Increased elastase release by CF neutrophils is mediated by tumor necrosis factor-alpha and interleukin-8

Clifford Taggart1, Raymond J. Coakley1, Peter Greally2, Gerry Canny2, Shane J. O'Neill1, and Noel G. McElvaney1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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)-alpha and interleukin (IL)-8 in CF BAL fluid. We found that subsequent NE release from CF PBNs was reduced significantly when TNF-alpha and IL-8 were removed from CF BAL fluid. When TNF-alpha 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-alpha 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 were Delta F508 homozygotes and were colonized with Pseudomonas species. All received standard CF therapy, but none had an active exacerbation at the time of the study. The patients with moderately severe pulmonary bronchiectasis were selected from outpatient clinics. They were all negative for the Delta F508 deletion and had normal sweat tests. Childhood infection was the etiology in all cases. The mean forced expiratory volume in 1 s was 50 ± 5% of the predicted value. All were clinically stable and free of infective exacerbation for at least 6 wk at the time of the study. All patients had normal arterial blood gases. This study was performed under a protocol approved by the Institutional Review Board (OLHSC).

Neutrophil 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)-alpha and interleukin (IL)-8 (R&D Systems, Abingdon, UK) had been added. For this experiment, mouse anti-human TNF-alpha 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-alpha 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-alpha 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-alpha and IL-8 in CF BAL Fluid

Levels of TNF-alpha and IL-8 in CF BAL fluid were measured with commercially available quantitative ELISA kits (R&D Systems).

Activation of NE Release by Dual-Cytokine Stimulation

Neutrophils from normal, bronchiectatic, and CF individuals were activated with TNF-alpha (10 ng/ml) for 5 min followed by IL-8 (100 ng/ml) for 30 min at 37°C. After this time, the cells were spun down, and the supernatants and cell lysates were kept as before for the measurement of NE.

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-alpha Receptor Types I and II and IL-8 Receptor Types A and B 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-treated neutrophils were then incubated with 10 µl of anti-TNF-alpha receptor types I and II (TNFRI/II)-FITC or anti-IL-8 receptor types A and B (IL-8RA/B)-FITC (R&D Systems) for 30 min at 4°C. After two washes in wash buffer, the cells were fixed with Cell-Fix (Becton Dickinson). 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. Data were stored and subsequently analyzed with LYSIS II software (Becton Dickinson).

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Measurement of TNF-alpha and IL-8 in CF BAL Fluid

Quantitation of TNF-alpha in CF BAL fluid revealed that this protein was present at 8.5 ng/ml. IL-8 was present at 67 ng/ml 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.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Neutrophil elastase (NE) in lysates of normal (Norm) and cystic fibrosis (CF) neutrophils and in normal and CF supernatants after stimulation with opsonized Escherichia coli. To determine total cellular NE, normal and CF neutrophils were lysed (PBS-Triton X-100) and analyzed for NE content by ELISA. To determine effect of opsonized bacteria on NE secretion, an equal number of normal and CF neutrophils were incubated with 20 µl of opsonized E. coli for 30 min at 37°C. Cells were centrifuged, and supernatant was retained for analysis of NE content by ELISA. Total NE content was the same in CF and normal neutrophils. Addition of opsonized E. coli to CF and normal neutrophils resulted in little increase in NE secretion over baseline.

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.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of CF bronchoalveolar lavage (BAL) fluid on NE release from normal and CF neutrophils. Normal and CF neutrophils were preincubated with CF BAL fluid for 45 min at 37°C followed by resuspension in medium or incubation of normal and CF neutrophils with opsonized E. coli. After this time, cells were pelleted, and supernatant was removed for analysis of NE content by ELISA. In presence of CF BAL fluid alone, CF neutrophils released significantly more NE than normal neutrophils (* P < 0.05). When opsonized E. coli was added to neutrophils after preincubation with CF BAL fluid, CF neutrophils released almost twice as much NE as normal neutrophils (** P < 0.01).

