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Laboratory of Immunology, Institute of Hematologic Research, National Academy of Medicine, and
Department of Microbiology, Buenos Aires University School of Medicine, Buenos Aires, Argentina
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Extracellular acidosis is a condition commonly associated to a variety of physiological and pathological situations. It has been reported that urine pH values in distal tubules and bladder can fall below 5.5 under physiological conditions (7). It has also been shown that the mean crevicular pH varies from moderately basic (7.7) to mildly acidic (6.5) (8). Other studies have demonstrated that severe tissue ischemia induces extracellular acidosis (9, 10). Measurements of pH achieved in drainage fluids as well as in situ in different tissues have demonstrated that interstitial acidification is a common feature associated with inflammatory processes, where extracellular pH (pHo)3 values as low as 6.1 have been documented (11, 12, 13, 14, 15). On the other hand, exhaustive researches have shown that tumor microenvironments are usually more acidic than the normal ones, with values of pH ranging from 5.8 to 7.6, both in human and rodent malignant tissues (16, 17).
Considering the widespread distribution of acidic microenvironments, it is surprising that to date the effect of interstitial acidosis on neutrophil physiology remains to be resolved. Early studies have shown that acidic pHo per se increases neutrophil locomotion (3, 6, 18). Many studies have also examined the effect of pHo on the activation of the respiratory burst (5, 19, 20, 21). These works have shown that O2- production induced either by opsonized zymosan (Zy), FMLP, or PMA is substantially inhibited at acidic pHo (5, 19, 20, 21). By contrast, studies performed some years ago in our laboratory have shown that acidic pHo markedly enhances oxygen-dependent cytotoxic responses elicited by conventional agonist such as precipiting immune complexes (pIC), Zy, FMLP, and Con A (22). These apparent contrasting findings may be explained considering that the production of reactive oxygen species other than O2- could be increased under extracellular acidic conditions and that these species were responsible for the cytotoxic effect.
The current study was undertaken to determine the effect of extracellular acidification on neutrophil functions. The results to be presented suggest that acidic pHo is in itself able to induce neutrophil activation. Moreover, extracellular acidic conditions enhance neutrophil proinflammatory responses triggered by conventional agonists. Our findings are consistent with the hypothesis that the abrupt decrease in the intracellular pH (pHi) observed after extracellular acidification in neutrophils suspended in bicarbonate-buffered medium, which does not occur in those suspended in bicarbonate-free medium, plays a key role in the stimulation of neutrophil responses. Our observations also show that extracellular acidosis delays neutrophil apoptosis and extends its functional lifespan. Taken together, these results support the notion that acidic pHo may intensify acute inflammatory responses by enhancing neutrophil functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents were obtained as follows: RPMI 1640 medium, dextran 266, FMLP, cytochrome c, superoxide dismutase from bovine erythrocytes (5000 U/mg of protein), amiloride, 4-aminoantipirine, horseradish peroxidase, phenol red, acridine orange, and ethidium bromide from Sigma (St. Louis, MO); Ficoll from Pharmacia Fine Chemicals (Uppsala, Sweeden); Hypaque from Winthrop Products (Buenos Aires, Argentine); carboxy-SNARF-1-AM and Fluo 3-AM from Molecular Probes (Eugene, OR); FCS from Life Technologies (Grand Island, NY), anti-CD18 mAb TS1/18 from American Type Culture Collection (Manassas, VA) and FITC-conjugated mouse anti-human myeloperoxidase (MPO) mAb MPO-7 from Dako Corporation (Carpinteria, CA). pIC were prepared at the equivalence zone with human IgG and rabbit IgG anti-human IgG, as we previously described (22). Zy was prepared by incubating 100 mg of zymosan with 10 ml of normal human serum for 30 min at 37°C.
Culture media
The standard medium employed throughout this study was bicarbonate-buffered RMPI 1640 supplemented with 1% FCS, adjusted to different pH values by the addition of distinct volumes of an isotonic HCl solution. Some assays were performed by employing bicarbonate-free PBS or isotonic PBS supplemented with 24 mM sodium bicarbonate (bicarbonate-supplemented PBS), both containing 1 mM CaCl2 and 1 mM MgCl2, and adjusted to the desired pH values by the addition of an isotonic HCl solution.
