Department of Medicine and Cardiovascular Research Institute, University of California, San Francisco, California 94143
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ABSTRACT |
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Neutrophils, eosinophils, and their
proinflammatory constituents are important mediators of airway disease,
and high levels of neutrophil proteases and eosinophil cationic protein
(ECP) are found in sputum from patients with cystic fibrosis (CF). To investigate whether neutrophil proteases or CF sputum causes eosinophil degranulation, purified eosinophils from atopic asthmatic subjects were
incubated for 2 h with neutrophil elastase, cathepsin G, and CF sputum,
and the release of ECP was measured. We found that the percent release
of ECP was higher after incubation with neutrophil elastase
(105 M) than with a buffer
control [6.1 ± 0.8 (SE) vs. 1.7 ± 0.1%; P < 0.003] and represented
>50% of the release caused by positive controls
[Ca2+ ionophore A-23187 (5 × 10
6 M) or
serum-coated Sephadex beads]. The release of ECP after incubation
with cathepsin G (2.3 ± 0.2%) and CF sputum (6.2 ± 2.0%) was
also significantly higher than that with a buffer control (P < 0.05). Neutralization of free
elastase activity with
1-proteinase inhibitor reduced
the mean percent degranulation of eosinophils by neutrophil elastase by
50% (P = 0.0004) and by CF sputum by 75% (P = 0.02). Preincubation of
eosinophils with cytochalasin B (10 mg/ml) and depletion of the
incubation medium of Ca2+ also
significantly attenuated degranulation of eosinophils incubated with
purified free neutrophil elastase or CF sputum
(P < 0.05). We conclude that
neutrophil proteases, especially neutrophil elastase, and elastase-rich
CF sputum cause degranulation of eosinophils in a mechanism partially
dependent on Ca2+ and actin filaments.
eosinophil cationic protein
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INTRODUCTION |
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NEUTROPHILS AND EOSINOPHILS play important roles in airway inflammation and tissue damage (18, 19, 28). Neutrophil elastase, a serine protease with broad target specificity, is a major constituent of the azurophil granules in neutrophils. Elevated levels of neutrophil elastase are found in sputum from patients with bronchiectasis, cystic fibrosis (CF), chronic bronchitis, and asthma (5, 7, 17, 24). In addition to damaging tissues by hydrolyzing many proteins associated with basal laminal, extracellular matrix, and cell-associated glycocalyxes (27), neutrophil elastase activates several types of cells, including endothelial (10), epithelial (16), and serous (21) cells. The eosinophil and its granule products are considered important mediators of airway inflammation, especially in asthma (4, 8). Degranulation of eosinophils can be induced by a variety of physiological and nonphysiological stimuli, including immunoglobulin, complement, platelet-activating factor, a Ca2+ ionophore, and serum- or IgA-coated Sephadex beads (1, 2, 12, 15, 26, 29).
Recent clinical studies suggested that neutrophil-eosinophil interactions may be important in the pathogenesis of airway disease. For example, although eosinophils are often considered to play a more important role than neutrophils in mediating asthmatic inflammation, Fahy et al. (6) recently found that both neutrophils and eosinophils are prominent cells in sputum from asthmatic subjects in acute exacerbation. Other investigators (25) have found immunohistochemical evidence of both neutrophil and eosinophil products in airway mucosal tissue from patients dying of acute severe asthma. In addition, although neutrophils are often considered the most important inflammatory cells mediating airway disease associated with CF and bronchiectasis, high levels of eosinophil cationic protein (ECP) have recently been reported in the sputum of patients with CF (14). These findings prompted us to explore possible mechanisms by which neutrophils might interact with eosinophils to mediate airway inflammation. Two lines of evidence suggested to us that neutrophil proteases might degranulate eosinophils. First, our group has already shown that neutrophil elastase and cathepsin G degranulate bovine serous gland cells (21), so we considered it possible that these proteases might also degranulate eosinophils. Second, it has recently been shown that eosinophils can be degranulated by the mast cell and basophil protease tryptase (11). Thus, in this study, we wanted to determine whether neutrophil elastase, cathepsin G, or the elastase-rich supernatants of CF sputum degranulate eosinophils.