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.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3.   Expression of Fc receptor type IIa (FcRII) and CD11b receptors (CD11) on normal and CF neutrophils after incubation with CF BAL fluid. Normal and CF neutrophils were incubated with CF BAL fluid for 45 min at 37°C, washed, and incubated with either anti-FcRII IgG (1 µl) or anti-CD11 IgG (5 µl) for 30 min at 4°C. Neutrophils labeled with anti-FcRII IgG were then incubated with FITC-labeled goat anti-mouse IgG for 30 min at 4°C. Cells were washed, fixed, and then analyzed for FcRII and CD11 expression by flow cytometry. There were no differences in FcRII or CD11 expression on normal or CF neutrophils after incubation with CF BAL fluid, suggesting that increased release of NE by CF neutrophils after exposure to CF BAL fluid is not due to increased binding or ingestion of opsonized bacteria.

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-alpha or IL-8 are added separately to CF BAL fluid followed by incubation with opsonized E. coli (for anti-TNF-alpha IgG, effect on NE release was 93.8 ± 15.4 to 95.1 ± 16.0 ng for CF and 51.3 ± 6.4 to 50.8 ± 10.7 ng for normal neutrophils; for anti-IL-8 IgG, effect on NE release was 93.8 ± 15.4 to 95.0 ± 8.3 ng for CF and 51.3 ± 6.4 to 51.2 ± 12.5 ng for normal neutrophils). The addition of an isotype control antibody to CF BAL fluid did not reduce NE release from CF (96.11 ± 8.17 ng) or control (51.28 ± 3.93 ng) neutrophils. However, the addition of anti-TNF-alpha and IL-8 IgGs together to CF BAL fluid followed by incubation with opsonized E. coli had the effect of reducing NE release from CF (93.8 ± 15.4 to 43.9 ± 6.0 ng; P < 0.05) and normal (51.3 ± 6.4 to 38.6 ± 7.2 ng) neutrophils. Intriguingly, the reduction in NE release is more pronounced from CF neutrophils (~53%) compared with that from control neutrophils (~25%). This indicates that the TNF-alpha and IL-8 components of CF BAL fluid act together to have a greater effect on the subsequent NE release from CF neutrophils when these cells are incubated with opsonized E. coli in comparison to the effect on NE release observed for control neutrophils treated in the same manner.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of blocking tumor necrosis factor (TNF)-alpha and interleukin (IL)-8 in CF BAL fluid with neutralizing antibodies to both cytokines. Anti-TNF-alpha IgG (12.5 µg/ml), anti-IL-8 IgG (12.5 µg/ml), and isotype control IgG (12.5 µg/ml) were incubated with CF BAL fluid for 30 min at room temperature. Untreated CF BAL fluid or CF BAL fluid plus anti-TNF-alpha IgG, IL-8 IgG, isotype control IgG, or anti-TNF-alpha plus IL-8 IgGs were added to neutrophils (1 × 106/ml) from normal (A) and CF (B) individuals for 45 min at 37°C. After this time, cells were spun down, washed twice in Hanks' balanced salt solution (HBSS), and resuspended in RPMI 1640 medium. Opsonized E. coli (20 µl) was then added to cells for a further 30 min at 37°C. Cells were then spun down, and supernatant was retained for NE determination. This revealed that addition of neutralizing antibodies to TNF-alpha , IL-8, or isotype control IgG in CF BAL fluid had no effect on NE release from normal or CF neutrophils after exposure to opsonized E. coli. However, neutralizing antibodies to TNF-alpha and IL-8 together in CF BAL fluid had the effect of reducing NE release from normal neutrophils incubated with opsonized E. coli by ~25%, whereas reduction in NE release from CF neutrophils was on the order of ~53% (* P < 0.05).

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-alpha and anti-IL-8 IgGs together.

