1 Pulmonary Research Division, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9; and 2 Our Lady's Hospital for Sick Children, Dublin 12, Ireland
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ABSTRACT |
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Cystic fibrosis (CF)
is a lethal, hereditary disorder characterized by a
neutrophil-dominated inflammation of the lung. We sought to determine
whether neutrophils from individuals with CF release more neutrophil
elastase (NE) than neutrophils from normal subjects. Our results showed
that peripheral blood neutrophils (PBNs) from normal subjects and
individuals with CF contained similar amounts of NE, but after
preincubation with CF bronchoalveolar lavage (BAL) fluid,
significantly more NE was released by CF PBNs, a release that was
amplified further by incubation with opsonized Escherichia
coli. To determine which components of CF BAL fluid stimulated this
excessive NE release from CF PBNs, we repeated the experiments after
neutralization or immunoprecipitation of tumor necrosis factor
(TNF)- and interleukin (IL)-8 in CF BAL fluid. We found that
subsequent NE release from CF PBNs was reduced significantly when
TNF-
and IL-8 were removed from CF BAL fluid. When TNF-
and IL-8
were used as activating stimuli, CF PBNs released significantly greater
amounts of NE compared with PBNs from control subjects and individuals
with bronchiectasis. These results indicate that CF PBNs respond
abnormally to TNF-
and IL-8 in CF BAL fluid and react to opsonized
bacteria by releasing more NE. This may help explain the increased NE
burden seen in this condition.
secretion; inflammation; proteases; cytokines
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INTRODUCTION |
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CYSTIC FIBROSIS (CF) is an autosomal recessive disorder caused by mutations of the CF transmembrane conductance regulator (CFTR) gene, a 27-exon, 250-kb segment of chromosome 7 at q31 (17, 25, 26). The major cause of mortality and morbidity in patients with CF is lung disease from chronic pulmonary insufficiency, characterized by a neutrophil-dominated inflammation on the respiratory epithelial surface (5, 20). Extensive research has shown that elevated levels of proteases released from neutrophils, most significantly neutrophil elastase (NE), overwhelm the antiprotease defenses of the lung, thus rendering the epithelium susceptible to proteolytic attack and destruction (3, 23). NE, a powerful proteolytic enzyme, is capable of impairing host defense, injuring bronchial epithelial cells, and destroying most of the components of the lung extracellular matrix (20, 22).
The enormous NE burden in the CF lung may be due to infection caused by microorganisms such Staphylococcus aureus or Pseudomonas aeruginosa. However, active NE has been detected in the lungs of very young infants with CF even before the onset of bacterial colonization or infection (2, 4, 19). Although these elevated levels of NE may be due to the increased neutrophil burden on the CF epithelial surface, there are also data to suggest that CF neutrophils differ from normal neutrophils. Stimulated neutrophils from individuals with CF have been shown to release significantly more oxidants (32) and shed significantly less L-selectin compared with those from control subjects (27). This raises the question as to whether the increased NE on the respiratory epithelial surface in CF is due to exaggerated NE secretion by the CF neutrophil. Furthermore, correlation between the CFTR mutation and lung inflammation has been suggested, with dysregulation of cytokine production by CF epithelial cells postulated as causal factors for the sustained inflammation associated with CF (6, 7). These elevated levels of proinflammatory cytokines may act to exaggerate NE secretion from CF neutrophils.
To evaluate this hypothesis, we compared NE release from peripheral blood neutrophils (PBNs) of CF individuals and control subjects. We exposed these cells to the various stimuli found in the milieu of the CF lung. After this, we examined the role of proinflammatory cytokines, shown by these experiments to be centrally involved in NE secretion from CF neutrophils, and compared these effects with those observed for control neutrophils. We also evaluated PBNs from individuals with long-term, non-CF bronchiectasis to ensure that any changes we found were not due to a chronic pulmonary inflammatory stimulus.
