Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, San Francisco, California 94143-0130
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ABSTRACT |
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We examined the effect of the neutrophil chemoattractants interleukin (IL)-8 and N-formyl-methionyl-leucyl-phenylalanine on goblet cell (GC) degranulation in guinea pigs. Chemoattractants caused time-dependent neutrophil recruitment and GC degranulation in vivo. NPC 15669 (an inhibitor of leukocyte infiltration) prevented both responses, implicating neutrophils. ICI 200,355 (an inhibitor of neutrophil elastase and proteinase-3) or secretory leukocyte protease inhibitor (an inhibitor of elastase but not of proteinase-3) abolished IL-8-induced GC degranulation, implicating elastase. Incubating tracheal segments with IL-8 plus neutrophils caused GC degranulation in vitro, an effect due to activation of the neutrophils themselves (and not an effect present in the supernatant). Chemoattractant increased surface staining of elastase and the cleavage of elastase-specific fluorogenic substrate by neutrophils. Pretreatment with anti-intercellular adhesion molecule-1, anti-CD18, or anti-CD11b antibody inhibited the chemoattractant-induced GC degranulation in vitro, implicating adhesion molecules. These studies suggest that chemoattractants cause neutrophil-dependent GC degranulation involving adhesive interactions between cells, with elastase activity occurring at the cell interface, causing GC secretion. The findings, reproduced in human airways, suggest novel methods of therapeutic intervention.
airways; secretagogue; neutrophil chemoattractants; intercellular adhesion molecule-1; CD11b/CD18
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INTRODUCTION |
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AIRWAY HYPERSECRETION is a major cause of symptoms in cystic fibrosis (1), bronchiectasis (6), and chronic bronchitis (34), but the mechanisms of hypersecretion remain unknown. Neutrophils are recruited into the airways in these diseases (1, 26), and responsible chemoattractants are present (14, 18, 22). This suggests that neutrophils, chemoattractants, and hypersecretion could be associated. Purified neutrophil elastase has been shown to be a potent secretagogue in airway submucosal glands (33) and in goblet cells (GCs) (2, 15, 17, 35) in various species including humans (28). This provides a mechanism for hypersecretion by a neutrophil product. However, it is unclear whether intact neutrophils cause hypersecretion in vivo, and if they do, what mechanisms are involved. We hypothesized that chemoattractants in the airway not only cause neutrophil recruitment but also cause elastase to move from azurophilic granules in the cytoplasm to the neutrophil surface (21). On contact with GC, adhesion molecules on the surfaces cause elastase release in a confined space of close intercellular contact, resulting in GC degranulation.
To investigate neutrophil-dependent GC degranulation, we studied guinea pigs (a species that normally has many GCs in the airway epithelium) (36), and we confirmed the results in human airways that contained GCs. First, we delivered two neutrophil chemoattractants, interleukin (IL)-8 and N-formyl-methionyl-leucyl-phenylalanine (fMLP) into the trachea of guinea pigs in vivo, and we studied the recruitment of neutrophils and degranulation of GCs in the tracheal epithelium. Next, we designed studies in the guinea pig trachea in vitro to determine the mechanisms involved in neutrophil-dependent GC degranulation. We examined the roles of neutrophils, chemoattractants, and adhesion molecules in mediating GC degranulation. We also examined the possible translocation of neutrophil elastase to the neutrophil surface by chemoattractants, a movement that might bring elastase into close contact with the GC surface.
Some of these results have been reported in abstract form (35).
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METHODS |
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The experimental animal protocol was approved by the Committee on Animal Research of the University of California, San Francisco. Male Dunkin-Hartley outbred guinea pigs weighing 400-600 g (Simonsen Laboratories, Gilroy, CA) were used. They were maintained in a temperature-controlled (21°C) room with standard laboratory food and water freely available.