Immunoprecipitation of TNF-alpha and IL-8 From CF BAL Fluid

To ensure that the reduction in NE release from CF and normal neutrophils observed in the presence of both anti-TNF-alpha and anti-IL-8 IgGs was not due to an immunosuppressive effect of the antibody-antigen complexes on neutrophil function, we immunoprecipitated TNF-alpha and IL-8 from CF BAL fluid separately and together. To do this, the same antibodies used in the blocking experiments were used again. However, on this occasion, the antibody-antigen complexes formed were removed from the CF BAL fluid with protein A/G (depletion of TNF-alpha and IL-8 was confirmed by measuring the levels of both cytokines with ELISA). Incubation of neutrophils with CF BAL fluid treated in this manner gave similar results as those observed in the blocking experiments. The results in Fig. 5 show that NE release from normal (A) and CF (B) neutrophils is not affected when TNF-alpha or IL-8 is immunoprecipitated separately from CF BAL fluid followed by incubation with opsonized E. coli (for TNF-alpha removal, effect on NE release was 83.1 ± 7.7 to 83.8 ± 8.9 ng for CF and 46.75 ± 10.4 to 45 ± 8.66 ng for normal neutrophils; for IL-8 removal, effect on NE release was 83.1 ± 7.7 to 79 ± 8.1 ng for CF and 46.75 ± 10.4 to 43 ± 7.6 ng for normal neutrophils). However, the immunoprecipitation of TNF-alpha and IL-8 together from CF BAL fluid followed by incubation with opsonized E. coli had the effect of reducing NE release from CF (83.1 ± 7.7 to 37.8 ± 4.4 ng; P < 0.05) and normal (46.75 ± 10.4 to 35.8 ± 6.4 ng) neutrophils as observed in the blocking experiments. As before, the reduction in NE release is more pronounced from CF neutrophils (~55%) compared with that from control neutrophils (~24%). These results confirm those of the blocking experiments that showed that the TNF-alpha and IL-8 components of CF BAL fluid act together to have a greater effect on the subsequent NE release from CF neutrophils. Once again, NE activity was higher for the CF cell supernatants than for the normal supernatants and correlated closely with antigenic NE amounts (r = 0.76).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of immunoprecipitating TNF-alpha and IL-8 in CF BAL fluid with antibodies to both cytokines and protein A/G. Anti-TNF-alpha IgG (12.5 µg/ml) and anti-IL-8 IgG (12.5 µg/ml) were incubated with CF BAL fluid for 30 min at room temperature. CF BAL fluid treated with protein A/G or from which TNF-alpha , IL-8, or TNF-alpha plus IL-8 had been immunoprecipitated together was added to neutrophils (1 × 106/ml) from normal (A) and CF (B) individuals for 45 min at 37°C. After this time, cells were spun down, washed twice in HBSS, and resuspended in RPMI 1640 medium. Opsonized E. coli (20 µl) was then added to cells for a further 30 min at 37°C. Cells were then spun down, and supernatant was retained for NE determination. This revealed that immunoprecipitation of TNF-alpha or IL-8 from CF BAL fluid had no effect on NE release from normal or CF neutrophils after exposure to opsonized E. coli. However, immunoprecipitation of TNF-alpha and IL-8 together from CF BAL fluid incubated with opsonized E. coli had the effect of reducing NE release from normal neutrophils by ~24%, whereas reduction in NE release from CF neutrophils was on the order of ~55% (* P < 0.05).

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-alpha and IL-8 receptors, we measured the cell surface expression of TNFRI/II and IL-8RA/B on control and CF neutrophils. The data in Fig. 6 show no differences in TNFRI (control, 8.60 ± 0.29 MCF; CF, 8.34 ± 0.47 MCF; P = 0.31), TNFRII (control, 9.96 ± 0.28 MCF; CF, 10.12 ± 0.22 MCF; P = 0.31), IL-8RA (control, 60.67 ± 0.89 MCF; CF, 59.49 ± 1.11 MCF; P = 0.5), or IL-8RB (control, 98.66 ± 1.21 MCF; CF, 100.67 ± 0.98 MCF; P = 0.31) after incubation with CF BAL fluid. This suggests that exposure to CF BAL fluid does not increase cell surface receptor expression of TNF-alpha and IL-8 receptors by neutrophils from CF patients compared with those from control subjects.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 6.   Expression of TNF receptor types I and II (TNFRI and TNFRII, respectively; A) and IL-8 receptor types A and B (IL-8RA and IL-8RB, respectively; B) on normal and CF neutrophils after incubation with CF BAL fluid. Normal and CF neutrophils were incubated in medium plus CF BAL fluid for 45 min at 37°C, washed, and incubated with either anti-TNFRI- or anti-TNFRII-FITC IgG (10 µl) or anti-IL-8RA- or IL-8RB-FITC IgG (10 µl) for 30 min at 4°C. Cells were washed, fixed, and then analyzed for TNFRI, TNFRII, IL-8RA, and IL-8RB expression by flow cytometry. There were no differences in TNF-RI, TNFII, IL-8RA, or IL-8RB expression on normal or CF neutrophils after incubation with CF BAL fluid, suggesting that increased release of NE by CF neutrophils after exposure to CF BAL fluid is not due to upregulated cell surface expression of TNF-alpha or IL-8 receptors.