Preparation of human neutrophils
Blood samples were obtained from healthy donors by venipuncture of the forearm vein at the Instituto de Investigaciones Hematológicas Blood Bank. Neutrophils were isolated from heparinized human blood samples by dextran sedimentation and Ficoll-Hypaque gradient centrifugation, as described (23). Contaminating erythrocytes were removed by hypotonic lysis. Unless otherwise stated, after washing, the cell pellets (>96% of neutrophils on May Grunwald/Giemsa stained cytopreps) were resuspended in RPMI 1640 supplemented with 1% heat-inactivated FCS, previously adjusted to the desired pH values. In short incubations, cells were placed in a water bath at 37°C (15–30 min). In longer cultures (18 h), cells were maintained at 37°C in a humidified atmosphere either with 5% CO2 (for cells suspended in medium at pH 7.4) or 7% CO2 (for cells suspended in medium at lower pH values) to maintain the desired pHo. In some experiments, after the hypotonic lysis step, cells were suspended either in bicarbonate-free PBS or in bicarbonate-supplemented PBS, containing 1 mM CaCl2 and 1 mM MgCl2.
Acidification of the culture medium
In most of the experiments performed throughout this work,
extracellular acidification was achieved by suspending cell pellets in
media previously adjusted to the desired pH values. However, in some
experiments, acidification of cells suspended in media at
pHo 7.4 was accomplished by the addition of a precalculated
volume of isotonic HCl solution (see Figs. 1
, 8, and 9). By this
experimental approach, after the addition of the HCl solution to
neutrophil suspended in pH 7.4 medium, and until complete
pHo equilibration is attained, different cell fractions
might be instantaneously exposed to pHo values even lower
than the desired pHo. Hence, in these cases, control
experiments were also performed in which cell pellets were suspended in
media previously adjusted to the desired pH value. Similar results were
obtained by both methods.
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Flow cytometric assays were performed in a FACScan argon laser flow cytometer (Becton Dickinson Immunocytometry System, San Jose, CA). Data were analyzed by employing CellQuest software (Becton Dickinson).
Calcium measurements
Changes in intracellular free calcium concentration
([Ca2+]i) were measured using Fluo 3-AM as
previously described (24). Neutrophils, suspended at a concentration of
5 x 106 cells/ml in RPMI 1640 with 4 µM Fluo 3-AM,
were incubated during 30 min at 30°C. Then, cells were washed three
times with RPMI 1640 medium and resuspended at 5 x
106 cells/ml in RPMI 1640 supplemented with 5% FCS.
Aliquots of 50 µl each were then added to 450 µl of 37°C RPMI
1640 medium containing 5% FCS (pH 7.4). The prewarmed sample was
immediately loaded onto the flow cytometer and fluorescence was
recorded during 
25 s. Then, the medium was acidified by the addition
of a predetermined volume of isotonic solution of HCl to adjust its pH
to the desired value, and the fluorescence was recorded during an
additional 40 s. A gate based on forward and side scatter was used
to exclude debris, whereas a time-based gate was used to divide the
original data file and separate cells according to the time at which
their fluorescence in FL1 was measured. Measurements of fluorescence in
samples suspended at pH 7.4 were used to establish a marker at FL1
fluorescence channel number greater than that exhibited by at least
97% of these resting cells. This marker was then used to determine the
relative percentage of activated cells. Cells that raised their
[Ca2+]i to higher levels than that shown by
97% of resting cells in response to the change in the pHo
were considered to be activated. Alternatively, some measurements were
performed employing neutrophils loaded with Fluo 3-AM, washed, and
suspended in bicarbonate-free PBS supplemented with 1 mM
CaCl2.