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MATERIALS AND METHODS |
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Study Population
Ten children with CF and ten age- and sex-matched control subjects were evaluated for the study, and, in addition, 10 non-CF bronchiectatic patients served as inflammatory controls to the CF population for some of the experiments. The CF individuals attended Our Lady's Hospital for Sick Children (OLHSC; Dublin, Ireland). The mean age of the children was 8 ± 4 yr (range 4-12 yr), and the mean forced expiratory volume in 1 s was >50% of the predicted value. CF was diagnosed by standard criteria including sweat tests and genotyping. All the CF patients studied wereNeutrophil Isolation
Neutrophils were isolated from heparinized (10 U/ml; Sarstedt) venous blood. Briefly, density gradient centrifugation was carried out in Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) to separate the red cell pellet containing the neutrophil population from the lymphocytes. The neutrophils were separated from the erythrocytes by sedimentation in a 3% dextran solution. Residual erythrocytes were lysed by treating the cell pellet with hypotonic saline solution followed by addition of an equal volume of hypertonic saline and finally by washing in Hanks' balanced salt solution (HBSS; Sigma Aldrich, Poole, UK). The isolated neutrophils were resuspended in RPMI 1640 medium (Sigma Aldrich) and counted. Cell viability was confirmed by trypan blue dye exclusion.Priming and Activation of Neutrophils
Neutrophils were resuspended in RPMI 1640 medium at 1 × 106 cells/ml, and a sample of 250 µl of cells was used for each experiment unless stated otherwise.Activation with various stimuli. The cells were incubated with phorbol 12-myristate 13-acetate (PMA; 500 nM), formyl-methionyl-leucyl-phenylalanine (fMLP; 400 nM), or opsonized Escherichia coli (20 µl; prepared for Becton Dickinson by Orpegen Pharma, Heidelberg, Germany) for 30 min at 37°C. The stimuli were chosen because PMA is a protein kinase C agonist and therefore mimics the actions of a number of inflammatory cytokines, fMLP is a secreted bacterial product capable of activating neutrophils, and opsonized E. coli is a phagocytosable organism representative of the opsonized microorganisms almost invariably present in the lungs of individuals with CF. The samples were centrifuged at 200 g for 10 min at 4°C, and the supernatant was removed for determination of NE. The cell pellet was resuspended in 0.1% Triton X-100 in PBS and kept for NE determination.
Incubation with CF bronchoalveolar lavage fluid. Bronchoalveolar lavage (BAL) fluid was obtained from CF individuals with the standard guidelines set out by Klech and Pohl (18). CF BAL fluid (50 µl) was added to each cell suspension and incubated for 45 min at 37°C. The cells were then centrifuged at 200 g for 10 min at 4°C, and the supernatant was removed and discarded. The neutrophils were washed twice in HBSS to remove all remnants of NE activity already present in the CF BAL fluid. The cells were then resuspended in medium or medium containing 20 µl of opsonized E. coli for 30 min at 37°C. An incubation period of 30 min with suitable stimuli has been previously shown to be optimal for NE release from neutrophils (8, 11). The samples were centrifuged as before, the supernatants were retained for measurement of NE, and the lysates were resuspended in PBS-0.1% Triton X-100 for NE determination. Because NE may have been released from both sets of neutrophils during incubation with CF BAL fluid, neutrophil cell lysates were retained after incubation with CF BAL fluid to determine how much NE was released. The cells were lysed with 0.2% Triton X-100 in PBS, and NE content was estimated by ELISA.