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MATERIALS AND METHODS |
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Eosinophil isolation. Eosinophils were
purified from the peripheral blood of nine atopic asthmatic subjects
(age 22-53 yr) with mild clinically stable asthma who were using
only -adrenergic agonists to control their asthma symptoms. Each
subject donated 60 ml of blood on one or more occasions. All subjects
signed consent forms approved by the University of California, San
Francisco (UCSF) Committee on Human Research.
Eosinophils were isolated from whole blood with a two-step method whereby granulocytes were first isolated by Percoll gradient centrifugation and neutrophils were then removed with immunomagnetic beads (9). Briefly, heparinized blood was diluted 1:3 with phosphate-buffered saline (PBS; Cell Culture Facility, UCSF), layered on an isotonic Percoll solution (density 1.082/ml, pH 7.2; Pharmacia Biotech, Uppsala, Sweden), and centrifuged at 400 g for 30 min at 20°C. The supernatant and monocytes at the interface were then carefully removed, and the granulocytes at the second interface were transferred to new tubes. Erythrocytes were lysed by two cycles of exposure to sterile water. Isolated granulocytes were washed two times with Ca2+-free Hanks' balanced salt solution (HBSS) containing 0.5% BSA (Sigma, St. Louis, MO). Anti-CD16 antibody-conjugated magnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany) were added to the resuspended granulocytes. After 35 min of incubation at 4°C, the cells were loaded onto the separation column positioned in the magnetic cell sorting magnetic field (Miltenyi Biotec). Eosinophils were eluted in ice-cold Ca2+-free PBS with 1% BSA. An aliquot of the cells was cytocentrifuged (Shandon Cytospin, Shandon, Pittsburgh, PA) and stained with the Leukostat staining system (Fisher Diagnostics, Pittsburgh, PA) to determine cell purity from a cell count of at least 400 cells. Eosinophils always represented >99% of the eluted cells. Eosinophil viability as assessed by exclusion of trypan blue was always >98.5%. The purified eosinophils were washed two times in PBS with 1% BSA and immediately used for experiments.
Sputum collection and processing. Sputum samples were collected from patients with CF who were attending the outpatient department for routine care or who were hospitalized with acute exacerbations of CF-related lung disease. All subjects were between the ages of 20 and 45 yr and had CF documented by abnormal sweat chloride concentrations and typical clinical profiles. The subjects signed consent forms approved by the UCSF Committee on Human Research.
Sputum was processed within 1 h of collection. The sputum volume was
determined, and an equal volume of PBS was added. The diluted sputum
was briefly mixed gently with a vortex mixer before the addition of two
volumes of 0.01% dithiothreitol (DTT) in saline solution (10%
Sputalysin, Behring Diagnostics, Somerville, NJ). The sample was then
incubated in a shaking water bath at 37°C for 15 min, with removal
for gentle mixing with a transfer pipette at 5-min intervals. The
homogenized sputum was centrifuged at 10,000 g for 20 min, and the supernatant was
aspirated and stored at 70°C for later incubation and analysis.
Neutrophil elastase. Neutrophil elastase derived from human sputum was purchased from Elastin Products (Owensville, MO). According to the manufacturer, the purity of this neutrophil elastase is >95% as assessed by SDS-PAGE. However, we carried out SDS-PAGE on the elastase preparation and found that ~30% of the material had a lower molecular weight than that of neutrophil elastase. Suspecting that this lower-molecular-weight material represented partial degradation products of elastase, we transferred the electrophoresed proteins to nitrocellulose and performed immunoblotting with a polyclonal antibody directed against human neutrophil elastase (Elastin Products). We found that both the higher- and lower-molecular-weight bands were immunoreactive, suggesting that the latter were derived from partial degradation of elastase, likely because of autolysis. As in other reported elastase preparations, the higher-molecular-weight material partially resolved into three bands, which are known to arise from variable N-glycosylation.
We measured the specific activity of the neutrophil elastase in the chromogenic assay system (see Neutrophil elastase assay) using the substrate and conditions methods for determining the specific activity reported by Stein (23). We found that the specific activity of the neutrophil elastase was 87% of that of the highly purified neutrophil elastase preparation used by Stein. The SDS-PAGE and immunoblotting data presented above, together with the data showing the preparation to be 87% active on the basis of the specific activity of a highly purified preparation reported in the literature, suggested that the elastase from Elastin Products, although partially autolyzed, was pure or nearly so.