Neutrophil shape change
Cell pellets containing 2.5 x 106 neutrophils were suspended in 1 ml of RPMI 1640 medium with 1% FCS, previously adjusted to the desired pH value and incubated in a shaking water bath during 5 min at 37°C. In some cases, FMLP was employed as agonist. Cells were then centrifuged, suspended in PBS and fixed by the addition of an equal volume of 0.5% glutaraldehyde in PBS. On the other hand, in kinetic studies, neutrophil shape change was evaluated by flow cytometry simultaneously with pHi changes after the addition of isotonic HCl solution to cells suspended in pH 7.4 medium. The shape change was measured as the shift in the forward light scatter parameter. Results were expressed as mean forward scatter (MFSC) values.
CD18 expression
Neutrophils (2 x 106) were suspended in 2 ml of RPMI 1640 medium containing 1% FCS, previously adjusted to the desired pH value. They were stimulated or not with FMLP and incubated in a shaking water bath at 37°C for the indicated time period. Then, cells were washed, fixed with 0.5% paraformaldehyde in PBS during 30 min at 4°C, and washed with PBS. Finally, cells were stained with anti-CD18 mAb and FITC-conjugated goat IgG anti-mouse IgG. Fluorescence was recorded by flow cytometry. Results were expressed as the mean fluorescence intensity (MFI) in arbitrary fluorescence units.
Hydrogen peroxide generation
Hydrogen peroxide production was measured by a modification of the oxidation of phenol red assay previously described (25). This assay was chosen because it is not influenced by pH variations of the reaction mixture (our unpublished observations). A buffered phenol red solution containing 116 mM NaCl, 24 mM NaHCO3, 10 mM potassium phosphate buffer, pH 7.0, 5.5 mM dextrose, 0.28 mM phenol red, was mixed with bicarbonate-buffered RPMI 1640 medium containing 1% FCS at a 2:1 ratio, and, immediately before starting the assay, 8.5 U/ml of horseradish peroxidase was added. In the course of the experiments, pellets containing 5 x 106 neutrophils were suspended in 1 ml of the reaction mixture previously adjusted to the desired pH values. Then, 1 mM NaN3 was added to block H2O2 consumption by MPO and catalase, and the production of H2O2 was triggered either by FMLP, Zy, or pIC. After a 120-min incubation at 37°C, cells were centrifuged and 900 µl of the supernatants were collected and added to tubes containing 100 µl of 1 mM NaOH. Absorbance at 610 nm was determined in a Hewlett Packard 8452A spectrophotometer. Results, calculated by interpolating the sample absorbance on a linear regression curve obtained from known concentrations of H2O2, were expressed as nmol H2O2 produced per 5 x 106 neutrophils per 120 min.
MPO release
The release of MPO to the extracellular medium was evaluated by using 4-aminoantipyrine as chromogen, as previously described (26). Briefly, pellets containing 2.5 x 106 neutrophils were resuspended in 1 ml RPMI 1640 medium containing 1% FCS previously adjusted to the desired pH value. Then, cells were stimulated with pIC or Zy and incubated in a steady water bath during 30 min at 37°C. After this period, cells were centrifuged and the supernatants were recovered and placed in an incubator with 5% CO2 to equilibrate their pH at 7.4. In a 3-ml cuvette, 1.4 ml of 2.5 mM 4-aminoantipyrine in 170 mM phenol was mixed with 1.5 ml of freshly prepared 1.7 mM H2O2 and incubated 4 min at 25°C. An aliquot of 100 µl of the supernatant was added and the increase in A510 measured for 5 min with continuous stirring at 25°C in a Hewlett Packard (Waldbronn, Germany) model 8452A spectrophotometer. The amount of MPO released was calculated by interpolating the sample activities on a linear regression curve obtained from known concentrations of horseradish peroxidase. MPO attached to neutrophil surface after stimulation under acidic and control pHo conditions was evaluated by flow cytometry by employing a FITC-conjugated mouse anti-human MPO mAb.