The neutrophils were also incubated with CF BAL fluid to which
neutralizing antibodies to tumor necrosis factor (TNF)- and interleukin (IL)-8 (R&D Systems, Abingdon, UK) had been added. For this
experiment, mouse anti-human TNF-
IgG and mouse anti-human IL-8 IgG
were added separately to 50 µl of CF BAL fluid at a final concentration of 12.5 µg/ml for 30 min at room temperature. This BAL
fluid sample was then added to the CF and control neutrophils followed
by washing and incubation with opsonized E. coli as outlined above. An isotype control IgG was also added to the CF BAL fluid at a
final concentration of 12.5 µg/ml and was then added to the CF and
control neutrophils followed by washing and incubation with opsonized
E. coli. The supernatants were retained for NE release as were
the cell lysates. In a separate experiment, anti-TNF-
and IL-8 IgGs
were added together to 50 µl of CF BAL fluid at a final concentration
of 12.5 µg/ml each for 30 min at room temperature. This BAL fluid
sample was then added to the CF and control neutrophils followed by
washing and incubation with opsonized E. coli. The supernatants were retained for measurement of NE release.
Finally, the neutrophils were incubated with anti-TNF- IgG and
anti-IL-8 IgG either separately or together as described above. The
antibody-antigen complexes were then removed by immunoprecipitation with protein A/G (30 µl; Calbiochem-Novabiochem, Nottingham, UK) for
2 h at 4°C. CF BAL fluid was also treated with protein A/G in the
same way. After this time, the protein A/G beads containing antibody-antigen complexes were removed by centrifugation at 13,000 rpm
for 2 min. The remaining BAL fluid supernatant was then added to CF and
control neutrophils followed by washing and incubation with opsonized
E. coli as outlined above. The supernatants were retained for
measurement of NE release.
Measurement of TNF- and IL-8 in CF BAL Fluid
Activation of NE Release by Dual-Cytokine Stimulation
Neutrophils from normal, bronchiectatic, and CF individuals were activated with TNF-Measurement of NE Activity in Cell Supernatants
NE activity in neutrophil supernatants was determined with the NE-specific substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Sigma). Liberation of p-nitroaniline was measured at 405 nm over a 5-min time period. NE activity in the supernatants was compared with active NE standard (Sigma).Measurement of NE Concentration by ELISA
Sheep anti-human NE IgG (Serotech, Kidlington, UK) was diluted 1:1,000 in 0.1 M carbonate buffer, pH 9.6, and 100-µl aliquots were loaded onto a 96-well plate (Immulon 2, Dynatech, Chantilly, VA) and left overnight at 4°C in a humidified chamber. The plates were washed with 200-µl aliquots of PBS-0.05% Tween (PBS-T) three times, and at the end, 100-µl aliquots of PBS-T were pipetted onto the plate. Samples were preincubated with phenylmethylsulfonyl fluoride (Sigma Aldrich) to prevent active NE in the samples from degrading the NE-specific antibodies used in the ELISA and applied to the plate in duplicate 100-µl aliquots. NE standard (250 ng/ml), also inactivated with phenylmethylsulfonyl fluoride, was applied to the plate in duplicate 100-µl aliquots. The standard and samples were then diluted 1:2 across the plate and left at room temperature for 2 h. After this time, the plates were washed as before, and rabbit anti-NE IgG (Calbiochem-Novabiochem) diluted 1:1,000 in PBS-T was loaded onto the plate in 100-µl aliquots and left at room temperature for 1 h. Finally, goat anti-rabbit horseradish peroxidase IgG (DAKO, High Wycombe, UK) diluted 1:2,000 in PBS-T was pipetted onto the plate in 100-µl aliquots and left at room temperature for 1 h. After a final wash, 100 µl of the peroxidase substrate o-phenylenediamine was loaded into each well, and the color was left to develop. After development, the reaction was stopped with 50 µl of 2 M H2SO4 and read at 410 nm with a microtiter plate reader (Bio-Tek, Southampton, UK). Absorbance values were converted to actual NE concentrations by four-parametric logistic fit of the data with the ImmunoFit version 2.0 software package (Beckman Instruments, Fullerton, CA).Measurement of Fc Receptor Type IIa and CD11b/18 Receptor Densities on Neutrophils
