Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California, San Francisco, California 94132-0130
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ABSTRACT |
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Mucus hypersecretion
contributes to the morbidity and mortality in acute asthma. Both T
helper 2 (Th2) cytokines and epidermal growth factor receptor (EGFR)
signaling have been implicated in allergen-induced goblet cell (GC)
metaplasia. Present results show that a cascade of EGFR involving
neutrophils is implicated in interleukin (IL)-13-induced mucin
expression in GC. Treatment with a selective EGFR tyrosine kinase
inhibitor prevented IL-13-induced GC metaplasia dose dependently and
completely. Instillation of IL-13 also induced tumor necrosis
factor- protein expression, mainly in infiltrating neutrophils.
Control airway epithelium contained few leukocytes, but intratracheal
instillation of IL-13 resulted in time-dependent leukocyte recruitment
by IL-13-induced IL-8-like chemoattractant expression in airway
epithelium. Pretreatment with an inhibitor of leukocytes in the bone
marrow (cyclophosphamide) or with a blocking antibody to IL-8 prevented
both IL-13-induced leukocyte recruitment and GC metaplasia. These
findings indicate that EGFR signaling is involved in IL-13-induced
mucin production. They suggest a potential therapeutic role for
inhibitors of the EGFR cascade in the hypersecretion that occurs in
acute asthma.
airway epithelium; mucus hypersecretion; epidermal growth factor receptor activation; T helper 2 cytokine; interleukin
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INTRODUCTION |
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MUCUS HYPERSECRETION FROM hyperplastic goblet cells (GC) causes airway mucus plugging in peripheral airways of rodents (3) and contributes to mortality in asthma (1, 5, 25). Recent studies implicate the T helper 2 (Th2) cytokines interleukin (IL)-4 and IL-13 in allergen-induced GC metaplasia in sensitized mice (10, 15, 32). IL-13 is a cytokine that is secreted by activated Th2 cells and has immunoregulatory activities that overlap those of IL-4 (6). Several observations implicate Th2 cytokines in GC metaplasia: 1) pretreatment with a neutralizing IL-4 receptor (IL-4R) antibody prevented allergen-induced GC metaplasia (13); 2) allergen-induced GC metaplasia does not occur in signal transducer and activator of transcription (STAT)-6-deficient mice, which have impaired IL-4R signaling (19); 3) IL-4 transgenic mice, which specifically express IL-4 in the airways, develop GC metaplasia (23); and 4) IL-4 instillation into airways induces the differentiation of epithelium into mucous glycoconjugate-containing GC (7, 10). The mechanism that mediates these effects of IL-4 and IL-13 on airway epithelial cell differentiation remains unknown.
Epidermal growth factor receptor (EGFR) signaling also has been
implicated in GC metaplasia (28). EGFR is known to be
upregulated by the proinflammatory cytokine tumor necrosis
factor- (TNF-
), which is increased in lungs in
hypersecretory diseases including asthma (26, 28, 30).
Stimulation with TNF-
induces EGFR expression in human epithelial
cells in culture and in rats in vivo (28). Activation of
EGFR by its ligands (28) or by oxidative stress
(27) results in mucin production. Activated neutrophils release oxygen-free radicals (11, 31), which cause mucin
synthesis via EGFR transactivation, and these effects were blocked by
an EGFR tyrosine kinase inhibitor and also by antioxidants
(27). EGFRs are only weakly expressed in airways in
pathogen-free animals and in healthy humans, but EGFRs are expressed in
the airway epithelium in asthma (2, 28). The mechanisms of
this EGFR cascade in asthma remain unknown.
We hypothesized that IL-13 induces GC metaplasia by activating an EGFR cascade, resulting in mucin expression. EGFR activation could occur via neutrophils recruited into the airways by IL-13. To test this hypothesis, we examined the role of EGFR activation on IL-13-induced mucus production by instilling IL-13 into pathogen-free rats, and we examined the effect of EGFR tyrosine kinase inhibitors on IL-13-induced GC growth. Because we found that IL-13 caused neutrophil recruitment by IL-13-induced IL-8-like chemoattractant on airway epithelium, we examined the effect of preventing leukocyte recruitment by pretreatment with cyclophosphamide or with a blocking antibody to IL-8- on IL-13-induced GC growth.