Quantitation of cellular apoptosis and viability by fluorescence microscopy
Neutrophils (5 x 106) were resuspended in 2 ml RPMI 1640 medium containing 1% FCS, previously adjusted to the desired pH value, and cultured during 18 h at 37°C. Then, apoptosis was quantitated using the fluorescent DNA-binding dyes acridine orange (100 µg/ml), to determine the percentage of cells that had undergone apoptosis, and ethidium bromide (100 µg/ml), to differentiate between viable and nonviable cells, as previously described (27, 28). With this method, nonapoptotic cell nuclei show variations in fluorescent intensity that reflect the distribution of euchromatin and heterochromatin. By contrast, apoptotic nuclei exhibit highly condensed chromatin that is uniformly stained by acridin orange. To assess the percentage of cells showing morphological features of apoptosis, at least 200 cells were scored in each experiment.
Cytotoxic assay
Neutrophils were cultured during 18 h at 37°C in RPMI
1640 medium previously adjusted to pH 6.7 or 7.4. Then, cells were
washed, suspended in pH 7.4 RPMI 1640 medium, and employed as effector
cells in cytotoxic assays. The assays were performed as we previously
described (29). Briefly, neutrophils (2 x 105) were
stimulated by pIC, FMLP, and Con A in the presence of 2 x
105 51Cr-labeled chicken RBC. After incubation
for 18 h at 37°C, the culture plate was centrifuged, the
radioactivity of supernatants and pellets measured in a 
counter,
and the percentage of lysis determined. Spontaneous release was always
<6%.
Measurement of pHi
Measurement of pHi was performed using carboxy-SNARF-1-AM as previously described (30). Neutrophils (5 x 106/ml in PBS) were loaded with 10 µM carboxy-SNARF-1-AM during 30 min at 37°C, washed in PBS, and resuspended in the same buffer at 5 x 106 in 100 µl. Then, 10 µl of the cell suspension were added to 2 ml of the indicated medium at pH 7.4, and allowed to equilibrate at 37°C during 20 min in a steady water bath. Then, the time courses of changes in internal pH was evaluated before and after the addition of a precalculated volume of isotonic HCl solution to adjust the pHo to 6.7. Assays were performed by flow cytometry, with excitation at 488 nm and emission analysis at FL2 and FL3. Ten thousand events were collected. The pHi was estimated from the ratio of emission intensities at the two wavelengths, standardizing by comparison with the fluorescence intensity ratios of cells whose pHi values were fixed by incubation with nigericin (10 µM) in high-potassium buffers, as previously described (31).
Statistical analysis
Data were analyzed using Wilcoxon nonparametric paired test, in which p values < 0.05 were considered statistically significant.
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Results |
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It is well established that the [Ca2+]i
is increased upon neutrophil stimulation with different agonists such
as FMLP, LPS, and pIC (32). In the present study, Fluo 3-AM was used to
determine whether extracellular acidification was able to induce
calcium mobilization. With this aim, different volumes of an isotonic
HCl solution were added to the sample tubes at pH 7.4 to adjust the
pHo to the desired values. Fig. 1
, which shows the dot plot data from a
single donor, illustrates the pattern of
[Ca2+]i changes after extracellular
acidification. A fraction of neutrophils exhibited a rapid transient
increase in [Ca2+]i, followed by a gradual
decrease to the resting [Ca2+]i levels, 40
s after the addition of the HCl solution (Fig. 1, A–C).
Similar results were observed when extracellular acidification was
achieved by an alternative procedure, i.e., by suspending neutrophils
in culture medium previously adjusted to pH 6.7 (not shown). As
expected, no differences in fluorescence emission were observed when
pHo was maintained at pH 7.4 throughout the experiment
(Fig. 1D).
As Fig. 1E shows, similar cellular activation levels
(expressed as the percentage of the neutrophil population that
underwent an increase in [Ca2+]i above the
threshold we defined as baseline) were documented at pHo
values comprised in the range 6.5–7.0. A gradation of responses was
observed in the intervening pHo range between 7.0 and 7.2,
while no activation was observed at pHo 7.3 (Fig. 1E). It is important to point out that the activation
observed at acidic pHo is presumably underestimated because
previous works have shown that the affinity of Fluo 3-AM for calcium
markedly declines as pH decreases from 7.4 to 5.5, with a twofold
increase in the Kd of Fluo 3-AM for calcium from pH 7.4 to
6.5 (33, 34).