A sample (100 µl) of cells resuspended in RPMI 1640 medium was preincubated with 20 µl of CF BAL fluid for 45 min at 37°C. The neutrophils were then washed with HBSS twice and resuspended in medium. BAL fluid-treated neutrophils were then incubated with 5 µl of anti-CD11b/18-PE (Becton Dickinson, Mountain View, CA), 1 µl of anti-Fc receptor type IIa (FcRIIa; Medarex, Annandale, NJ), or 5 µl of isotype control IgG for 30 min at 4°C. After two washes in wash buffer, the cells incubated with anti-CD11b/18-PE were fixed with Cell-Fix (Becton Dickinson). Those neutrophils incubated with anti-FcRIIa were resuspended in RPMI 1640 medium and probed with FITC-labeled goat anti-mouse IgG (DAKO) for 30 min at 4°C. After this time, the cells were washed and fixed as before. Receptor binding of the respective antibodies was quantified by flow cytometry. Flow cytometry analysis was performed on a FACScan flow cytometer (Becton Dickinson) with a 488-nm air-cooled argon laser. A total of 10,000 gated neutrophils were discriminated from lymphocytes with forward versus side (90°) light-scatter characteristics. Fluorescence light emission was collected with a 520-nm band-pass filter in the case of FITC-labeled IgG or a 580-nm band-pass filter in the case of PE-labeled IgG. Data were stored and subsequently analyzed with LYSIS II software (Becton Dickinson).Measurement of TNF- Receptor Types I and II and
IL-8 Receptor Types A and B Densities on Neutrophils
Statistical Evaluation
Data were analyzed with the GraphPad Prism software package (GraphPad Software, San Diego, CA). Results are expressed as means ± SE and were compared with ANOVA, Student's two-tailed t-test (paired or unpaired), or nonparametric tests such as Kruskal-Wallis with Dunn's post hoc analysis as indicated. Differences were considered significant when the P value was 0.05 or less. ![]() |
RESULTS |
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Measurement of TNF- and IL-8 in CF BAL Fluid
Total NE and NE Release From Control and CF PBNs
The total NE for PBNs isolated from control subjects and individuals with CF is shown in Fig. 1. The mean NE value for control PBNs was 220 ± 63 ng compared with 221 ± 41 ng for individuals with CF (p = 0.33). To evaluate NE release from stimulated cells, three stimuli were used to activate the CF and control neutrophils. PBNs were activated with fMLP, PMA, and opsonized E. coli. Stimulation resulted in extremely low NE release from both the CF and control neutrophils with fMLP (CF, 0.41 ± 0.09 ng; control, 0.85 ± 0.41 ng) and PMA (CF, 2.85 ± 0.72 ng; control, 0.97 ± 0.3 ng). Activation with the strongest stimulant, opsonized E. coli, resulted in <2% release of the total neutrophil NE complement (CF, 3.37 ± 1.32 ng; control, 3.22 ± 1.12 ng; Fig. 1). This suggests that PBNs are not primed for activation before they enter the lung in either individuals with CF or control subjects.
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Incubation With CF BAL Fluid
To evaluate NE release in conditions similar to those encountered in the CF lung, CF and control PBNs were primed in CF BAL fluid. This was followed by washing and resuspension in medium and measurement of spontaneous NE release. The results shown in Fig. 2 show NE release from CF BAL fluid-primed cells for both CF individuals and control subjects. This result is higher than that obtained for NE release with fMLP, PMA, or opsonized E. coli. However, NE release for the CF group (33.2 ± 5.1 ng) was significantly higher than that in the control group (23.9 ± 6.1 ng; P < 0.05), suggesting that although the CF BAL fluid may markedly enhance NE release from both the CF and control neutrophils, the CF cells have an intrinsic propensity to secrete more NE. NE release from both sets of neutrophils during the incubation period in CF BAL fluid was determined by measuring the NE content of the cell lysates after this period (and subtracting it from the mean NE content of both sets of cells as shown in Fig. 1). This revealed that only a small amount of NE (<20 ng/250,00 cells) was released from the cells during incubation with CF BAL fluid. Therefore, it is unlikely that a greater amount of NE was released from the normal neutrophils compared with that from CF neutrophils during incubation with CF BAL fluid.