Effect of TNF-alpha and IL-8 on NE Release From Neutrophils

TNF-alpha and IL-8 have previously been shown to stimulate NE release from neutrophils (11). To assess the effect of a dual stimulus of TNF-alpha and IL-8 on NE release from CF compared with control neutrophils, the cells were incubated with both cytokines and NE release was measured in the supernatant. We also isolated neutrophils from non-CF bronchiectatic patients and incubated these with TNF-alpha and IL-8 to ascertain NE release. The rationale for enlisting non-CF bronchiectatic patients was to determine whether chronic pulmonary inflammation per se could result in increased NE release from PBNs isolated from CF individuals.

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).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of TNF-alpha and IL-8 on NE release from activated normal, bronchiectatic, and CF neutrophils. Neutrophils from normal, CF, and bronchiectatic individuals were activated to release NE with TNF-alpha (10 ng/ml) for 5 min followed by IL-8 (100 ng/ml) for 30 min at 37°C. Cells were then pelleted, and supernatant was evaluated for NE content by ELISA. CF neutrophils released significantly more NE than neutrophils from normal and bronchiectatic individuals (* P < 0.01), suggesting that dual action of these cytokines in CF BAL fluid is important in stimulating CF neutrophils to release more NE than in normal or bronchiectatic individuals.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 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-alpha 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-alpha 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-alpha 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-alpha -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-alpha 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-alpha 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-alpha 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-alpha 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 HCO-3/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-alpha 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-alpha 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-alpha 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Armstrong, D. S., K. Grimwood, J. B. Carlin, R. Carzino, J. P. Gutierrez, J. Hull, A. Olinsky, E. M. Phelan, C. F. Robertson, and P. D. Phelan. Lower airway inflammation in infants and young children with cystic fibrosis. Am. J. Respir. Crit. Care Med. 156: 1197-1204, 1997[Abstract/Free Full Text].

2.   Balough, K., M. McCubbin, M. Weinberger, W. Smits, R. Ahrens, and R. Fick. The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis. Pediatr. Pulmonol. 20: 63-70, 1995[ISI][Medline].

3.   Berger, M., R. J. Soerensen, M. F. Tosi, D. G. Dearborn, and G. Doring. Complement receptor expression on neutrophils at an inflammatory site, the Pseudomonas-infected lung in cystic fibrosis. J. Clin. Invest. 84: 1302-1313, 1989[ISI][Medline].

4.   Birrer, P., N. G. McElvaney, A. Rudeberg, C. W. Sommer, C. Liechti-Gallati, R. Kraemer, R. Hubbard, and R. G. Crystal. Protease-antiprotease imbalance in the lungs of children with cystic fibrosis. Am. J. Respir. Crit. Care Med. 150: 207-213, 1994[Abstract].

5.   Boat, T. F., M. J. Welsh, and A. L. Beaudet. Cystic fibrosis. In: The Metabolic Basis of Inherited Disease, edited by C. R. Schriver, A. L. Beaudet, W. S. Sly, and D. Valle. New York: McGraw-Hill, 1989, p. 2649-2680.

6.   Bonfield, T., M. Konstan, and M. Berger. Altered respiratory epithelial cell cytokine production in cystic fibrosis. J. Allergy Clin. Immunol. 104: 72-78, 1999[ISI][Medline].

7.   Bonfield, T. L., J. R. Panuska, M. W. Konstan, K. A. Hilliard, J. B. Hilliard, H. Ghnaim, and M. Berger. Inflammatory cytokines in cystic fibrosis lungs. Am. J. Respir. Crit. Care Med. 152: 2111-2118, 1995[Abstract].