Stimulation of neutrophil shape change by acidic pHo
After appropriate stimulation, neutrophils undergo transient shape
changes, which can be detected by analyzing variations in their light
scattering properties by flow cytometry (35). To ascertain whether
acidic pHo was also able to stimulate cellular shape
changes, neutrophils were cultured during 5 min at 37°C in media
adjusted to different pH values, then they were fixed and analyzed by
flow cytometry. Neutrophils suspended in medium adjusted to pH 6.7
showed higher forward light scattering values than cells suspended at
pHo 7.4 (inset of Fig. 2).
Moreover, a hydrogen ion concentration-dependent increase in the
forward light scatter parameter was observed, with maximal shape
changes at lower pH values (Fig. 2).
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To determine whether acidic pHo was able to induce the
up-regulation of neutrophil adhesion-promoting receptors for
endothelial cells, the expression of the ß2 integrin CD18
was evaluated. Kinetic assays indicated that CD18 expression at
pHo 6.7 peaked at about 5 min and started to decline after
15 min of incubation (Fig. 3). As
expected, the expression of CD18 at pH 7.4 was not significantly
modified over the period of time evaluated (Fig. 3).
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The ability of acidic pHo to induce H2O2 production and MPO release (a measurement of neutrophil degranulation) was also determined. Neither H2O2 generation nor MPO release could be detected either under acidic or control pHo conditions.
Enhancement by acidic pHo of neutrophil responses induced by conventional agonists
Additional experiments indicated that extracellular acidic
conditions were also able to enhance neutrophil responses triggered by
agonists. Data in Table I show that
acidic pHo markedly increased calcium mobilization
triggered by 0.1 nM FMLP, while moderately augmented the response
induced by 1 nM FMLP. Acidic conditions also increased the shape change
response and the up-regulation of the expression of CD18 to every FMLP
concentration tested (Table I). However, these differences were found
statistically significant only for 10 and 1 nM FMLP concentrations.
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, neutrophils stimulated either with
FMLP, pIC, or Zy during 120 min under extracellular acidic conditions
generated markedly higher amounts of H2O2 than
those stimulated in pH 7.4 medium. Additional experiments indicated
that the release of MPO elicited by either pIC or Zy was also
significantly higher when neutrophils were stimulated at acidic
pHo (Fig. 5A).
Because MPO is a highly charged enzyme that once released binds with
high avidity to cell membranes (2), the increased MPO activity detected
in the supernatants of neutrophils incubated in acidic pH medium could
be due to a diminished binding of MPO to the neutrophil membrane. To
rule out this possibility, the MPO attached to the cell surface after
stimulation under acidic and control pHo conditions was
evaluated by flow cytometry. No differences in the fluorescence
emission signal were observed (Fig. 5B).
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Different stimuli that induce neutrophil activation also extend
its lifespan through the inhibition of apoptosis (36). Therefore, we
investigated whether acidic pHo, besides triggering
neutrophil activation, was also able to modulate neutrophil survival.
Apoptotic rates of cells cultured during 18 h at 37°C in media
adjusted to different pH values were evaluated by fluorescence
microscopy. Extracellular acidosis induced a significant inhibition of
neutrophil apoptosis without inducing cellular necrosis. Cell integrity
was always >94% as judged by the exclusion of both trypan blue and
ethidium bromide dyes (Fig. 6).
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Considering the ability of acidic pHo to delay
neutrophil apoptosis, we then evaluated whether the prevention of
apoptosis was associated with an extension of neutrophil lifespan. To
test this possibility, neutrophils were cultured during 18 h at
37°C in media adjusted to either pH 6.7 or 7.4. Then, they were
washed, resuspended in pH 7.4 medium, and employed as effector cells in
cytotoxic assays. Results in Fig. 7
indicate that neutrophils cultured under acidic conditions exhibited a
markedly higher cytotoxic potential than those cultured at pH 7.4.
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In an attempt to determine the underlying mechanisms of neutrophil
stimulation by acidic pHo, we evaluated the time course of
changes in the pHi upon the addition of HCl solution to the
extracellular medium, employing carboxy-SNARF-1-AM-loaded neutrophils.
