1 Pulmonary Research Division, Beaumont Hospital, Dublin 9; and 2 Our Lady's Hospital for Sick Children, Crumlin, Dublin 10, Ireland
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ABSTRACT |
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Cystic fibrosis (CF) is a condition
characterized by neutrophil-mediated lung damage and bacterial
colonization. The physiological basis for reported functional
alterations in CF neutrophils, including increased release of
neutrophil elastase, myeloperoxidase, and oxidants, is unknown. These
processes are, however, regulated by intracellular pH
(pHi). We demonstrate here that pHi regulation is altered in neutrophils from CF patients. Although resting
pHi is similar, pHi after acid loading and
activation (N-formyl-methionyl-leucyl-phenylalanine and
phorbol 12-myristate 13-acetate) is more acidic in CF cells than in
normal cells. Furthermore, patients with non-CF-related bronchiectasis
handle acid loading and activation in a fashion similar to subjects
with normal neutrophils, suggesting that chronic pulmonary inflammation
alone does not explain the difference in pHi. This is
further supported by data showing that normal neutrophils exposed to
the CF pulmonary milieu respond by increasing pHi as opposed to decreasing pHi as seen in activated CF
neutrophils. These pHi differences in activated or
acid-loaded CF neutrophils are abrogated by ZnCl2 but not
by amiloride and bafilomycin A1, suggesting that passive
proton conductance is abnormal in CF. In addition, DIDS, which inhibits
HCO3/Cl
exchange, causes alkalinization
of control but not of CF neutrophils, suggesting that anion transport
is also abnormal in CF neutrophils. In summary, we have shown that
pHi regulation in CF neutrophils is intrinsically abnormal,
potentially contributing to the pulmonary manifestations of the condition.
human; inflammation; acid load; anion exchanger; passive proton conductance
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INTRODUCTION |
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CYSTIC FIBROSIS
(CF) is a common, lethal, inherited condition caused by
mutations of the CF transmembrane conductance regulator (CFTR) gene
(12, 27). Although CF is a multisystem
disorder, its most serious manifestations are progressive lung
parenchymal destruction and chronic pulmonary bacterial colonization
(8, 10). The lung destructive process in CF
is mediated predominantly by neutrophils and is characterized by a
marked disruption of the protease-antiprotease (5) and
oxidant-antioxidant (9, 29) balances in the
respiratory epithelial lining fluid. In particular, neutrophil elastase
(NE), an omnivorous protease released by neutrophils, plays a major
role in the progressive degradation of the pulmonary connective tissue
matrix (23). However, the relationship between mutation of
the CFTR gene and neutrophilic pulmonary inflammation is incompletely
understood. The early establishment of inflammation in apparently
noninfected CF lungs (5, 19) and the enhanced
inflammatory response of the noninfected F508 deletion mouse model
of CF (17) suggest a fundamental abnormality of
inflammatory control in the condition. This supposition is reinforced
by the observations of increased NE release from blood neutrophils of
stable CF patients (34) and of enhanced release of
myeloperoxidase (MPO) and MPO-derived oxidants from neutrophils and
monocytes of CF individuals (26, 37). The
genetic basis of these immune phenomena is suggested by increased
MPO-derived oxidant production from individuals who are heterozygotes
for the
F508 deletion. Other changes in neutrophil effector
responses in CF, including abnormal shedding of L-selectin, have also
been reported (30).
The physiological basis for these functional changes is obscure. Altered intracellular pH (pHi) regulation would, however, provide a potential, common mechanistic pathway contributing to diverse functional alterations in these cells. Activated inflammatory cells are intensely metabolic, generating huge quantities of acid equivalents mandating a capacity for prodigious proton translocation, by several mechanisms (2, 13, 32). Regulated intracellular proton trafficking in the face of metabolic acid generation and an acidic inflammatory milieu is essential for optimal cell effector functions (3, 4). In neutrophils, these pH-sensitive functional responses include triggering of secretion in azurophil granules (containing NE and MPO), oxidant production, and microbe killing. Critically, acidification of the cytoplasmic compartment of neutrophils is associated with an increased release of azurophil granule contents via Ca2+-independent activation of phospholipase D (15). In addition, the functional competence of a wide spectrum of other neutrophil effector responses is dependent on tightly controlled pHi because most cellular enzyme systems are pH dependent (28), and these responses (including induction of secretion) are also modulated by decrements in extracellular pH (36).