In vivo studies. Specific pathogen-free male Hartley guinea pigs (400-600 g) were anesthetized with pentobarbital sodium (35 mg/kg ip; Anthony Products, Arcadia, CA) and allowed to breathe spontaneously. Drugs were delivered into the trachea via a 22-gauge Angiocath catheter (Becton Dickinson, Sandy, UT), with a high-intensity illuminator (FiberLite, Dolan Jenner Industries, Lawrence, MA) for visualization. The animals were placed in a heated chamber, and body temperature was maintained at 37°C. At preselected times during the study, the heart was exposed, a blunt-ended needle was inserted from the apex of the left ventricle into the ascending aorta, and the systemic circulation was perfused with 1% paraformaldehyde at a pressure of 120 mmHg. The trachea was then removed and placed in 4% paraformaldehyde for 24 h. After fixation, the tracheal segments were divided into two pieces (upper trachea and lower trachea) cut longitudinally along the membranous portion, dehydrated, and embedded in JB-4 plus monomer solution A. Each section (4 µm thick) was placed on a slide and stained with 3,3'-diaminobenzidine (Sigma, St. Louis, MO) to visualize neutrophils that had migrated into the epithelium. The slides were then stained with Alcian blue and periodic acid Schiff (PAS) and counterstained with hematoxylin. Slides were observed at ×400 magnification with an Axioplan microscope equipped with a Plan-NEOFLUAR ×40/0.75 objective lens.
Protocol of in vivo study. To
determine the effect of neutrophils that migrate into the airway
epithelium on GC degranulation, two potent neutrophil chemoattractants,
fMLP and IL-8, were chosen to stimulate neutrophil migration to the
tracheal epithelium. IL-8
(107 M, 100 µl; Genzyme,
Cambridge, MA) or fMLP (10
7
M, 100 µl) was delivered into the tracheal lumen. The trachea was
removed 1, 2, or 4 h after injection. Sterile PBS (100 µl) was
injected into the trachea as a control. Subsequent studies examining
the effects of inhibitors were performed with IL-8 as the
chemoattractant. The dose dependence of IL-8-induced GC degranulation and neutrophil recruitment was examined with concentrations of IL-8 at
10
8,
10
7, and
10
6 M. They were examined 2 h after instillation of IL-8 because preliminary studies showed that
profound degranulation occurred by this time. To determine the
relationship between neutrophil recruitment and GC degranulation, we
examined the effect of an inhibitor of neutrophil recruitment, NPC
15669 (kindly provided by Scios-Nova, Mountain View, CA). The dose of
NPC 15669 used was determined from previously published data (19). To
determine the relationship of neutrophil elastase and GC degranulation, we examined the effect of an elastase inhibitor, ICI 200,355 (generously provided by Zeneca Pharmaceuticals Group, Wilmington, DE).
The dose of ICI 200,355 used was estimated from preliminary studies with the inhibitor to prevent GC degranulation after intratracheal instillation of elastase
(10
7 M). Secretory
leukocyte protease inhibitor (SLPI) was generously provided by Dr. John
R. Hoidal. The animals were pretreated with either NPC
15669 (10 mg/kg iv 1 h before) or ICI 200,355 (700 µg/kg iv 30 min
before), and then IL-8 (10
7
M, 100 µl) was instilled into the trachea. The trachea was removed 2 h after IL-8 injection.
Quantification of GC degranulation. To analyze GC degranulation, we determined the volume density of Alcian blue-PAS-stained mucosubstances on the mucosal surface epithelium with a semiautomatic imaging system. We chose this morphometric analysis because of its reproducibility and practicality in performing studies in vivo. The stained slides were examined with an Axioplan microscope (Zeiss, Thornwood, NY), which was connected to a video camera control unit (DXC7550MD, Sony of America, Park Ridge, NJ). Images of the tracheal epithelium were recorded from 10 consecutive high-power fields with a phase-contrast lens at ×400 with an IMAXX video system (PDI, Redmond, WA). The intracellular mucin in superficial epithelial secretory cells appeared as oval-shaped purple granules of varying sizes. We measured the Alcian blue-PAS-positive stained area and total epithelial area, and we express the data as the percentage of the total area stained by Alcian blue-PAS. The analysis was performed on a power Macintosh 9500/120 computer (Apple Computer, Cupertino, CA) with the public domain National Institutes of Health Image program (available from the Internet by anonymous FTP from zippy.nimh.gov or on floppy disk PB95-500195GEI, National Technical Information Service, Springfield, VA).