Results of seven experiments indicated that pHi of resting
neutrophils suspended in RPMI 1640 medium adjusted to pH 7.4 averaged
7.15 ± 0.04. The addition of HCl-isotonic solution to adjust the
pHo to 6.7 elicited a reproducible pattern of changes in
the pHi (Fig. 8A).
As early as 15 s after extracellular acidification (the earliest
time point analyzed), an abrupt fall in the pHi of
0.46 ± 0.06 U was observed (n = 7), which
persisted over the course of 240 s recorded (Fig. 8A).
A similar drop in the pHi was observed when extracellular
acidification was achieved by an alternative procedure, i.e., by
suspending neutrophils in culture medium previously adjusted to pH 6.7
(not shown). As expected, the pHi of neutrophils maintained
in medium adjusted to pH 7.4 remained unaffected over the time analyzed
(n = 7) (Fig. 8A).
The above described experiments were conducted with neutrophils
suspended in RPMI 1640 medium, which contains physiological
concentrations of sodium bicarbonate, to provide culture conditions as
close as possible to those found in vivo (37). We then reasoned that
the abrupt drop in the pHi observed after extracellular
acidification may be due to the reaction of the H+ supplied
by the acid load with the bicarbonate present in the culture medium
(HCO3- + H+ 
CO2 +
H2O), resulting in the production of CO2. This
compound would rapidly diffuse into the cell, causing an overproduction
of intracellular protons (CO2 + H2O
HCO3- + H+) (37). Considering this
hypothesis, we investigated the role of sodium bicarbonate in the
intracellular acidification induced by external acidification.
Experiments were conducted employing neutrophils suspended either in
bicarbonate-free PBS or isotonic bicarbonate-supplemented PBS. The
addition of isotonic-HCl solution to neutrophils suspended in
bicarbonate-free PBS slightly reduced the pHi over the
240 s analyzed (0.11 ± 0.06 U, n = 5) (Fig. 8B). By contrast, the addition of HCl to neutrophils
suspended in bicarbonate-supplemented PBS induced an abrupt fall in the
pHi (0.38 ± 0.05 U, n = 4) (Fig. 8C), similar to that observed upon extracellular
acidification of neutrophils suspended in RPMI 1640 medium. As
expected, the pHi of neutrophils maintained at pH 7.4,
either in bicarbonate-free or bicarbonate-supplemented PBS, remained
unaffected (n = 5) (Fig. 8, B and
C).
Role of intracellular acidification in neutrophil activation induced by extracellular acidification
To define the biochemical basis of neutrophil activation in
response to extracellular acidification, the time relationship between
the pHi and the light scattering responses elicited by the
addition of isotonic HCl solution to the extracellular medium was
examined simultaneously in cells suspended either in RPMI 1640 medium,
bicarbonate-free or bicarbonate-supplemented PBS, both containing 1 mM
CaCl (Fig. 8). It was found a close correlation between both
parameters. Thus, the abrupt drop in the pHi observed in
neutrophils suspended either in RPMI 1640 medium or
bicarbonate-supplemented PBS after extracellular acidification was
accompanied by cellular shape changes (Fig. 8, A and
D, C and F). Conversely, the slight
reduction in the pHi of neutrophils suspended in
bicarbonate-free PBS was associated with a modest alteration in the
light scattering properties (Fig. 8, B and E).