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Neutrophils were also incubated with opsonized bacteria after preincubation with CF BAL fluid. This was to determine how CF and control cells respond to phagocytosable particles in an environment similar to that encountered by neutrophils in the CF lung exposed to opsonized microorganisms. Once again, NE release was higher in both groups (Fig. 2) compared with the results obtained with stimuli alone or cells pretreated with CF BAL fluid only. NE release was markedly increased in the CF group compared with control group (CF, 91.6 ± 11.7 ng; control, 48.1 ± 1.6 ng; P < 0.01) and represented ~42% of the total NE complement compared with only 22% for the control neutrophils. These results show that in conditions similar to those found in the CF lung, neutrophils from individuals with CF can release up to twice as much NE as control neutrophils.
In all experiments, the NE activity in each supernatant was also measured with a NE-specific substrate. The results obtained revealed that more active NE was present in CF samples than in normal samples and correlated very closely with the results obtained for antigenic NE levels in each supernatant (r = 0.8).
Evaluation of FcRIIa and CD11b/18 Receptor Expression on Neutrophils After Incubation in CF BAL Fluid
To evaluate whether the increased E. coli-stimulated NE release, noted in CF neutrophils after incubation with CF BAL fluid, was due to increased expression of receptors involved in the binding and internalization of opsonized bacteria, we measured FcRIIa and CD11b/18 receptor expression on control and CF neutrophils. The data shown in Fig. 3 show no differences in CD11b/18 receptor [control, 1,005 ± 131 mean channel fluorescence (MCF); CF, 962 ± 96 MCF; P = 0.42] or FcRIIa (control, 1,129 ± 38 MCF; CF 1,029 ± 90 MCF; P = 0.27) expression after incubation with CF BAL fluid. This suggests that exposure to CF BAL fluid does not increase receptor binding or ingestion of opsonized E. coli by neutrophils from CF patients compared with those from control subjects.
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Evaluation of Factors in CF BAL Fluid Involved in Stimulating NE Release
To ascertain the role of proinflammatory cytokines in CF BAL fluid in exaggerating the response of CF neutrophils to opsonized E. coli, neutralizing antibodies to these stimuli were incubated with CF BAL fluid to abolish their activity. The results shown in Fig. 4 show that NE release from normal (A) or CF (B) neutrophils is not affected when neutralizing antibodies to either TNF-
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As before, NE activity in each sample was also measured and found to
correlate well with the antigenic levels of NE determined (r = 0.73). This revealed that NE activity was decreased only in the cells
treated with anti-TNF- and anti-IL-8 IgGs together.
Immunoprecipitation of TNF- and IL-8 From CF BAL
Fluid
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Evaluation of TNFRI/II and IL-8RA/B Expression on Neutrophils Before and After Incubation With CF BAL Fluid
To evaluate whether the increased NE release noted in CF neutrophils after incubation with CF BAL fluid was due to increased expression of TNF-
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Effect of TNF- and IL-8 on NE Release From
Neutrophils
As in the case of preincubation with CF BAL fluid, PBNs from CF
patients released more NE than those from normal subjects, and normal
subjects had a profile similar to that of bronchiectatic patients
(control, 29.6 ± 4.6 ng; bronchiectatic, 35.1 ± 7.1 ng; CF, 61.3 ± 6.4 ng; P < 0.01; Fig.
7). NE activity also correlated very
closely to the antigenic NE values that were determined (r = 0.88).
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DISCUSSION |
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This study shows that neutrophils from individuals with CF secrete
supranormal levels of NE when exposed to stimuli similar to those found
in the CF lung. This is despite the fact that the total complement of
NE in the CF neutrophil is the same as that in normal neutrophils.