Eosinophil degranulation. Eosinophils
were suspended at a concentration of 1 × 106 cells/ml in HBSS supplemented
with 2% BSA. Subsequent degranulation experiments were done with
100-µl aliquots of this cell suspension. The control medium was
HBSS-2% BSA. An additional negative control was Sputalysin (containing
0.01% DTT). As positive controls for eosinophil degranulation,
serum-coated Sephadex beads (Pharmacia Biotech) prepared according to
the method of Winqvist et al. (29) and the
Ca2+ ionophore A-23187 (5 × 106 M; Calbiochem, San
Diego, CA) were used. In addition, the effects of neutrophil elastase
and cathepsin G (Elastin Products) were tested. To observe the effect
of CF sputum on eosinophil degranulation, equal volumes of eosinophil
suspension and supernatants from CF sputum samples (final dilution
after processing was 1:8) were incubated. A different sputum sample was
used in each experiment. All experiments were conducted in duplicate.
In a preliminary experiment, we examined the percent release of ECP from eosinophils incubated with neutrophil elastase for 30, 60, and 120 min. The percent release at these time points was 2.9, 3.1, and 6.3%, respectively. Thus, for all subsequent experiments, incubations were for 120 min at 37°C in polypropylene tubes. Reactions were terminated by centrifugation for 5 min at 800 g at room temperature. The cell-free supernatants were collected. The cell pellets were extracted in 0.3% cetyltrimethylammonium bromide for 1 h, then centrifuged at 1,000 g. Cell-free supernatants and extracts of pellets were assayed for ECP. ECP levels in the cell pellets were measured in parallel with the supernatants from the cell pellets lysed with 0.3% cetyltrimethylammonium bromide.
The percent release of ECP (%ECPrel) after stimulation was calculated according to the equation %ECPrel = ECPstim/ECPtotal × 100, where ECPstim is the ECP level in the cell-free supernatant after incubation with a stimulus and ECPtotal is the total cellular ECP content as determined by adding the ECP level in the cell-free supernatant to that in the pellet from a parallel negative control. Because CF sputum contained high levels of ECP at baseline, the release of ECP from eosinophils by CF sputum was corrected for by subtracting the ECP in the CF sputum from the total ECP release stimulated by the same sputum sample.
To investigate the role of actin filaments on any protease-induced
eosinophil degranulation, eosinophils were first incubated with
cytochalasin B (10 mg/ml) for 15 min before the addition of the
protease stimuli. In addition, to test the relative roles of catalytic
and noncatalytic neutrophil elastase activities on any protease-induced
eosinophil degranulation, 100 µl of neutrophil elastase
(n = 5 experiments) and
100 µl of the supernatant from CF sputum
(n = 9 experiments) were preincubated
with 10 µl of 1-proteinase
inhibitor (
1-PI;
10
4 M) at 37°C for 15 min before the eosinophil suspension was added. Finally, to test the
role of Ca2+ in any
protease-induced degranulation, a series of degranulation experiments
were performed in Ca2+-free
HBSS-2% BSA.
ECP assay. ECP was measured with a commercially available radioimmunoassay that has a lower limit of detection of 2 ng/ml (Pharmacia Diagnostics, Fairfield, NJ). The effects of Sputalysin on the radioimmunoassay for ECP was tested. The 80-ng ECP standard was diluted onefold, twofold, and fourfold in the assay diluent and in 10% Sputalysin, and ECP concentrations similar to the predicted ECP concentrations were obtained with both the assay buffer diluent and the Sputalysin diluent (data not shown).
Neutrophil elastase assay. To
determine the free elastase activity of samples preincubated with
1-PI and in the sputum samples from the patients with CF, a chromogenic substrate largely specific for
human neutrophil elastase
(methoxysuccinyl-L-alanyl-alanyl-prolyl-L-valyl-4-nitroanilide) was used as previously described (7). Briefly, the release of
chromophore was detected at 410 nm after 10 µl of sample were mixed
with 3 ml of substrate solution in 1-methyl-2-pyrrolidinone and
Tris-NaCl buffer (1:100). The standard and positive controls were
purified sputum neutrophil elastase. All measurements were carried out
in duplicate. Sputalysin did not affect the neutrophil elastase assay;
Sputalysin was added to the substrate solution to yield a final
concentration of DTT of 0.025%, and the activity of three
concentrations of neutrophil elastase (0.1, 0.2, and 0.3 mg/ml) was not
changed compared with the buffer control (data not shown).