Finally, the effect of extracellular acidification on calcium
mobilization was evaluated in neutrophils suspended in bicarbonate-free
PBS. As shown in Fig. 9, no changes in
[Ca2+]i levels were observed at any
pHo values analyzed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Taking into account that acidic pHo is a common feature of several inflammatory sites where neutrophils are recruited from circulation, it is surprising that its impact on the development of inflammatory processes remains uncertain. Early studies showed that acidic pHo stimulates per se neutrophil locomotion (18). Nahas et al. demonstrated that the rate of neutrophil locomotion as a function of pH presents a bimodal distribution, with peak velocities at pHo 6.5 and 7.4 (3), while Zigmond and Hargrove observed that human and rabbit neutrophils, placed in a pH gradient between 5.0 and 8.0, orient their locomotion toward the acidic pHo (6). In a recent report, Serrano et al. demonstrated that neutrophil CD18 expression is markedly increased under hypercarbic acidic conditions (20% CO2) (38). The authors also found that, despite acidosis which notably decreases ICAM-1 expression on human aortic endothelial cells, it significantly enhances neutrophil adhesion to these cells, promoted by the up-regulated expression of CD18 (38). Although performed by employing a different experimental setting, our findings indicating that acidic pHo increases the expression of neutrophil CD18 are in agreement with Serrano’s studies. Moreover, in experiments designed to determine whether pHo affects neutrophil transendothelial migration, we observed an increase in spontaneous neutrophil locomotion through HUVEC monolayers under acidic conditions (A. Trevani, unpublished results). Taking into account that neutrophil shape change and its adhesive interaction with endothelial cells are essential steps in the process of granulocyte migration to sites of inflammation, our data suggest that extracellular acidic pH may promote neutrophil recruitment at inflammatory sites. Two mechanisms might account for the shape change observed at acidic pHo: 1) cytoeskeleton reorganization that involves changes in the ratio of polymeric (F) to monomeric (G) actin; and 2) cell swelling induced by ionic movement and water uptake, which depends, at least in part, on the activation of the Na+/H+ antiport (39). Indeed, previous studies have demonstrated that the activation of this antiport, which occurs at low pHi, is involved in the cell swelling necessary for efficient neutrophil migration (39). In this regard, we observed that neutrophil shape change promoted by acidic pHo was substantially prevented by 1 mM amiloride, an inhibitor of the Na+/H+ antiport, supporting a role for the antiport in this response (A. Trevani, unpublished results). Additional experiments are required to determine whether the shape change response induced by extracellular acidification also involves changes in the ratio of polymeric (F) to monomeric (G) actin.
In the absence of appropriate stimuli, neutrophils rapidly undergo characteristic changes indicative of programmed cell death or apoptosis, including cell shrinkage, nuclear chromatin condensation, and DNA fragmentation into nucleosome-length fragments (40, 41). Apoptosis, which represents an alternative fate to necrosis, not only determines neutrophil uptake by macrophages, but also is associated with a loss of neutrophil functions, such as chemotaxis, phagocytosis, stimulated shape change, degranulation, and respiratory burst (41, 42). A variety of stimuli that induce neutrophil activation, such as C5a, FMLP, and LPS, have been described to prolong lifespan via inhibition of apoptosis (43, 44, 45). We here demonstrated that cellular activation induced by external acidification is able to delay spontaneous neutrophil apoptosis. Moreover, this delay in apoptotic rates is associated with an extension of neutrophil functional lifespan.
The addition of isotonic-HCl solution to neutrophils suspended in RPMI
1640 medium induced an abrupt drop in their pHi, which
persisted over the recorded period. By contrast, previous reports have
shown only slight changes in the pHi of resting cells after
extracellular acidification (19, 20, 46, 47). These disparate results
can be attributed to differences in the experimental conditions of each
study. Thus, our experiments were conducted in media containing
bicarbonate to provide conditions as close as possible as those in
vivo. Therefore, the immediate fall in the pHi observed
after extracellular acidification may be caused by the rapid diffusion
into the cell of CO2 originated from the reaction of
protons with the bicarbonate present in the culture medium. This would
in turn result in the overproduction of intracellular protons (37, 48).
Conversely, the above mentioned studies were conducted using
HEPES-buffered media without bicarbonate. In this case, the addition of
HCl may lead to titration of the extracellular HEPES buffer without
significantly affecting the pHi. A role for bicarbonate in
the drop of the pHi after extracellular acidification is
supported by the fact that the addition of HCl solution to neutrophil
suspensions in bicarbonate-free PBS induced only a slight decrease in
the pHi (Fig. 8B), while external acidification
of neutrophils suspended in bicarbonate-supplemented PBS caused an
abrupt fall in the pHi (Fig. 8C).