8.   Brandt, E., F. Petersen, and H.-D. Flad. Recombinant tumor necrosis factor-alpha potentiates neutrophil degranulation in response to host defense cytokines neutrophil-activating peptide 2 and IL-8 by modulating intracellular cyclic AMP levels. J. Immunol. 149: 1356-1364, 1992[Abstract/Free Full Text].

9.   Burguyne, R. D., and A. Morgan. Regulated exocytosis. Biochem. J. 293: 305-316, 1993[ISI][Medline].

10.   Coakley, R., C. Taggart, N. O'Regan, G. Canny, S. O'Neill, and N. G. McElvaney. Dysregulation of pH (pHi) in peripheral blood neutrophils in cystic fibrosis (Abstract). Am. J. Respir. Crit. Care Med. 157: A258, 1998.

11.   Csernok, E., M. Ernst, W. Schmitt, D. F. Bainton, and W. L. Gross. Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo. Clin. Exp. Immunol. 95: 244-250, 1994[ISI][Medline].

12.   Dewald, B., M. Thelen, and M. Baggiolini. Two transduction sequences are necessary for neutrophil activation by receptor agonists. J. Biol. Chem. 263: 16179-16184, 1988[Abstract/Free Full Text].

13.   DiMango, E., A. J. Ratner, R. Bryan, S. Tabibi, and A. Prince. Activation of NF-kappa B by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells. J. Clin. Invest. 101: 2598-2605, 1998[Abstract/Free Full Text].

14.   Dosanjh, A. K., D. Elashoff, and R. C. Robbins. The bronchoalveolar lavage fluid of cystic fibrosis lung transplant recipients demonstrates increased interleukin-8 and elastase and decreased IL-10. J. Interferon Cytokine Res. 18: 851-854, 1998[ISI][Medline].

15.   Elsner, J., V. Kaever, A. Emmendorffer, T. Breidenbach, M.-L. Lohmann-Mathes, and J. Roesler. Heterogeneity in the mobilization of cytoplasmic calcium by human polymorphonuclear leucocytes in response to fMLP, C5a, and IL-8/NAP-1. J. Leukoc. Biol. 51: 77-83, 1992[Abstract].

16.   Kammouni, W., C. Figarella, S. Marchand, and M. Merten. Altered cytokine production by cystic fibrosis tracheal gland serous cells. Infect. Immun. 65: 5176-5183, 1997[Abstract].

17.   Kerem, B.-S., J. M. Rommens, J. A. Buchanan, D. Markiewicz, T. K. Cox, A. Chakravarti, M. Buchwald, and L.-C. Tsui. Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073-1080, 1989[ISI][Medline].

18.   Klech, H., and W. Pohl. Technical recommendations and guidelines for bronchoalveolar lavage (BAL). Report of the European Society of Pneumology Task Group. Eur. Respir. J. 2: 561-585, 1989[ISI][Medline].

19.   Konstan, M. W., and M. Berger. Current understanding of the inflammatory process in cystic fibrosis: onset and etiology. Pediatr. Pulmonol. 24: 137-142, 1997[ISI][Medline].

20.   Konstan, M. W., K. A. Hilliard, T. M. Norvell, and M. Berger. Bronchoalveolar lavage findings in cystic fibrosis patients with clinically mild lung disease suggest ongoing infection and inflammation. Am. J. Respir. Crit. Care Med. 150: 448-454, 1994[Abstract].

21.   Kronborg, G. Lipopolysaccharide (LPS), LPS-immune complexes and cytokines as inducers of pulmonary inflammation in patients with cystic fibrosis and chronic Pseudomonas aeruginosa lung infection. APMIS Suppl. 50: 1-30, 1995.

22.   McElvaney, N. G., and R. G. Crystal. Proteases and lung injury. In: The Lung, edited by R. G. Crystal, J. B. West, P. F. Barnes, and E. Weibel. New York: Lippincott-Raven, 1997, p. 2205-2218.