Counting of neutrophils. We stained the slides with 3,3'-diaminobenzidine and then counterstained them with toluidine blue. Neutrophils seen as myeloperoxidase-positive blue cytoplasmic cells were counted in 20 consecutive high-power fields of the epithelial layer (from the basement membrane to cell apices) from each of the upper and lower tracheal sections at ×400 magnification.
In vitro studies. The trachea was removed from just below the larynx to the carina, the connective tissue was removed, and the trachea was divided into 3 segments of 10-11 cartilage rings each. The segments were kept in sterile PBS at 4°C until the time of study to prevent tissue damage. Tracheal segments were bathed in DMEM-Ham's F-12 medium containing HEPES buffer (25 mM) and incubated at 37°C in a 5% CO2 water-jacketed incubator (Forma Scientific, Marietta, OH). Each segment was incubated with control medium alone or with a stimulus. At the end of the in vitro studies, the tracheal segments were prepared similar to the in vivo studies.
Isolation of neutrophils. In limited studies, we isolated guinea pig neutrophils. However, because of the difficulty of obtaining sufficient guinea pig neutrophils, we used human neutrophils purified from peripheral blood obtained from healthy human donors. The neutrophil isolation was performed by standard techniques of Ficoll-Hypaque gradient separation, dextran sedimentation, and hypotonic lysis of erythrocytes. The cells were routinely >95% viable by trypan blue dye exclusion. To prevent endotoxin contamination, all solutions were passed through a 0.1-µm filter. All buffers and media used for the in vitro studies were assayed for endotoxin with a commercially available Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD).
Protocol of in vitro studies. In
previous studies (2, 15, 17, 35), purified neutrophil elastase was
shown to cause secretion in GCs; in preliminary experiments, we
confirmed the ability of neutrophil elastase to degranulate guinea pig
GCs in vitro. The Alcian blue-PAS-stained area in the control state
(14.2 ± 0.8%) was decreased by neutrophil elastase
(107 M for 1 h) to 2.7 ± 0.4% (P < 0.001;
n = 5 segments). When the epithelium was preincubated with ICI 200,355 (10
5 M) for 30 min, the
area staining with Alcian blue-PAS was no longer decreased after
neutrophil elastase (stained area, 13.7 ± 1.1%;
P > 0.05). These studies provided
positive controls for the studies with intact neutrophils. To elucidate
the mechanism of neutrophil chemoattractant-induced GC degranulation
found in vivo, we examined the area of epithelium that stained with
Alcian blue-PAS under various conditions in vitro. Preliminary studies showed that 106 neutrophils were
sufficient to cause GC degranulation. Isolated tracheal segments were
incubated with neutrophils (106
cells/ml) alone, IL-8 (10
7
M) alone, fMLP (10
8 M)
alone, IL-8 plus neutrophils, or fMLP plus neutrophils for 1 h. As a
control, one of the tracheal segments was fixed immediately after
removal with 4% paraformaldehyde, and a second segment was incubated
for 1 h with DMEM-Ham's F-12 medium alone. The study groups were
prepared similarly. To examine the mechanism of GC degranulation caused
by chemoattractant plus neutrophils, we studied neutrophils that had
been incubated with IL-8
(10
7 M) for 1 h and then
washed with sterile PBS to avoid contamination with the supernatant
(e.g., IL-8 or released elastase). Then either the neutrophils
("activated") or the supernatant was added to the tracheal
segment. To study the contribution of elastase released from
neutrophils on GC degranulation in the airways, the tissues and
neutrophils were preincubated with an elastase inhibitor, ICI 200,355 (10
5 M), or with SLPI
(10
5 M) for 30 min, and
then activated neutrophils were added to the tracheal tissue. To
determine whether neutrophil adhesion molecules are involved in GC
degranulation, we preincubated neutrophils with human blocking
antibodies to
2-integrins,
including anti-CD11a [lymphocyte function-associated antigen
(LFA)-1
], anti-CD11b [macrophage-1
(Mac-1)], or anti-CD18 (LFA-1
, 10 µg/ml; Biosource, Camarillo, CA) for 30 min, and then we
incubated the treated neutrophils with tracheal segments at 37°C in
a 5% CO2 water-jacketed incubator for 1 h and fixed the tissue. In other experiments, we preincubated tracheal segments with human anti-intercellular adhesion molecule-1 (ICAM-1; 50 µg/ml; Genzyme, Cambridge, MA) for 30 min. Neutrophils were then added, and the segments were incubated for 1 h, after which
the tissue was fixed.