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METHODS |
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Animals. Specific pathogen-free, male, F344 Fischer rats, weighing 220-240 g, were purchased from Simonsen Laboratories (Gilroy, CA). The animals were housed in pathogen-free rooms and maintained on laboratory chow with free access to food and water. The Committee on Animal Research, University of California, San Francisco approved all procedures. Five animals were studied in each group.
Effect of selective inhibitor of EGFR activation on IL-13-induced GC metaplasia. Studies were first performed in rats in vivo and showed that IL-13 induces GC metaplasia in rat tracheal epithelium. Animals were anesthetized with pentobarbital sodium (Nembutal, 50 mg/kg ip; Abbott Laboratories) and allowed to breathe spontaneously. Vehicle (PBS) or IL-13 was instilled intratracheally via a 20-gauge Angiocath catheter (Becton Dickinson, Sandy, UT) through the mouth while the laryngeal area was visualized using a high-intensity illuminator (FiberLite; Dolan Jenner Industries, Lawrence, MA). The carinal tissues were examined 48 h after instillation of IL-13. Various concentrations of IL-13 (recombinant murine IL-13; 5, 50, 100, and 500 ng/rat; R&D Systems, Minneapolis, MN) were instilled into the trachea in 200 µl of PBS. Sterile PBS (200 µl) was instilled into the tracheae of control animals.
For examination of the relationship between IL-13-induced GC metaplasia and activation of EGFR, animals were pretreated 1 day before instillation of IL-13 and daily thereafter with a selective EGFR tyrosine kinase inhibitor (BIBX1522, 1-30 mg · kgRole of leukocyte recruitment in IL-13-induced GC metaplasia. In preliminary studies, we noted that IL-13 causes leukocyte recruitment into the airways. We hypothesized that leukocyte recruitment results from IL-13-induced chemoattractant release from epithelium and that this recruitment is involved in the IL-13-induced EGFR cascade, leading to GC metaplasia. Groups of animals were euthanized 4, 8, 16, 24, and 48 h after instillation of IL-13 (500 ng), and leukocytes were counted in airway tissue.
For evaluation of the role of leukocytes in IL-13-induced GC metaplasia, rats were pretreated with an inhibitor of leukocytes in the bone marrow [cyclophosphamide (18); Sigma, St. Louis, MO] or with a blocking antibody to IL-8 (rabbit anti-human IL-8 antibody; Biosource, Camarillo, CA). Cyclophosphamide (100 mg/kg ip) was given 5 days before instillation of a single dose of IL-13 (500 ng), and a second injection of cyclophosphamide (50 mg/kg ip) was given 1 day before instillation of IL-13. In another series of studies, we instilled IL-8 antibody (10 µg/rat) intratracheally along with IL-13; we repeated the instillation of anti-human IL-8 blocking antibody at 12-h intervals until the animals were euthanized. The antibody alone had no effect on GC production in control rats (data not shown).Tissue preparation. Animals were euthanized with a lethal dose of pentobarbital sodium (200 mg/kg ip), and the systemic circulation was perfused with 1% paraformaldehyde in diethylpyrocarbonate (Sigma)-treated PBS via the left ventricle. For frozen sections, carinal tissues were removed, placed in 4% paraformaldehyde overnight, and then placed in 30% sucrose for cryoprotection. The tissues were embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA). For plastic or paraffin sections, tissues were placed in 4% paraformaldehyde overnight, dehydrated with ethanol, and embedded in JB-4 plus monomer solution A (Polysciences, Warrington, PA) or in paraffin. The embedded tissues were cut as cross sections 4 µm thick and placed on glass slides.
Quantification of GC metaplasia. In all studies, the carina was examined to obtain consistent sampling. We measured Alcian blue (AB)-periodic acid-Schiff (PAS)-positive areas and total epithelial area, and we express the results as the percentage of AB-PAS area to total epithelial area. The stained slides were examined with a light microscope (Axioplan, Zeiss) that was connected to a video camera (3CCD; Sony, Park Ridge, NJ) and to a control unit (DXC7550MD, Sony) and then a color digital image capture board (IMAXX; PDI, Redmond, WA) and a color monitor (Multisync XV17; NEC, Tokyo, Japan). Images of the airway epithelium were recorded from six consecutive high-power fields with a phase- contrast lens at ×400. The analysis was performed with the public domain NIH IMAGE program (developed at the National Institutes of Health and available by anonymous FTP from zippy.nimh.gov. or floppy disk from the National Technical Information Service, Springfield, VA, part number PB95-500195GEI).