23.   McElvaney, N. G., R. C. Hubbard, P. Birrer, M. S. Chernick, D. B. Caplan, M. M. Frank, and R. G. Crystal. Aerosol alpha 1-antitrypsin treatment for cystic fibrosis. Lancet 337: 392-394, 1991[ISI][Medline].

24.   Petersen, F., J. Van Damme, H.-D. Flad, and E. Brandt. Neutrophil-activating polypeptides IL-8 and NAP-2 induce identical signal transduction pathways in the regulation of lysosomal enzyme release. Lymphokine Cytokine Res. 10: 35-41, 1991[ISI][Medline].

25.   Riordan, J. R., J. M. Rommens, B.-S. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak, J. Zielenski, S. Lok, N. Plavsic, J.-L. Chou, M. L. Drumm, M. C. Iannuzzi, F. S. Collins, and L.-C. Tsui. Identification of the cystic fibrosis gene: cloning and characterization of the complementary DNA. Science 245: 1066-1073, 1989[ISI][Medline].

26.   Rommens, J. M., M. C. Iannuzzi, B.-S. Kerem, M. L. Drumm, G. Melmer, M. Dean, R. Rozmahel, J. L. Cole, D. Kennedy, N. Hidaka, M. Zsiga, M. Buchwald, J. R. Riordan, L.-C. Tsui, and F. S. Collins. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245: 1059-1065, 1989[ISI][Medline].

27.   Russell, K. J., J. McRedmond, N. Mukherji, C. Costello, V. Keatings, S. Linnane, M. Henry, M. X. Fitzgerald, and C. M. O'Connor. Neutrophil adhesion molecule surface expression and responsiveness in cystic fibrosis. Am. J. Respir. Crit. Care Med. 157: 756-761, 1998[Abstract/Free Full Text].

28.   Schumann, M. A., C. C. Leung, and T. A. Raffin. Activation of NADPH-oxidase and its associated whole-cell H+ current in human neutrophils by recombinant human tumor necrosis factor alpha  and formyl-methionyl-leucyl-phenylalanine. J. Biol. Chem. 270: 13124-13132, 1995[Abstract/Free Full Text].

29.   Simchowitz, L., L. C. Fischbein, I. Spilberg, and J. P. Atkinson. Induction of transient elevation in intracellular levels of adenosine-3',5'-cyclic monophosphate by chemotactic factors: an early event in human neutrophil activation. J. Immunol. 124: 1482-1491, 1980[Free Full Text].

30.   Tabary, O., J. M. Zahm, J. Hinnrasky, J. P. Couetil, P. Cornillet, M. Guenounou, D. Gaillard, E. Puchelle, and J. Jacquot. Selective up-regulation of chemokine IL-8 expression in cystic fibrosis bronchial gland cells in vivo and in vitro. Am. J. Pathol. 153: 921-930, 1998[Abstract/Free Full Text].

31.   Tosi, M. F., H. Zakem, and M. Berger. Neutrophil elastase cleaves C3bi on opsonized Pseudomonas as well as CR1 on neutrophils to create a functionally important opsonin receptor mismatch. J. Clin. Invest. 86: 300-308, 1990[ISI][Medline].

32.   Witko-Sarsat, V., R. C. Allen, M. Paulais, A. T. Nguyen, G. Bessou, G. Lenoir, and B. Deschamps-Latscha. Disturbed myeloperoxidase-dependent activity of neutrophils in cystic fibrosis homozygotes and heterozygotes, and its correction by amiloride. J. Immunol. 157: 2728-2735, 1996[Abstract].

33.   Yoshimura, K., N. Nakamura, B. C. Trapnell, C. S. Chu, W. Dalemans, A. Pavirani, J. P. Lecocq, and R. G. Crystal. Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin. Nucleic Acid Res. 19: 5417-5421, 1991[Abstract].

34.   Yuo, A., S. Kitagawa, I. Suzuki, A. Urabe, T. Okabe, M. Saito, and F. Takaku. Tumor necrosis factor as an activator of human granulocytes. Potentiation of the metabolisms triggered by the Ca2+-mobilizing agonists. J. Immunol. 142: 1678-1684, 1989[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 278(1):L33-L41
0002-9513/00 $5.00 Copyright © 2000 the American Physiological Society