The biochemical basis of neutrophil activation in response to extracellular acidification has not been defined yet. However, our findings suggest a role for intracellular acidification in neutrophil activation induced by extracellular acidosis because only neutrophils that exhibited an abrupt drop in the pHi (i.e., neutrophils suspended in bicarbonate-buffered media) underwent significant shape changes and rises in their [Ca2+]i. Other studies have also indicated that intracellular acidification plays a role in neutrophil activation. In this regard, it has been shown that the intracellular acidification that occurs after the engagement of chemotactic factor-receptors is responsible for the induction of neutrophil shape change (49, 50). Moreover, a role for intracellular acidification as a second messenger for the initiation of chemotaxis, has also been postulated (49).
Our findings also suggest that extracellular acidic conditions not only trigger neutrophil activation, but also modulate the functional profile of neutrophils in response to stimulation with different agonists. In this regard, we have observed that either calcium mobilization, shape change response, and up-regulation of the CD18 expression induced by FMLP were all significantly increased when evaluated in acidic pH media. These results contrast with those reported by Leblebicioglu et al. (21), who found a moderate and nonsignificant reduction in calcium transients in pH 6.7 medium. These differences may be explained considering that those experiments were performed in bicarbonate-free medium and employing a high FMLP concentration (100 nM), conditions that could mask the ability of acidic pHo to enhance this response.
We also found that acidic pHo enhances H2O2 generation induced by conventional agonists. These results contrast with previous reports showing that extracellular acidic pHo inhibits the generation of O2- triggered by different stimuli. In this regard, Gabig et al. observed that O2- production induced by Zy substantially declined as pHo fell from 7.5 to 6.0, with values at pH 7.0, pH 6.5, and pH 6.0 being about 80%, 50% and 11% those at pH 7.5, respectively (5). Similar results were reported in separate works by Simchowitz, Ahlin et al., and Leblebicioglu et al., who found that acidic pHo markedly reduces the amount of O2- generated by FMLP-activated neutrophils (19, 20, 21). In agreement with these studies, we also found that neutrophil O2- production in response to FMLP (10–100 nM) was markedly inhibited at acidic pHo, even though the reaction took place in bicarbonate-buffered medium (our unpublished results). The apparent discrepancy between the effects of acidic pHo on O2- production and H2O2 generation cannot be easily explained. However, an increased rate of dismutation of O2- at acidic pHi values may account for these results. In support of this hypothesis, previous studies have demonstrated that spontaneous dismutation of O2- occurs more rapidly at acidic pH (51). Moreover, findings reported by Tonetti et al. (52) indicated that lowering pHi in 0.1–0.3 U by the addition of short chain carboxylic acids to the culture medium induces not only a significant decrease in the O2- recovery but also an increase in the H2O2 generated by stimulated neutrophils. The fact that acidic pHo favors H2O2 generation by stimulated neutrophils may also explain our previous findings indicating an enhancement of oxygen-dependent cytotoxic responses elicited by pIC, FMLP, and Zy under extracellular acidic conditions (22).
The current work has been developed under an experimental setting that resembles physiologic conditions, where the pairing CO2/HCO3- is the most important extra- and intracellular buffering system (53). Taking into account the essential role that neutrophils play in acute inflammatory processes and considering that interstitial acidic pH characterizes most inflammatory microenvironments, our results support the notion that extracellular acidosis may intensify the acute inflammatory responses by enhancing neutrophil activity and extending its functional lifespan.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Analía S. Trevani, Laboratorio de Inmunología, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina. E-mail address:
3 Abbreviations used in this paper: pHo, extracellular pH; pHi, intracellular pH; [Ca2+]i, intracellular free calcium concentrations; pIC, precipiting immune complexes; Zy, opsonized zymosan; Fluo 3-AM, Fluo 3 acetoxymethyl ester; carboxy SNARF-1-AM, carboxy SemiNaphtoRhodaFluor acetoxymethyl ester; MFSC, mean forward scatter; MFI, mean fluorescence intensity; MPO, myeloperoxidase.
Received for publication May 27, 1998. Accepted for publication January 14, 1999.
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RII activity. Immunology 82:632.[Medline]
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