Studies of airways in lungs removed from patients at lung transplantation. Human bronchial segments were obtained from four patients at the time of lung transplantation and were chosen because their surface epithelium contained many GCs (mean control, 22.8 ± 2.1%). Two patients had idiopathic pulmonary fibrosis, one had cystic fibrosis, and one had chronic obstructive lung disease. After removal, the lung was bathed immediately in PBS, and the bronchi were removed and divided into pieces containing five to six cartilage rings. Bronchial segments were placed in DMEM-Ham's F-12 medium containing HEPES buffer (25 mM) in a 12-well dish that was incubated at 37°C in a 5% CO2 water-jacketed incubator. Each segment was incubated with medium alone, neutrophils alone, IL-8 alone, or IL-8 plus neutrophils for 30 min. In one series of experiments, the neutrophils activated by IL-8 were separated from the supernatant (activated neutrophils) before they were incubated with the airway tissue. To assess the contribution of neutrophil adhesion molecules to neutrophil-dependent GC degranulation in human tissues, neutrophils were preincubated with anti-CD18 for 30 min before activated neutrophils were added to the tracheal segments. After incubation, all segments were fixed and stained similar to the animal studies.
Immunocytochemical localization of neutrophil
elastase. Neutrophils were isolated as described in
Isolation of neutrophils. We examined whether the neutrophil chemoattractants fMLP and IL-8 (n = 4 subjects/groups) caused the
surface expression of neutrophil elastase using immunocytochemical
staining with an antibody to neutrophil elastase. We coated eight-well
chamber slides with fibronectin (5 µg/cm2) for 1 h at room
temperature. Neutrophils were then added for varying times (2-15
min) either alone or with fMLP
(108 M) or IL-8
(10
8 M) at 37°C in a
5% CO2 water-jacketed incubator.
The concentration of chemoattractants
(10
8 M) chosen was based on
previously reported results (20). The cells (2 × 105 cells/well) were fixed with
PBS containing 3% paraformaldehyde and 0.5% glutaraldehyde (pH 7.4)
for 3 min and then were washed two times in Hanks' balanced salt
solution (21). To stain for elastase on the surface of neutrophils, PBS
containing 2% normal goat serum and 100 mM levamisole was used as a
diluent for the antibodies. The cells were incubated with mouse
monoclonal antibody to human neutrophil elastase (1:5,000; Dako,
Carpinteria, CA) for 45 min at 4°C, then washed three times with
PBS to remove the excess of primary antibody. The cells were then
incubated with biotinylated horse anti-mouse immunoglobulin (Vector
Laboratories, Burlingame, CA) at a 1:200 dilution for 1 h at room
temperature. Bound antibody was visualized according to standard
protocols for the avidin-biotin-alkaline phosphatase complex method
(ABC Kit, Vector Laboratories). As a control for the specificity of the
antibody, a 100-fold excess by weight of human purified neutrophil elastase (Elastin Products, Owensville, MO) was used to preabsorb the
antibody at room temperature for 2 h. This preabsorbed antibody preparation was used to stain neutrophils that were incubated with fMLP
for 15 min as described above.
