHi in the regulation of neutrophil
apoptosis following phagocytosis. There are few previous studies
in this regard relating to neutrophils and related cells, and their
results are conflicting.6,13,32,33 The role of cytosolic acidification in
promoting the apoptotic cascade appears to be more established in
other cell types. Critical for the inception of mitochondriadependent apoptosis, which has been described in neutrophils,40 is
activation of the proapoptotic caspase-3 by mitochondrial cytochrome C (cytC). This is maximal at pH values less than 6.8 but
minimal at physiologic pH.22 This finding is consistent with the pHi
values that we detected in neutrophils under conditions in which
apoptosis was either activated or suppressed. In our study, significant acceleration of apoptosis occurred only when pHi during the
first hour fell below 6.8, and even higher rates of apoptosis
accompanied more profound and sustained falls in pHi (high
bacterial loading), whereas increases in pHi (low bacterial loading)
were paralleled by inhibition of apoptosis. Our studies suggest that
pHi actively modulates apoptosis, rather than simply paralleling it,
because manipulation of pHi (by targeted inhibition of NHEs,
V-ATPases, or proton conductance channels) after phagocytosis
acidified pHi and accelerated apoptosis. The relationship between
pHi and apoptosis is likely to be complex. Reports that extracellular
acidification inhibits constitutive neutrophil apoptosis may reflect
differential pH sensitivity of constitutive versus phagocytosisassociated apoptotic pathways. For example, the insertion of the
proapoptotic Bax protein into the mitochondrial cell membrane of
neutrophils (an event occurring upstream of cytC release) is
favored under alkaline conditions and appears to be inhibited at
acidic pHi.41 Phagocytosis may induce signals that overcome the
pH dependence of Bax insertion, leading to a cascade of events
downstream with an alternative pH sensitivity. This scenario would
be teleologically advantageous because acidification of pHi in an
acidic milieu would protect neutrophils from constitutive apoptosis
(before they have usefully ingested bacteria), but accelerate
apoptosis after successful bacterial ingestion (in cells that have
exhausted their useful capacity, but pose a threat to host tissues if
they undergo necrosis). Alternatively, it has been suggested that
constitutive neutrophil apoptosis is independent of mitochondria,
in which case lack of a proapoptotic effect of cytosolic and
extracellular acidification would be an anticipated finding.40 Our
use of pharmacologic inhibition of individual mechanisms of
proton extrusion might be problematic because these inhibitors
may have pH-independent effects on apoptotic pathways. However, the weight of evidence from our experiments, particularly the
pH AND CELL DEATH IN PHAGOCYTIC NEUTROPHILS
3389
close correlation between pHi and apoptosis in the absence of these
pharmacologic inhibitors, favors a role for pHi in regulating
apoptosis in neutrophils when considered in toto.
pHi is likely to be an important modulator, rather than an
initiator, of agonist-mediated neutrophil apoptosis. Reactive oxygen intermediates have been implicated in the instigation of
neutrophil apoptosis.42,43 However, this relationship is apparently
not a simple one because oxidants do not appear to be involved in
neutrophil apoptosis in some situations,44 and proinflammatory
signals, such as FMLP and IL-8, activate the generation of reactive
oxygen intermediates but also inhibit apoptosis. Oxidants may be
an initial signal for cytC release, with downstream events modulated significantly by pHi. This is consistent with our observation
that low-density bacterial exposure increased the generation of
reactive oxygen intermediates but alkalinized pHi and suppressed apoptosis.