Ultimately, pHi reflects a balance between metabolic acid
production and a variety of pumps and channels that are implicated in
net proton translocation across cell membranes. These include Na+/H+ exchange, proton-translocating
ATPases, HCO3/Cl
exchangers, and
passive proton conductance channels (13, 28, 32). It has been demonstrated that pH regulatory function
may be altered in vitro by cytokines and bacterial products such as lipopolysaccharide (LPS), which is abundant in the lungs of patients with CF (25, 33). However, the manner in
which components of the inflammatory milieu combine to collectively
affect pH regulation and the way in which it may be altered in vivo by
disease states has not been delineated.
In this study, we hypothesized that pHi regulation may be altered in neutrophils from CF patients due to either the effects of the chronic inflammatory process or the fundamental abnormalities of the cells themselves, which may result in altered effector function (30, 34, 37). We investigated this hypothesis by assessing pHi regulation in resting, activated, and acid-loaded neutrophils from patients with CF and comparing it with neutrophils from normal control subjects and patients with non-CF-related bronchiectasis (to ensure that any differences were not nonspecific manifestations of chronic pulmonary inflammation). Finally, because the inflammatory milieu contains substances that can affect neutrophil pH regulation, we also investigated the effect of activated mononuclear cells and their secretory products, CF bronchoalveolar lavage fluid, and LPS on pHi regulation in neutrophils.
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MATERIALS AND METHODS |
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Study Population
Forty-one patients with CF (24 men and 17 women, mean age 16 ± 4.5 yr) known to be homozygous for theIsolation of Neutrophils
Neutrophils were isolated from heparinized peripheral venous blood. Blood was layered onto cell separation medium (Ficoll-Paque, research grade; Pharmacia Biotech, Uppsala, Sweden) and centrifuged for 30 min at 1,250 rpm. The neutrophil-rich layer was mixed with 3% dextran-saline solution (Sigma-Aldrich, Poole, UK) in the presence of autologous plasma to sediment red blood cells. Remaining erythrocytes were than subjected to brief hypotonic lysis (0.2% saline; Sigma-Aldrich) twice, after which the cells were washed twice, counted, and resuspended in cell culture medium at a concentration of 1 × 106/ml. The neutrophil population was at least 95% pure by morphological assessment cytospin preparations and Giemsa staining. Cell viability was at least 95% as assessed by trypan blue dye exclusion under light microscopy and propidium iodide exclusion with flow cytometry. Cell viability was similar 1 h after stimulation in all groups. The cells were cultured in RPMI 1640 medium containing NaHCO3 (Sigma Cell Culture, Poole, UK) and supplemented with 0.3 g/l of L-glutamine and 25 mM HEPES (pH 7.35).Determination of pHi
The cells were washed twice and resuspended in phosphate-buffered saline (Sigma-Aldrich) at 1 × 106/ml. The cells were loaded with the fluorescent probe Spiro [7H-benzo[c]xanthane-71'(3H)isobenzofuran]-ar'-carboxylic acid, 3-(acetyloxy)-10-(dimethylamino)-3'-oxo-(acetyloxy)methyl ester, or carboxy seminaphthorhodafluor (SNARF; Molecular Probes Europe, Leiden, The Netherlands), at a concentration of 1.68 µM for 30 min at 37°C in 5% CO2. Cytoplasmic esterases limit the distribution of this probe to the cytoplasmic, but not to the granular, compartments of the cell. The cells were than washed twice in RPMI 1640 medium (with HCO3), resuspended at a concentration of 5 × 105/ml, and stored at 37°C in 5% CO2 in polypropylene tubes (to reduce adherence) until an experimental run was commenced. At this time, the cells were transferred to sterile polyethylene flow cytometry tubes (Becton Dickinson Labware, Lincoln Park, NJ). Transfer of the specimen from a 5% CO2 environment to the cytometer sample nozzle took <3 s and measurement of pHi took a further 1-1.5 s, after which that sample was immediately reequilibrated with 5% CO2. We evaluated whether this 3- to 4-s exposure to ambient CO2 could affect the reliability of our findings by studying the rate of pHi change in neutrophils exposed to ambient CO2 for varying lengths of time. pHi rose over time, but no significant alteration was detected for the first minute (data not shown). pHi in loaded cells was measured with flow cytometry (FACScan, Becton Dickinson, Mountain View, CA) At each time point, at least 5,000 cells were analyzed. The probe was stimulated with an argon-laser light at a frequency of 488 nm, and the ratio