Effect of chemoattractants on cleavage of specific
fluorogenic substrate by membrane-bound neutrophil
elastase. Activity of neutrophil elastase was
quantified with an elastase-specific fluorogenic substrate that cannot
gain access to intracellular enzyme in neutrophils (20). Cells (1 × 106) were incubated
without chemoattractant (Hanks' balanced salt solution only) or with
IL-8 or fMLP (each at 108
M) for 30 min in test tubes, and then the supernatants were removed. The cells were fixed with PBS containing 3% paraformaldehyde and 0.5%
glutaraldehyde (pH 7.4). This procedure does not abolish the catalytic
activity of membrane-bound elastase (21). Fixed neutrophils,
supernatant, or purified elastase
(10
10 to
10
6 M) was incubated in
duplicate with the fluorogenic substrate methoxysuccinyl-Ala-Ala-Pro-Val-7-amino-4-trifluoromethylcoumarin (200 µM) in 0.1 M HEPES buffer containing 0.15 M NaCl for 25 min at room
temperature. Samples were then measured for the liberated 7-amino-4-trifluoromethylcoumarin with a fluorescence measurement system (Millipore, Bedford, MA).
Statistics. All data are expressed as means ± SE. One-way analysis of variance was used to determine statistically significant differences between groups. Scheffé's F-test was used to correct for multiple comparisons when statistical significances were identified in the analysis of variance. A probability of <0.05 for the null hypothesis was accepted as indicating a statistically significant difference.
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RESULTS |
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In vivo studies. To determine whether
neutrophil recruitment results in GC degranulation, we instilled the
neutrophil chemoattractants fMLP and IL-8 (each at
107 M, 100 µl)
intratracheally in guinea pigs (n = 5 animals/group). The chemoattractants caused neutrophil recruitment
(Fig. 1,
bottom) and GC degranulation
(Fig. 1, top) in a
time-dependent fashion. Degranulation was observed at a time when
neutrophils in the epithelium were in close contact with GCs. GC
degranulation induced by IL-8 was examined and shown to be dose
dependent (area of epithelium stained with Alcian blue-PAS:
10
8 M, 8.7 ± 1.1%;
10
7 M, 3.7 ± 0.7%;
10
6 M, 2.8 ± 0.5%;
n = 5 animals). Neutrophil recruitment
was also dose dependent
(10
8 M, 12.5 ± 3.8 neutrophils/mm epithelium;
10
7 M, 18.9 ± 5.2 neutrophils/mm epithelium;
10
6 M, 21.7 ± 4.5 neutrophils/mm epithelium; n = 5 animals/group).
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Pretreatment with NPC 15669 [10 mg/kg iv; a molecule that
prevents leukocyte migration (12)] 1 h before the instillation of
IL-8 (107 M) inhibited
IL-8-dependent neutrophil recruitment (Fig.
2,
bottom) and GC degranulation (Fig.
2, top); treatment with vehicle only (saline) was without effect.
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When we pretreated animals for 30 min with ICI 200,355 (700 µg/kg iv;
an inhibitor of neutrophil elastase), GC degranulation induced by IL-8
(107 M) was prevented (Fig.
2, top), but there was no effect on
neutrophil recruitment (Fig. 2,
bottom); the vehicle for ICI 200,355 (saline) was without effect (n = 4 animals/group).
In vitro studies. To dissect further
the underlying mechanisms of neutrophil-dependent GC degranulation, we
studied guinea pig tracheal segments in vitro
(n = 4 animals/group). First, we studied factors involved in chemoattractant-mediated GC degranulation. Incubation of tracheal segments for 1 h with medium alone, with IL-8
(107 M) alone, with fMLP
(10
8 M) alone, or with
neutrophils (106 cells/ml) alone
was without effect. However, when tissues were incubated with
chemoattractant plus neutrophils
(106 cells/ml) together, GC
degranulation occurred (Fig. 3). In limited studies, we confirmed these results using guinea pig neutrophils (data
not shown).
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To determine whether GC degranulation was due to the release of
elastase-containing granules by the neutrophils and the subsequent diffusion of elastase to the GC or whether elastase on the surface of
the neutrophils was the cause of the degranulation, we incubated neutrophils with chemoattractants for 1 h. First, neutrophil
chemoattractants plus neutrophils were incubated with tracheal tissue
and caused GC degranulation. Then we harvested neutrophils that had
been incubated with IL-8 (activated neutrophils) and washed them with sterile PBS to avoid further contamination with IL-8. The
activated neutrophils caused GC degranulation, whereas the supernatant
was without effect (Fig. 3). The GC degranulation caused by activated neutrophils was inhibited by preincubation for 30 min with ICI 200,355 (105 M) or with SLPI
(10
5 M; Fig. 3), suggesting
that elastase played a role in the degranulation.