Furthermore, by inhibiting the actions of two cytokines, TNF- and
IL-8, in CF BAL fluid, one can decrease NE release from these cells in
the presence of opsonized particles to levels observed for control
neutrophils treated in a similar manner. This suggests that the
increased NE secretion observed in the lungs of individuals with CF is
due, in part, to the combined actions of TNF-
and IL-8 in enhancing
the response of CF neutrophils to opsonized particles. NE release from
CF neutrophils exposed to these cytokines is greater than that in
control neutrophils and greater than NE release from neutrophils of
bronchiectatic patients. The latter finding suggests that increased NE
release from CF neutrophils is not due purely to changes induced by
chronic airway inflammation.
It has been assumed that elevated levels of NE in the CF lung are due to increased neutrophil number rather than any inherent abnormality in the CF neutrophil (1, 20). However, increased myeloperoxidase and oxidant release from CF neutrophils has previously been described (30), and it has recently been shown that stimulated neutrophils from CF patients shed less L-selectin than neutrophils from control and bronchiectatic individuals (27). Furthermore, analysis of BAL fluid from individuals with CF after lung transplantation has shown that NE and IL-8 remain significantly elevated compared with those in BAL fluid from non-CF transplantees (14). Thus although the CFTR defect in bronchial epithelial cells may be "cured" as a result of transplantation, NE levels remain elevated, perhaps due to excess NE secretion. In addition, neutrophil-stimulating factors, including cytokines, have been associated with the CFTR defect in epithelial cells. Basal cell expression of IL-6 and IL-8 is significantly higher in cultured human tracheal gland serous cells from individuals with CF compared with that from control subjects, and on stimulation with Pseudomonas aeruginosa lipopolysaccharide (LPS), CF cells express even more IL-6 and IL-8 than their control counterparts (16). Clearly, the ability of unstimulated CF epithelial cells to produce large amounts of proinflammatory cytokines (13, 30) in conjunction with the hyperactive secretory response of CF neutrophils demonstrated in this study can both initiate and propagate a severe cycle of inflammation on the epithelial surface.
The question arises as to how preincubation with CF BAL fluid and
subsequent incubation with opsonized particles leads to this increase.
We initially investigated the ability of CF BAL fluid to upregulate the
CD11b receptors and FcRIIa, which are involved in binding and ingestion
of opsonized particles. However, analysis of these receptors revealed
that their number did not differ between CF and control neutrophils
after incubation with CF BAL fluid, suggesting that increased ingestion
of opsonized particles by CF neutrophils does not occur. Due to the
fact that NE release was markedly higher after preincubation with CF
BAL fluid, we also investigated the components of CF BAL fluid that might be responsible for priming CF neutrophils. Various
proinflammatory modulators in CF BAL fluid were inhibited by the use of
neutralizing antibodies or inhibitors. Inhibitors of NE and LPS, both
of which are present in CF BAL fluid and have been shown to be
proinflammatory (3, 21, 31), did not reduce NE release from CF or
control neutrophils after incubation with opsonized E. coli
(data not shown). Initial efforts to neutralize either TNF- or IL-8
in CF BAL fluid, both of which were present at elevated levels,
followed by incubation with opsonized E. coli had no effect on
reducing NE release from either CF or control neutrophils. However,
when antibodies to TNF-
and IL-8 were added together to CF BAL fluid followed by incubation with opsonized E. coli, this had the
result of decreasing NE release from the control neutrophils by
~25%, but interestingly, NE release from the CF neutrophils was
decreased by >50%. This was not related to increased IL-8- and
TNF-
-receptor density because there was no difference in TNFRI/II
and IL-8RA/B densities on the surface of CF and control neutrophils
before and after incubation with CF BAL fluid. Recombinant TNF-
and IL-8 also increased NE release from CF and normal neutrophils, comparable to the results obtained with CF BAL fluid.