Lactate dehydrogenase assay. The levels of lactate dehydrogenase (LDH) in the supernatants after incubation were determined with a commercially available bioassay (Sigma). Briefly, the formation of NADH was determined spectrophotometrically (340 nm) after 50 µl of sample were mixed with 1 ml of prewarmed substrate. The standard was purified LDH (Sigma).
Statistical analysis. Data are expressed as means ± SE. Paired two-tailed Student's t-tests were used to compare the effects of different stimuli on eosinophil degranulation. An exception was the data for CF sputum, which were not normally distributed and for which the Wilcoxon signed rank test was employed. Spearman's rank order test was used to determine correlations between data. Probability values of <0.05 by two-tailed tests were considered significant.
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RESULTS |
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Effects of neutrophil proteases on eosinophil
degranulation. Neutrophil elastase caused degranulation
of purified eosinophils (Table 1, Fig.
1). The threshold concentration for this effect was
107 M and was dose
dependent up to 10
5 M, the
highest concentration tested. Serum-coated Sephadex beads and the
Ca2+ ionophore A-23187 (the
positive controls) induced degranulation of 11.1 and 7.4%,
respectively, of total intracellular ECP (Table 1, Fig.
2). At
10
5 M, neutrophil elastase
induced degranulation of 6.1% of total intracellular ECP, which is
82% of the degranulation induced by the
Ca2+ ionophore and 55% of
degranulation induced by the serum-coated Sephadex beads (Fig. 2).
Cathepsin G caused a small but significant degranulation of eosinophils
(Fig. 2). The mean percent degranulation of eosinophils induced by the
negative controls [HBSS-2% BSA and Sputalysin (containing 0.01%
DTT)] was <1%; in six experiments, Sputalysin-induced
degranulation of eosinophils (0.83 ± 0.62%) was not significantly
greater than that induced by HBSS-2% BSA (0.56 ± 0.35%).
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Preincubation of eosinophils with cytochalasin B for 15 min reduced eosinophil degranulation caused by neutrophil elastase by 50% (Fig. 3). Removal of Ca2+ from the incubation medium reduced eosinophil degranulation caused by neutrophil elastase by almost 40% (Fig. 3).
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The catalytic activity of neutrophil elastase
(105 M) after preincubation
with
1-PI
(10
4 M) was completely
inhibited, and there was significant but incomplete reduction in the
extent of degranulation of eosinophils stimulated with this mixture
compared with stimulation with free elastase (Fig. 3).
Characteristics of the CF sputum
samples. The total cell count in the 15 CF sputum
samples was very high. After correction for the eightfold processing
dilution factor, the mean (±SE) total cell count was 4.9 × 107 cells/ml, and 98 ± 0.5%
of these cells were neutrophils. Only 0.4 ± 0.1% of the cells were
eosinophils. The free neutrophil elastase activity in the diluted
sputum supernatant was 1.6 ± 0.4 × 105 M (2.0 ± 0.5 × 10
6 M before
correcting for the processing dilution factor of 1:8). The level of ECP
in these samples was 5,040 ± 1,040 ng/ml (630 ± 130 ng/ml
before correcting for the eightfold processing dilution factor).
Effect of CF sputum on eosinophil
degranulation. The supernatants of the CF sputum
samples induced degranulation of 6.2% of total intracellular ECP
(without correcting for the eightfold processing dilution factor),
which was similar to the eosinophil degranulation induced by purified
neutrophil elastase. The free elastase activity of the diluted CF
sputum (2.4 ± 0.7 × 106 M;
n = 9 samples) was completely
inhibited after incubation with
1-PI
(10
4 M), and no significant
degranulation of eosinophils by CF sputum was observed after
preincubation with
1-PI (Fig.
4).