Immunocytochemical localization of neutrophil
elastase. Our results suggest that elastase bound to
neutrophils but not released into the free medium causes the
degranulation of GC. Therefore, we studied the effect of a
chemoattractant (fMLP) on the expression of elastase on the surface of
neutrophils in vitro. We incubated isolated neutrophils alone or with a
neutrophil chemoattractant (fMLP or IL-8,
108 M) on
fibronectin-coated chamber slides. The chemoattractant caused the
surface expression of neutrophil elastase in a time-dependent fashion.
In neutrophils incubated alone, adherent cells did not change shape and
did not stain for elastase over a period of 15 min. When neutrophils
were incubated with fMLP or IL-8, neutrophil "spreading" began
within 2 min, but cell surface staining for neutrophil elastase was not
yet evident. Surface staining of neutrophil elastase was present at 5 min, increased at 10 min, and was maximal at ~15 min. The surface
staining of neutrophil elastase appeared to be stronger on one side of
many neutrophils (Fig. 4 shows a typical
study with fMLP).
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Effect of chemoattractants on cleavage of specific fluorogenic substrate by membrane-bound neutrophil elastase. To determine whether membrane-bound elastase is available in an active form when neutrophils are activated by neutrophil chemoattractants, we measured the elastase activity of neutrophils with a fluorogenic substrate that is specific for elastase. When neutrophils were incubated for 30 min with a chemoattractant (IL-8 or fMLP), cleavage of the substrate by catalytically active membrane-bound elastase increased strikingly. Little evidence of activity of elastase was present in the supernatant (Fig. 5).
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Effect of pretreatment of anti-adhesion molecule
antibodies on GC degranulation caused by neutrophils plus IL-8 in
vitro. Pretreatment of guinea pig airway tissue with
anti-ICAM-1 antibody inhibited GC degranulation significantly (Fig.
6). Similarly, pretreatment of neutrophils
with an anti-CD11b (Mac-1) or anti-CD18 (LFA-1) antibody inhibited
IL-8-mediated GC degranulation. However, anti-CD11a (LFA-1
) antibody
had a much smaller effect (Fig. 6).
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Incubation of IL-8 and neutrophils with GCs in human airways in vitro. In bronchial segments from the lungs of four patients studied at the time of lung transplantation (and selected because the airways contained GCs), GCs occupied a considerable percentage of the area of epithelium in the control state (Fig. 7). Like guinea pigs, incubation with IL-8 alone or with neutrophils alone was without effect. Incubation with IL-8 plus neutrophils or incubation of neutrophils activated by IL-8 and then washed to remove the IL-8 led to marked GC degranulation. Preincubation with an anti-CD18 antibody inhibited the degranulation caused by activated neutrophils (Fig. 7). Photomicrographs of bronchial epithelium containing GCs removed from a patient are shown in Fig. 8. When segments were incubated with neutrophils alone or with IL-8 alone, degranulation did not occur. However, when bronchial segments were incubated with IL-8 plus neutrophils, GC degranulation occurred. Similar effects were found in the bronchi of each of the four patients studied.
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DISCUSSION |
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The present study shows that chemoattractant-induced recruitment of neutrophils into the trachea in guinea pigs by fMLP and IL-8 (4, 9) results in profound GC degranulation. Neutrophil chemoattractant-induced GC degranulation was inhibited by NPC 15669, a molecule in which the primary action is to inhibit leukocyte movement (7). These results implicate neutrophils in chemoattractant-induced GC degranulation.