How might our findings be integrated into a model of neutrophil
behavior in inflammatory microenvironments? Optimally, neutrophil survival at inflammatory sites should be lengthy enough to
facilitate ingestion of the maximum number of bacteria before an
orderly process of cell death is instigated, thus restricting harm to
host tissues. Although neutrophils were reported to undergo
apoptosis after E coli ingestion,18 other studies suggested inhibition
of apoptosis at low pathogen-to-phagocyte ratios, but induction of
necrosis at higher ratios.19,20 This report refines previous observations on the effects of bacterial load on neutrophil apoptosis,
suggesting that neutrophil survival is promoted by ingestion of
relatively few bacteria. Where bacterial density in vivo is low (in
mild or early infection, for instance), neutrophil lifespan would be
prolonged, increasing the probability of useful subsequent phagocytosis if bacterial density subsequently increases. Because the
capacity of neutrophils to kill bacteria is limited by their inability to
regenerate intracellular microbicidal substances (such as lysosomal
granule contents), it is desirable that apoptosis is triggered when
the ingestion of a larger number of bacteria exhausts these stores.
Because many diseases involve apparently dysregulated neutrophil
inflammation, a limited ability of homeostatic mechanisms to
support a favorable mode of cell death under all pathologic
conditions is hardly surprising. In this respect, although we found
that these processes were efficient at physiologic extracellular pH,
they were compromised in acidic environments (pH 5.8), where
significant necrosis (instead of apoptosis) occurred on exposure to
bacteria at high density. Although the exceptional buffering capacity of the plasma compartment means that the tissue interstitium
will generally not acidify to this degree (even allowing for
anticipated local proton production due to immune-cellular and
bacterial respiration), situations may arise in which microenvironmental pH does fall significantly. It has been established that
serious and poorly resolving bacterial infections (eg, lung abscesses and pulmonary empyemas) are associated with very low
pH, and that low pH under these circumstances is a marker of tissue
destruction.1 The inability of neutrophils to regulate pHi while
ingesting bacteria in such a milieu may contribute to the poorly
resolving and necrotizing nature of these infections by promoting
neutrophil necrosis. How the host manipulates micro-milieu pH in
response to infection and inflammation is largely unknown and
merits scrutiny, particularly in relation to sites that are not in
continuity with the well-buffered plasma and interstitial compartments, such as the luminal epithelial surfaces of the pulmonary,
gastrointestinal, and urogenital tracts. Regulation of the surface
liquid pH on these epithelia may have significant effects on
neutrophil function and survival. This may be particularly pertinent
3390
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
COAKLEY et al
in the case of CF. Lung disease in this condition is characterized by
severe and unremitting bacterial infection and parenchymal lung
destruction, which are mediated in large part by neutrophil-derived
proteolytic and oxidant species. Lack of a CFTR (cystic fibrosis
transmembrane conductance regulator)–dependent apical epithelial
bicarbonate conductance has been hypothesized to cause hyperacidified airway surface liquid.45 Our results predict that in this
abnormally acidic milieu, bacterial ingestion may induce neutrophil necrosis, rather than apoptosis, and thus promote lung
parenchymal degradation. Although we demonstrated that the net
effect of CF ELF was to increase proton extrusion and inhibit
apoptosis in phagocytosing neutrophils, this effect was insufficient
to prevent significant necrosis of neutrophils when pHo was low
and bacterial density was high, a circumstance that likely replicates
the situation in vivo. This would contrast with the situation in the
non-CF lung, where the antiapoptotic effect of the inflammatory
microenvironment coupled with the ability of normal epithelia to
alkalinize surface liquid may protect neutrophils therein from the
deleterious effects of low pH. In keeping with this notion, apoptosis
is suppressed in lung neutrophils from patients with pneumonia.21
Manipulation of the inflammatory microenvironmental pH may
therefore provide a means of attenuating tissue destruction in
necrotizing infections.
In conclusion, we have demonstrated that following phagocytosis in vivo, a precarious balance between cytosolic proton loading
and extrusion likely exists. The resultant changes in pHi modulate
cell death in neutrophils in a manner likely to be relevant in disease
pathology and potentially amenable to therapeutic manipulation.
Acknowledgment
We thank Dr Barbara Grubb for her useful suggestions with regard
to this manuscript.
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