of emitted fluorescence at 580 nm to that at 630 nm was calculated and correlated with a specific pH value with a linear regression analysis of calibration values. During sample acquisition, characteristic side- and forward-scatter characteristics were used to confine analysis to a homogenous population of neutrophils, excluding clusters and debris. An in situ calibration was performed in neutrophils from all subjects studied. Calibration was performed with a modification of the method of Thomas et al. (35). In brief, after being loaded, the cells were washed and resuspended in HEPES-buffered solutions containing 135 mM KCl (Sigma-Aldrich) and 10 µM nigericin (Sigma-Aldrich) at a varying known pH between 6.5 and 7.8. The K+ ionophore nigericin equilibrates H+ concentration across the outer cell membrane when internal and external K+ concentrations approximate one another. The ratio of emitted fluorescence in the red and blue regions of the spectrum, once stabilized, was measured, and a calibration curve was generated. Ratiometric measurements reduce errors due to photobleaching, cell thickness, and instrument stability as well as leakage and nonuniform loading of the indicator. Calibration was initially performed in 13 normal individuals over a pH range of 6.5-7.8 to validate the technique. Individual calibrations for the experiments were performed over a pH range of 6.5-7.5. Calibration was repeated in stimulated cells from all groups of patients to ensure that calibration was not altered in patient groups compared with control groups. The calibration curve is shown in Fig. 1. This is a sigmoid dose-response curve similar to that described by others (7).
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Cellular Activation and Determination of Relative Roles of Proton-Extrusive Mechanisms
During experimentation, the cells were activated by exposure to phorbol 12-myristate 13-acetate (PMA; 0.625 µM; Sigma-Aldrich) or N-formyl-methionyl-leucyl-phenylalanine (fMLP; 400 nM; Sigma-Aldrich). PMA activates protein kinase C-dependent signaling pathways in common with a number of cytokines, and fMLP is a bacterially derived substance that activates cells by interaction with a specific cell surface receptor. The respiratory epithelial lining surface of the lung in CF is known to contain bacterial by-products and increased concentrations of cytokines. Physiological activation of neutrophils by cytokines or bacterial products is associated with the generation of metabolic acid. Both PMA and fMLP have been demonstrated to cause metabolic acid generation and proton extrusion in human neutrophils, making them suitable for studies of pHi regulation (31). Under these circumstances, cytoplasmic acidification may be prevented by activation of cellular processes that result in net proton extrusion. These processes include proton-translocating ATPases (sensitive to bafilomycin A1), Na+/H+ exchange (sensitive to amiloride), and passive proton conductance (via ZnCl2-sensitive NADPH oxidase-associated channels). pHi may also be modulated by Na+-independent and Na+-dependent HCO3Induction of a Cytoplasmic Acid Load
The inflammatory microenvironment is characterized by a low pH. Low extracellular pH is associated with passive proton loading the cytoplasm. A fall in pHi is prevented by proton extrusion. Neutrophils were subjected to an acid load by two methods. In the first, the cells were exposed to a 40 mM sodium propionic acid (Sigma-Aldrich) solution. This resulted in a rapid but nonsustained fall in pHi as previously noted (11), followed by spontaneous recovery and hyperrealkalinization. The propionate-exposed cells did exhibit some transient alteration in forward-scatter characteristics, but this had normalized fully by the time alkalinization was noted. A second technique of acid loading was utilized. This involved acidification of cells with a 1.34 µM solution of the K+ ionophore nigericin (Sigma-Aldrich), which generates an inwardly directed H+ flux in the presence of low extracellular K+ concentrations. The ionophore is then scavenged from the cell surface by introducing BSA (25 mg/ml; Sigma-Aldrich). The presence of BSA causes a fluorescence-quenching artifact, which is corrected by measuring the alteration in the fluorescence ratio of nonacidified cells from each individual in the presence of BSA and applying this correction to the fluorescence ratio in all acidified cells.Assessment of the Effect of Activated and Quiescent Mononuclear Cells, Their Secretory Products, and Components of the Inflammatory Milieu on pHi Regulation in Activated Neutrophils
Effect of CF bronchoalveolar lavage and LPS on pH regulation in
normal neutrophils.