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To determine the relationship among the level of free neutrophil elastase in CF sputum, the level of ECP in CF sputum, and the release of ECP from purified eosinophils induced by CF sputum, the correlation among these variables was examined. We found that the levels of neutrophil elastase correlated significantly with the concentration of ECP in CF sputum (r = 0.863; P = 0.001; n = 15 experiments; Fig. 5) but not with the ECP release induced by CF sputum (r = 0.51; P = 0.16; n = 9 experiments).
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Viability of purified eosinophils. To
determine whether eosinophils remained viable after the various 2-h
incubations, we tested the ability of eosinophils to exclude trypan
blue and measured the LDH levels in eosinophil supernatants. We found
no difference from the HBSS medium control in eosinophil viability.
Eosinophil viability after a 2-h incubation in HBSS was 99.1 ± 0.4%, and this was not significantly different after incubation with
neutrophil elastase (96.6 ± 1.2%), cytochalasin B (98.2 ± 0.8%), or 1-PI (97.7 ± 1.0%). The LDH level in eosinophil supernatants after a 2-h incubation
with HBSS was 0.9 ± 0.9 units/l, and this was not significantly
different after incubation with neutrophil elastase (2.2 ± 1.2 units/l), cytochalasin B (5.4 ± 4.0 units/l), or
1-PI (0.8 ± 0.8 units/l).
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DISCUSSION |
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The main findings of our study are that neutrophil elastase causes degranulation of eosinophils and that sputum from patients with CF also causes eosinophil degranulation, in large part due to free neutrophil elastase activity. Another neutrophil protease, cathepsin G, also caused eosinophil degranulation, but the effect was very small compared with that of neutrophil elastase. The neutrophil elastase-induced eosinophil degranulation was unlikely to be cytotoxic because 3-h incubations of eosinophils with neutrophil elastase were not associated with a reduction in eosinophil viability as assessed either by trypan blue exclusion or by release of LDH. The degranulation of eosinophils by neutrophil elastase was concentration dependent but was not dependent on the catalytic activity of elastase because degranulation was not completely inhibited.
The cell-free supernatant of sputum collected from patients with CF
also caused degranulation of eosinophils. The mechanism of eosinophil
degranulation by CF sputum is neutrophil protease activity of the
sputum because degranulation was completely inhibited by
1-PI and the size of the
degranulation effect was predictable from the measured free elastase
activity of the sputum. Our finding that the free elastase activity of
CF sputum was in the same range as the purified free elastase activity
levels required to induce eosinophil degranulation demonstrates that
the concentrations of free elastase required for eosinophil
degranulation are achieved in vivo.
CF sputum was characterized not only by high free elastase activity but also by very high neutrophil numbers and very low eosinophil numbers. Despite the paucity of eosinophils in the CF sputum, ECP levels were very high. Indeed, the ECP levels in the CF sputum were higher than the ECP levels we previously found (unpublished observations) in sputum from asthmatic subjects presenting to the emergency room in exacerbation. The cause of the high ECP levels in CF secretions is unknown, but our finding that neutrophil elastase causes eosinophil degranulation may be relevant, for it means that one consequence of high neutrophil elastase activity in CF airways is elastase-induced degranulation of eosinophils. This possibility is supported by our finding that the levels of neutrophil elastase in CF sputum correlated with the levels of ECP in the sputum and with the ECP release induced by CF sputum. The high ECP levels in CF sputum in the absence of high eosinophil numbers may still be explained by neutrophil elastase-induced eosinophil degranulation because degranulated eosinophils are difficult to identify morphologically (and so may be undercounted) or because the relevant degranulated eosinophils reside in the airway mucosa from where ECP (but not necessarily the degranulated eosinophils) may diffuse into the airway secretions.