Neutrophils store three proteases known to cause degranulation of airway secretory cells: elastase (2, 15, 17, 35), cathepsin G (33), and proteinase-3 (24, 25). In the present studies, pretreatment with ICI 200,355, a selective inhibitor of elastase and proteinase-3 but not of cathepsin G (32), prevented neutrophil chemoattractant-induced GC degranulation; SLPI, an inhibitor of elastase and cathepsin G but not of proteinase-3 (27), also prevented GC degranulation. These results implicate neutrophil elastase in neutrophil-dependent GC degranulation.
Purified neutrophil elastase is reported to be a potent secretagogue in airway submucosal glands (33) and GCs (2, 15, 17, 35) of various species (28) in vitro. When neutrophils are incubated with IL-8 in a test tube, elastase is not normally released into the supernatant (11). However, in the present in vitro studies, when we incubated neutrophils and a chemoattractant with guinea pig tracheal epithelium containing GCs in vitro, GC degranulation occurred. When neutrophils that had been preincubated with IL-8 (activated neutrophils) were washed free of IL-8 and then incubated with guinea pig epithelium, GC degranulation also occurred. Furthermore, the activated neutrophil-dependent GC degranulation was prevented by elastase inhibitors, implicating elastase in the response. From these results, we hypothesized that neutrophil chemoattractants cause elastase to move to the surface of neutrophils, making the elastase available for interaction with GCs.
When we isolated neutrophils and stained them with an antibody to elastase, very little staining of elastase occurred at the surface of resting neutrophils. However, when neutrophils were incubated with a chemoattractant, surface staining of elastase began in <5 min and was maximum at ~15 min. These results indicate that the chemoattractant had caused mobilization of elastase onto the neutrophil surface. Furthermore, when resting neutrophils were evaluated for cleavage of an artificial fluorogenic substrate specific for elastase, little cleavage occurred. However, when neutrophils were incubated with a chemoattractant, a substantial increase in substrate cleavage was found, indicating that surface-bound elastase expression had increased. These findings are compatible with the recent results of Owen et al. (20), who showed that neutrophils incubated with chemoattractants induced cleavage of elastase-specific substrates.
A recent study (21) reported that activation of neutrophils results in
the binding of elastase to the neutrophil plasma membrane, where it is
reported to escape from inhibition of elastase-mediated proteolytic
activity by inhibitors of elastase (e.g.,
1-antitrypsin) present in the
tissue environment. This study showed the importance of surface-bound
elastase and of the pericellular environment in determining the
enzymatic activity of elastase in tissues.
Because activated neutrophils (and not neutrophil supernatant) caused GC degranulation and because chemoattractants cause surface expression of elastase, we hypothesized that a close contact interaction between the surfaces of the neutrophils and GCs is required for GC degranulation. Three observations suggest that a close contact interaction is required: 1) GC degranulation was maximum at a time when large numbers of neutrophils had migrated into the epithelium and appeared physically close to GCs; 2) activated neutrophils (and not products secreted into the free medium) caused GC degranulation in vitro by a mechanism involving elastase. For this to occur, the neutrophils had to be in close contact with the GCs; and 3) neutrophils activated by a chemoattractant express elastase on their surfaces; this membrane-bound elastase is catalytically active. For this catalytically active elastase to cause degranulation, we hypothesize that close contact with the substrate (i.e., the GC) is required. Therefore, we examined the hypothesis that adhesion molecules on neutrophils and on epithelium were involved in neutrophil-dependent GC degranulation. Pretreatment of neutrophils with an anti-CD11b or anti-CD18 antibody inhibited chemoattractant-mediated GC degranulation, whereas an anti-CD11a antibody had only a small effect. Preincubation of tissues with an anti-ICAM-1 antibody also prevented GC degranulation. These results implicate adhesion molecules on neutrophils and GCs in neutrophil-dependent GC degranulation. The exact role of the adhesion molecules in making membrane-bound elastase available to the GCs remains unknown.
Although adhesion molecules on epithelial cells (e.g., ICAM-1) and on
neutrophils (e.g., Mac-1) have been characterized most extensively for
adhesion properties, these molecules are also recognized to be involved
in signal transduction in cells, especially in inflammatory responses.
For example, migrating neutrophils have been implicated in alveolar
epithelial cell damage via an adhesion-dependent mechanism (23).