Similar volumes (20 µl/ml) of CF bronchoalveolar lavage (BAL) fluid
or 1 µg/ml of Pseudomonas aeruginosa lipopolysaccharide (LPS; Sigma-Aldrich) were incubated (for 3 h at 37°C) with
freshly isolated normal neutrophils that were subsequently washed three times, resuspended in PBS, loaded with carboxy SNARF as described in
Determination of pHi and resuspended in
cell culture medium. Neutrophils were then activated with PMA or fMLP.
BAL fluid was derived from BAL performed according to standardized
conventional guidelines on CF patients free from exacerbation for at
least 6 wk and colonized with P. aeruginosa and on normal
control subjects. In brief, during fiber-optic 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 (20). Lavage fluid was filtered
through sterile gauze and centrifuged at 1,100 rpm for 10 min, and the
supernatant was stored at 80°C.
Effect of activated mononuclear cell-conditioned supernatants on pH regulation in normal neutrophils. Activated mononuclear cell supernatants were prepared from suspensions of autologous mononuclear cells activated with either 1) Pseudomonas-derived LPS (1 µg/ml for 4 h; Sigma) or 2) CF BAL fluid for 4 h at 37°C, after which the supernatants (conditioned media) were harvested.
Effect of quiescent and activated mononuclear cells on pH regulation in normal neutrophils. After being loaded with carboxy SNARF, the neutrophils from healthy donors were resuspended for 3 h at 37°C with autologous, unstained mononuclear cells that were either unstimulated or treated with CF BAL fluid (20 µl/ml) at a concentration ensuring a 5:1 ratio of neutrophils to monocytes. The cells were than activated with fMLP or PMA in the presence of the mononuclear cells as previously described in Cellular Activation and Determination of Relative Roles of Proton-Extrusive Mechanisms. Not only could mononuclear cells be distinguished from neutrophils on the basis of size and granularity, but fluorescence signals were also detected only from the population of neutrophils that had been loaded with carboxy SNARF.
Data Presentation and Statistical Analysis
All data are means ± SE. Statistical analysis was performed with GraphPad Prism software or Statistica software on a PC. Linear regression analysis was utilized for calibration and calculation of pHi. Gaussian distribution of pHi was tested with the Kolmogorov-Smirnov test. pHi over time in individual groups was analyzed with repeated-measures ANOVA with Bonferroni's post hoc correction. Differences over time between groups were compared with two-way ANOVA. ![]() |
RESULTS |
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Flow cytometry with carboxy SNARF produced accurate and reproducible measurements of pHi. The curve fit is sigmoid as shown in Fig. 1, which represents analysis of calibration values of 13 consecutive normal patients. This is similar to previously published data (7). Cellular activation with PMA resulted in minor displacement of the calibration linear regression line, but there was no detectable difference in the magnitude of displacement between control and patient groups. pHi was normally distributed in all groups.
pHi in Nonactivated Neutrophils From Control Subjects and Patients With CF and Non-CF-Related Bronchiectasis
There was no difference in resting pHi between CF patients and normal subjects (normal, 7.136 ± 0.016, n = 43; CF, 7.121 ± 0.019, n = 41; P = not significant), and pHi was stable in both groups over time. Resting pHi in neutrophils from patients with non-CF-related bronchiectasis was similar to that in the other groups (bronchiectasis, 7.046 ± 0.036, n = 10; P = not significant vs. CF patients and normal subjects).pHi in Activated Neutrophils From Control Subjects and CF Patients
Activation of cells with the chemotactic peptide fMLP was associated with rapid and profound alkalinization of pHi. The extent of alkalinization in CF neutrophils was significantly limited compared with that in normal cells and neutrophils derived from patients with non-CF-related bronchiectasis (see Fig. 2A). Activation of normal cells with the phorbol ester PMA (0.625 µM; see Fig. 2B) was associated with transient cytosolic alkalinization over several minutes followed by a sustained acidification. After stimulation with PMA, pHi in CF cells was more acidic than that in neutrophils from control subjects or from patients with non-CF-related bronchiectasis. This suggests that the differences noted in CF neutrophils were not nonspecific responses to chronic pulmonary inflammation. The cytoplasmic membrane integrity of neutrophils as assessed by exclusion of propidium iodide dye was maintained after stimulation with PMA and fMLP during the time period of the experiments. There were uniform pHi responses to stimulation in all groups. In all groups, subpopulations of neutrophils characterized by distinct pHi responses were not evident. In addition, where differences in mean pHi were apparent between CF patients and normal subjects, they were characterized by nonoverlapping histograms, suggesting that pHi in CF patients and normal subjects truly differs in the majority of cells (see Fig. 3).