The mechanism of degranulation of eosinophils is incompletely
understood but may involve
Ca2+-dependent mechanisms because
Ca2+ ionophore-induced
degranulation is completely inhibited in the absence of
Ca2+. In addition, degranulation
can be induced via
Ca2+-independent mechanisms
involving protein kinase C, diacylglycerol, and inositol
1,4,5-trisphosphate as evidenced by the degranulation of eosinophils by
phorbol 12-myristate 13-acetate, which directly stimulates protein
kinase C and which is not inhibited in the absence of
Ca2+ (12). In our experiments, we
found that depletion of Ca2+ in
the incubation medium reduced neutrophil elastase-induced degranulation
of eosinophils by 45%, indicating that
Ca2+ is not essential for this
effect. Interestingly, we also found that complete inhibition of
neutrophil elastase activity by
1-PI only reduced neutrophil
elastase-induced degranulation of eosinophils by 50%, indicating that
the effect of elastase on eosinophil degranulation is not entirely
dependent on its catalytic activity. However, the finding that
1-PI inhibits all of the
eosinophil-degranulating activity present in CF sputum (which, after
the addition of
1-PI, can be
presumed to contain elastase-inhibitor complexes) suggests that the
"nonspecific" effects seen with purified elastase and inhibitor
may be physiologically irrelevant. Our data do not allow us to
determine why the nonspecific effect is seen only with
purified enzyme and inhibitor. One possibility is that some of the
numerous substances in CF sputum with a potential to bind to and alter the activity of elastase (such as DNA, proteoglycans, and mucins) mask
the inherent eosinophil-degranulating activity of the naked elastase-inhibitor complex.
Cytochalasin B attenuated neutrophil elastase-induced eosinophil degranulation by 40%, which may be considered a paradoxical finding because cytochalasins are expected to enhance secretagogue-induced degranulation because they promote disaggregation of the intracellular actin network, which, in turn, will promote secretion by facilitating fusion of secretory granules with cell membranes (22). Indeed, cytochalasin B enhances the secretion of lysosomal enzymes by phagocytes and enhances eosinophil degranulation induced by formyl-methionyl-leucyl-phenylalanine (12). In contrast, however, in findings similar to our own, other investigators have found that cytochalasin B inhibits eosinophil degranulation induced by serum-, secretory IgA-, or IgG-coated Sephadex beads (12) and by the eosinophil granule proteins major basic protein and eosinophil peroxidase (13). These different effects of cytochalasin B on eosinophil degranulation may mean that the mechanism of eosinophil degranulation varies with the stimulus. For example, one possibility is that stimuli that are Ca2+ dependent are more likely to be enhanced by cytochalasins because an increase in intracellular Ca2+ activates gelsolin, which indirectly facilitates degranulation by causing disaggregation of actin (22). Alternately, cytochalasin B may have direct inhibitory effects on some agonists used to degranulate eosinophils.
Our data suggest that neutrophil elastase and elastase-rich CF sputum cause degranulation of eosinophils in a mechanism partially dependent on Ca2+ and actin filaments, but our experiments do not fully elucidate the mechanism. We speculate that the mechanism of action of elastase-induced degranulation may be similar to that of elastase-mediated degranulation of airway gland cells, which possibly involves mimicry of an endogenous metalloproteinase involved in stimulus-secretion coupling (20).
Our finding that neutrophil elastase degranulates eosinophils has relevance for airway diseases other than CF. For example, Fahy et al. (6) recently reported that both neutrophils and eosinophils are prominent in sputum samples from asthmatic subjects who present to the emergency room in acute exacerbation and that free neutrophil elastase activity is present in many of these asthmatic sputum samples. Thus, in asthma, where there is a well-documented excess of eosinophils in the airway epithelium and mucosa (3), it is possible that activation of these eosinophils could occur when asthmatic subjects are exposed to airway insults such as viral infection or ozone that induce airway neutrophilia.
In summary, we found that eosinophil degranulation can be induced by neutrophil elastase and elastase-rich CF sputum. We believe that this finding helps to explain the high levels of ECP in CF sputum, and we speculate that neutrophil elastase-induced eosinophil degranulation may be important in the pathogenesis of other airway diseases, especially asthma.
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ACKNOWLEDGEMENTS |
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We thank Hofer Wong for recruiting asthmatic subjects to donate blood, Jane Liu for technical assistance in establishing the protocols for eosinophil purification and measurement of eosinophil cationic protein, and William Raymond for performing the sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of the neutrophil elastase. We are also indebted to Homer Boushey for helpful advice and support.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-24136.
J. V. Fahy was a recipient of a Physician Scientist Award from the American College of Chest Physicians. G. H. Caughey was a recipient of a Career Investigator Award from the American Lung Association. H. Liu received fellowship training support from the World Health Organization.
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: J. V. Fahy, Box 0130, Univ. of California, San Francisco, 505 Parnassus Ave., San Francisco, CA 94143.
Received 24 April 1998; accepted in final form 15 September 1998.
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