Furthermore, the expression of these adhesion molecules on the surfaces
of epithelial cells and neutrophils are upregulated by cytokines (e.g.,
tumor necrosis factor-, interferon-
, and IL-1) (13, 37),
important modulators of inflammation. ICAM-1 is upregulated in the
bronchial mucosa of individuals with chronic obstructive pulmonary
disease (29), in infected intestinal epithelial cells (8), and in mice
during pneumonia (3). These results suggest that ICAM-1 plays a role in
other chronic inflammatory diseases.
The present results show that neutrophil chemoattractants cause neutrophil-dependent GC degranulation in guinea pig and human airways via a novel mechanism, mediated by neutrophil elastase, involving close contact between neutrophils and GCs. The chemoattractant plays several roles in this response, including 1) neutrophil recruitment, 2) mobilization of elastase to the surface membrane, and 3) increased expression of adhesion molecules (5). Thus chemoattractants such as IL-8, which is expressed in the airway epithelium during inflammation (10, 16), are especially important in the interaction of neutrophils with tissue substrates such as epithelial GCs.
Two issues remain unknown. First, the exact molecular mechanism of elastase-induced degranulation of secretory cells remains a mystery. The neutral serine proteinases, neutrophil elastase (33), and mast cell chymase (30) as well as cathepsin G (33) are the most potent secretagogues yet identified. However, although receptor-coupled secretion by agonists such as histamine occurs via cAMP-, protein kinase C-, and intracellular Ca2+ concentration-dependent pathways, degranulation induced by elastase does not involve identified second messengers (31). It has been suggested that elastase may substitute for or mimic the action of an endogenous metalloprotease that appears to be activated intracellularly during receptor-mediated secretion. Thus elastase may activate degranulation directly, bypassing the signal transduction mechanisms necessary for secretion caused by other agonists (31). The second unknown is the exact mechanism by which the interaction of adhesion molecules on GCs (i.e., ICAM-1) and on neutrophils (i.e., Mac-1) permits the surface-bound elastase on neutrophils to obtain "access" to GCs. There is evidence that the binding of elastase-containing granules on the neutrophil surface is due to charge interactions between the azurophilic granules and other structures on the neutrophil surface. We hypothesize that adhesive signals between the GC ICAM-1 and the neutrophil Mac-1 result in the generation of second messengers in the neutrophils. This causes changes in the conformation of the membrane-bound elastase (e.g., by neutralizing the charges), thereby releasing elastase from the neutrophil surface and allowing it to diffuse to the GC surface where it causes degranulation.
These studies describe a novel mechanism of neutrophil elastase-induced GC degranulation in guinea pigs. The findings were confirmed in human airways. The studies were performed in the trachea, where the epithelium does not occupy a substantial proportion of the cross-sectional area. Therefore, GC secretion in large airways might be anticipated to cause cough (due to hypersecretion) but not substantial airway obstruction. However, in peripheral airways, where disease causes a preponderance of GCs in the epithelium, degranulation could cause complete obstruction. This could result in marked pathophysiological changes.
Airway hypersecretion and neutrophil recruitment occur in chronic bronchitis, bronchiectasis, and cystic fibrosis (1, 6, 34). The present studies suggest a potent mechanism for linking these two phenomena. Furthermore, the studies suggest various strategies for therapeutic intervention.
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ACKNOWLEDGEMENTS |
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We thank Fraser Keith and Jeffrey Golden for providing the airway tissues, Walter Finkbeiner for assistance with pathological specimens, and Edward J. Campbell and Caroline A. Owen for helpful advice.
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FOOTNOTES |
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The work was supported in part by National Heart, Lung, and Blood Institute Program Project Grant HL-24136.
C. Agustí was supported in part by Hospital Clinic Barcelona and FUCAP-97, and the fellowship to L. O. Cardell was supported by the Will Rogers Memorial Fund.
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. A. Nadel, Cardiovascular Research Institute, Box 0130, Univ. of California, San Francisco, CA 94143-0130.
Received 7 January 1998; accepted in final form 23 April 1998.
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