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Realkalinization After Acid Loading in CF and Control Neutrophils
To confirm that the pHi differences in activated cells were due to alterations in net proton extrusion (as opposed to differences in magnitude of metabolic acid production), we analyzed the pH regulatory responses to a cytoplasmic acid load. The induction of a propionic acid load was associated with falls of a similar magnitude in pHi in CF and normal cells. However, the rate of recovery of pHi over the first 4 min in CF cells was less than that observed in normal cells (CF, 0.044 pH unit/min; normal, 0.068 pH unit/min; P < 0.05). Normal neutrophils underwent a significant hyperrealkalinization after recovery (see Fig. 4A). This did not occur in CF cells, however. After acid loading with nigericin (Fig. 4B), more marked falls in pHi were evident compared with those after propionic acid pulsing. The initial rate of recovery after scavenging of nigericin with BSA was similar between normal subjects and CF patients, but after recovery of pHi, hyperalkalinization was evident in normal subjects but not in CF patients (see Fig. 4B).
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Roles of Individual Mechanisms of Proton Extrusion
To elucidate the mechanism underlying the difference in pHi in activated and acid-loaded CF neutrophils, we analyzed the effects of inhibition of putative principal pHi regulatory mechanisms in activated human neutrophils.Effect of bafilomycin A1 on pHi in
PMA-stimulated neutrophils.
Bafilomycin A1 inhibits proton-translocating plasmalemmal
vacuolar proton ATPases, which are commuted to the cytoplasmic
membrane on activation of neutrophils and are known to contribute to
pHi regulation in neutrophils activated with PMA (see Fig.
5A) or fMLP
(24). Bafilomycin A1 did not
significantly affect pHi regulation in
PMA-activated neutrophils in our experiments. However, bafilomycin A1 did not abrogate the difference in pHi
regulation in CF and normal neutrophils, suggesting that
dysfunction of VATPases is not the mechanism of altered
pHi regulation in CF neutrophils.
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Effect of amiloride on pHi in PMA-stimulated neutrophils. Amiloride inhibits the exchange of external Na+ for internal H+ (see Fig. 5B). This is known to be critical for pHi regulation in activated and acid-loaded neutrophils. During stimulation with PMA, the presence of amiloride resulted in significant acidification in neutrophils from control subjects and CF patients. pHi in CF cells was more acidic than that in control cells, however, suggesting that impaired Na+/H+ exchange is not the basis of altered pHi regulatory responses in CF neutrophils.
Effect of ZnCl2 on pHi in PMA-stimulated neutrophils. ZnCl2 inhibits a passive proton conductance that can be induced in PMA-activated neutrophils (see Fig. 5C). This proton extrusion is closely related to NADPH oxidase activity. The presence of ZnCl2 in PMA-activated neutrophils was associated with a profound and sustained acidification in normal neutrophils. There was no significant difference in pHi regulation in PMA-activated neutrophils from normal subjects and CF patients in the presence of ZnCl2.
Effect of DIDS on pHi in PMA-stimulated neutrophils.
In normal, PMA-activated neutrophils, the presence of DIDS was
associated with a marked and significant alkalinization of pHi (see Fig. 6). This is
consistent with a PMA-activated, DIDS-sensitive HCO3 efflux. There was no significant alkalinization
of pHi in the presence of DIDS in CF neutrophils.
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Effect of preincubation with Pseudomonas LPS on pH regulation in activated normal neutrophils. Preincubation with P. aeruginosa-derived LPS (1 µg/ml for 3 h) in the presence of autologous serum before loading with carboxy SNARF did not have a significant direct effect on pHi regulation in neutrophils activated with either fMLP or PMA (data not shown).