With this as background, it can be postulated that the abnormality in
NE secretion from the CF neutrophil is most likely intracellular, involving one or more of the complex mechanisms governing
degranulation. This might involve a disturbance in the signal
transduction pathway leading from TNF- and/or IL-8 binding to the CF
neutrophil through to activation of protein kinase C and influx of
extracellular calcium. Chemoattractants such as IL-8 bind to their
receptor on neutrophils, and it is thought that this results in
activation of phospholipase C/D that, in turn, leads to the generation
of inositol trisphosphate and diacylglycerol. Inositol trisphosphate stimulates the release of calcium from intracellular stores, and diacylglycerol activates protein kinase C (12, 15, 34). The culmination
of these events is an influx of extracellular calcium and subsequent
oxidant burst and degranulation of azurophilic granules. Degranulation
of neutrophils has also been shown to be governed by cGMP and cAMP
levels, with cGMP promoting degranulation and cAMP preventing it (24,
29). A combination of TNF-
and IL-8 activation of neutrophils has
been shown to lead to a decrease in cAMP levels that results in
degranulation (8). The balance between cGMP and cAMP levels in
activated CF neutrophils might be altered in response to TNF-
and
IL-8 that may, in turn, lead to greater degranulation.
Another possible explanation stems from the fact that, on activation,
the cytosolic pH of CF neutrophils acidifies to a greater extent than
that of normal neutrophils (10). Increased acidification of the
cytosolic pH of neutrophils, as occurs during phagocytosis, is thought
to lead to an increase in phagosomal pH and increased secretion of
proteases including NE (9, 28). This disturbance in pH regulation in CF
neutrophils may also provide an explanation as to why NE secretion from
CF neutrophils is greater than that from control neutrophils. We have
found that although resting cytosolic pH is very similar in CF,
bronchiectatic, and control PBNs on stimulation with fMLP and PMA, CF
PBNs underwent a significant acidification that was not observed with
PBNs from control or bronchiectatic subjects (10). These pH differences
were not attenuated by amiloride and bafilomycin. Further experiments
with DIDS, which inhibits
HCO3/Cl
exchange,
caused alkalinization of activated control but not of CF neutrophils,
suggesting abnormal anion transport in CF cells (10). These results are
important in CF because neutrophil cytosolic acidification has been
previously shown to be associated with increased secretion of
azurophilic granule contents (9, 28). We have also shown that
experiments with a wide variety of physiological stimuli including CF
epithelial lining fluid, Pseudomonas LPS, and secretory
products of activated monocytes caused enhanced proton extrusion in
normal neutrophils that is in marked contrast to the values of lower
cytosolic pH observed in CF PBNs on activation. The question remains as
to why CF BAL fluid and TNF-
and/IL-8 might have specific effects on
CF neutrophils. At present, there is no obvious answer to this,
although a disturbance in the CF degranulation response may be related
to CFTR function or some other intrinsic abnormality in these cells. In
this regard, it should be noted that although CFTR mRNA has been
described in neutrophils, CFTR protein or cAMP-regulated
Cl
-channel activity has not (33).
In summary, we have shown that CF neutrophils act differently from
control neutrophils when exposed to a milieu such as that observed in
the CF lung, secreting nearly twice as much NE as its normal
counterpart. Blocking of the combined activities of TNF- and IL-8 in
CF BAL fluid returns NE secretion from CF neutrophils to levels
observed for normal neutrophils treated in the same way. This suggests
that TNF-
and IL-8 in CF BAL fluid play a significant role in the
priming and/or activation of CF neutrophils, which, in turn, behave
abnormally, resulting in exaggerated NE release and accounting, in
part, for the enormous NE burden and lung destruction observed in this condition.
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ACKNOWLEDGEMENTS |
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This work was supported by The Royal College of Surgeons in Ireland, The Health Research Board of Ireland, The Charitable Infirmary Charitable Trust, and the Higher Education Authority of Ireland.
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FOOTNOTES |
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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.
Address for reprint requests and other correspondence: N. G. McElvaney, Dept. of Medicine, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland (E-mail: respres{at}iol.ie).
Received 22 April 1999; accepted in final form 31 August 1999.
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