Effect of preincubation with CF BAL fluid on pH regulation in
activated normal neutrophils.
PMA activation of normal neutrophils pretreated with CF BAL fluid
resulted in a more alkaline pHi compared with that in
untreated normal neutrophils, suggesting priming of proton extrusion
by components of the CF inflammatory milieu (see Fig.
7A).
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Effect of the presence of Pseudomonas LPS- or CF BAL-treated monocyte-conditioned medium on pH regulation in activated normal neutrophils. PMA activation of normal neutrophils cultured in medium conditioned with either Pseudomonas LPS (Fig. 7B) or CF BAL fluid (Fig. 7C) resulted in a more alkaline pHi.
Effect of the presence of CF BAL fluid-activated autologous mononuclear cells on pHi regulation in normal neutrophils. Neutrophils cultured in the presence of CF BAL fluid-activated autologous mononuclear cells demonstrated a significantly more alkaline pHi on activation with PMA, again supporting a role for the mononuclear cells and their secretory products in the upregulation of proton extrusion in neutrophils (see Fig. 7D). Unstimulated autologous mononuclear cells did not affect pH regulation in activated neutrophils (data not shown).
These results suggest that our observed impairment of the capacity of CF neutrophils to alkalinize pHi on activation does not reflect a physiological effect of either bacterially derived immunomodulatory substances or paracrine effects of activated mononuclear cells. It suggests that the changes are due to more fundamentally altered neutrophil characteristics. ![]() |
DISCUSSION |
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This study shows that pHi regulation is altered in
neutrophils from individuals with CF. Although resting pHi
is similar, pHi after acid loading and activation is more
acidic in CF cells compared with that in cells from normal subjects or
individuals with non-CF-related bronchiectasis. This suggests that
chronic pulmonary inflammation alone does not explain the differences in pHi, a hypothesis further supported by the fact that
normal neutrophils exposed to the CF pulmonary milieu respond by
increasing pHi in contrast to decreasing pHi as
seen in activated CF neutrophils. These pHi differences in
activated or acid-loaded CF neutrophils are abrogated by
ZnCl2 but not by amiloride or bafilomycin A1, suggesting that passive proton conductance is abnormal in CF. In
addition, anion transport may also be abnormal in activated CF
neutrophils because DIDS, which inhibits
HCO3/Cl
exchange, causes alkalinization
of control but not of CF neutrophils.
Alterations in the magnitude and mechanisms of proton trafficking
in the cell may have functional consequences because pHi affects diverse effector responses. Previous research has demonstrated that neutrophils from CF patients release increased amounts of MPO
compared with neutrophils from normal subjects and CF heterozygotes (37). It has also been shown that increased release of NE
from neutrophils of CF patients compared with that from neutrophils of
normal subjects is abnormally regulated by the cytokines tumor necrosis
factor- and interleukin-8 (34). Such alterations in neutrophil effector function in CF neutrophils may be related to the
disturbed pHi response demonstrated in this paper and may account for the large NE burden demonstrated in the CF lung
(5, 23).
Our data suggest that alteration of Na+/H+
exchange is not the basis of abnormal pHi regulation in CF
neutrophils. Exudative normal neutrophils from the lungs of rabbits
after acid loading have been shown to have a
Na+/H+ antiporter-related defect in
pHi regulation (16). However, although
amiloride affected pHi in both normal and CF-activated neutrophils in this study, it did not abrogate differences between normal and CF cells, which were similar in magnitude to those observed
in its absence. H+-ATPase dysfunction is also not the basis
for abnormal pHi regulation in CF cells because bafilomycin
A1 did not affect pHi differences between CF
and normal PMA-activated neutrophils. The differences in
pHi regulation between CF and normal neutrophils were,
however, attenuated in the presence of ZnCl2, suggesting
that a defective function of the Zn2+-sensitive proton
conductance channels in CF cells could account for the differences
observed. However, the rapid and marked fall in pHi on PMA
stimulation in the presence of ZnCl2 may have had nonspecific effects on other pH regulatory mechanisms, masking differences between CF and normal cells. The data from the DIDS experiments is interesting in that it suggests a potential role for
DIDS-inhibitable HCO3 channels in activated normal
neutrophils, a role that is absent in CF neutrophils. The
alkalinization of pHi in the presence of DIDS in
metabolically active cells illustrates, we believe, a role for anion
exchangers in limiting the extent of the cytosolic alkalinization due
to other proton-extrusive mechanisms. Unopposed, the latter may result
in supraphysiological elevation of pHi. Outwardly directed
HCO3
flux may provide the physiological compensation
for this tendency. We have shown that DIDS does not increase
pHi in PMA-treated neutrophils when combined with
inhibitors of other proton extrusive mechanisms. On these occasions,
pHi falls significantly. This suggests that DIDS-inhibitable outwardly directed HCO3
flux is
active only when proton efflux is active and pHi is rising toward physiological levels, rendering the cell vulnerable to excessive alkalinization.
Although these experiments have illustrated perturbations of
pHi regulation in CF neutrophils, they have not
conclusively delineated the cause. Our observations could be explained
either by intrinsic abnormalities in CF neutrophils secondary to the CFTR defect or, alternatively, by the effects of chronic pulmonary inflammation. Against this latter hypothesis is the fact that this
study shows that neutrophils from individuals with chronic non-CF
bronchiectasis behave more like neutrophils from control individuals
than neutrophils from individuals with CF in pHi
regulation. Furthermore, exposure of normal neutrophils to CF BAL
fluid, Pseudomonas LPS, and secretory products of activated
mononuclear cells leads to increased net proton extrusion and a more
alkaline pHi in normal neutrophils in contrast to the
acidification seen in CF neutrophils. This reinforces the contention
that the pHi observations in CF cells are not a common
physiological response to chronic inflammation but represent specific
manifestations of altered response in CF. This raises the possibility
that the differences in pHi regulation in activated CF
neutrophils are intrinsic due to abnormalities in CFTR. Although the
highest density of expression of CFTR is on the apical surface of
secretory epithelial cells and mucous glands, "housekeeping gene"
features of CFTR would support its function in a broad variety of
cells. CFTR mRNA has been detected in neutrophils and other
inflammatory cells, although thus far, it has not been correlated with
abnormal regulation of Cl transport. Although the rates
of detected CFTR transcription may be low, this does not necessarily
mitigate against a functional role for it in these cells because
lymphocytes and lymphoid cell lines have low or undetectable levels of
CFTR mRNA yet display correctable defects in agonist-mediated
Cl
transport (14, 22). However,
it must be reiterated that functionally significant CFTR has not been
demonstrated to date in neutrophils. If present, though, a role for
CFTR in neutrophil pH-HCO3
homeostasis would be
rational on the basis of a previous report (18)
demonstrating CFTR function as a regulator of HCO3
transport in epithelial cells. In addition, abnormalities of subcellular pH have been described in
F508 mutant CFTR cells (1), and CFTR has been shown to be present and functional
in endosomes, regulating endosomal fusion processes (6,
21).
Such a demonstration would have major importance because it would further illustrate the nature of CF as a systemic disorder and not a condition in which the only major expressions are in the respiratory and gastrointestinal tracts. The impact on current concepts of gene therapy would also have to be examined. To date, gene therapy for CF has concentrated on correcting the bronchial epithelial manifestations of the disorder. If there is an intrinsic abnormality in the CF neutrophil, "normalization" of bronchial epithelium either by gene therapy or by lung transplantation will not "cure" the lung manifestations, many of which are mediated through neutrophils and their products. Adjunctive therapy such as antiproteases or antioxidants may also be required
In conclusion, we have presented data supportive of impaired pHi regulation in neutrophils from patients with CF, suggestive of abnormal anion or H+ trafficking, providing potential explanations for excessive neutrophil-mediated inflammatory responses and contributing to pulmonary parenchymal destruction in the condition.
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ACKNOWLEDGEMENTS |
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We acknowledge the invaluable assistance of Niamh O'Regan and Bridie McNulty with regard to the collection of clinical samples.
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FOOTNOTES |
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This study was funded 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.
Address for reprint requests and other correspondence: N. G. McElvaney, Dept. of Medicine, Beaumont Hospital, Dublin 9, Ireland (E-mail: respres{at}iol.ie).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 26 August 1999; accepted in final form 15 February 2000.
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