Immunohistochemical staining for MUC5AC, EGFR protein, TNF-,
and IL-8 in rat carinal epithelium.
PBS containing 0.05% Tween 20 and 2% normal goat serum was used as
diluent for the antibodies after endogenous peroxidase was blocked with
0.3% H2O2 in methanol. Sections were incubated with mouse monoclonal antibodies to EGFR (1:250; Calbiochem, San Diego,
CA) or to MUC5AC (clone 45 M1, 1:500; New Markers, Fremont, CA) or a
rabbit antibody to TNF-
(1:1,000; Genzyme, Cambridge, MA) overnight
at 4°C and washed with PBS to remove excess primary antibody.
For immunohistochemical localization of IL-8-like substance, we used
mouse anti-human IL-8 antibody (1:20; Biosource). The sections were
then incubated with biotinylated horse anti-mouse IgG (Vector
Laboratories, Burlingame, CA) at 1:200 dilution for 1 h at room
temperature. Bound antibody was visualized according to standard
protocols for avidin-biotin-peroxidase complex method. The measurement
of MUC5AC protein also utilized the same method used for quantitative
measurement of GC metaplasia in the epithelium.
Evaluation of leukocytes in airway tissue. Animals were euthanized 4, 8, 16, 24, and 48 h after instillation of IL-13 (500 ng), and leukocytes were counted in airway tissue. To evaluate the recruitment of neutrophils, we stained the sections with 3,3'-diaminobenzidine for neutrophils and then counterstained them with toluidine blue. Neutrophils seen as peroxidase-positive blue cytoplasmic cells were counted in six consecutive high-power fields of the epithelium (from the basement membrane to cell apices) in the carina. To evaluate the recruitment of eosinophils, we stained the sections with Luna's reagent.
Isolation and chemotaxis of human neutrophils.
Human neutrophils were purified from peripheral blood obtained from
healthy donors. Neutrophil isolation was performed by standard
techniques of Ficoll-Hypaque gradient separation, dextran sedimentation, and hypotonic lysis of erythrocytes. Cells were routinely >95% viable by trypan blue dye exclusion. To prevent endotoxin contamination, all solutions were passed through a 0.1-µm filter. Chemotactic activity was assessed in a 48-well microchemotaxis chamber (Neuroprobe, Cabin John, MD) utilizing the leading front technique. Migration was measured as net movement of neutrophils (µm)
through a nitrocellulose filter (pore size 3 µm) after 25 min at
37°C. The effect of IL-13 (1010, 10
9, and
5 × 10
9 M; recombinant human IL-13; R&D systems) is
expressed as the distance traveled compared with the random migration
of neutrophils incubated with RPMI 1640 medium (24).
Data analysis. All data are expressed as means ± SE. Statistical analysis performed with one-way ANOVA 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 ANOVA. P < 0.05 was accepted as indicating a statistically significant difference.
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RESULTS |
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Effect of IL-13 on GC metaplasia.
To confirm that IL-13 induces mucin production, IL-13 (5, 50, 100, and
500 ng/rat) was instilled into the trachea and tissues were examined
48 h later. In control rats, the airway epithelium contained
only sparse AB-PAS and MUC5AC staining (Figs.
1 and 2). IL-13 increased AB-PAS and MUC5AC
staining dose dependently. These results imply that IL-13 induces GC
metaplasia and MUC5AC mucin production in rat airway epithelium.
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Effect of a selective inhibitor of EGFR activation on IL-13-induced
GC metaplasia.
To examine the relationship between IL-13-induced GC metaplasia and
activation of EGFR, animals were pretreated with a selective EGFR
tyrosine kinase inhibitor (BIBX1522, 1-30
mg · kg1 · day
1). Control
rats showed little EGFR expression in airway epithelium, but
instillation of IL-13 increased EGFR expression (Fig.
3). Pretreatment with the selective EGFR
tyrosine kinase inhibitor BIBX1522 prevented IL-13-induced AB-PAS and
MUC5AC staining dose dependently and completely (Fig.
4). These findings implicate EGFR
activation in IL-13-induced mucin production.
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Expression of TNF- in rat airway tissue.
TNF-
has been shown to induce EGFR expression in airway epithelium
(20, 28). We examined the effect of instillation of IL-13
on TNF-
expression. In control rats, staining with TNF-
antibody
was minimal. Instillation of IL-13-induced TNF-
expression mainly in
infiltrating neutrophils, which were the main infiltrating cells in
IL-13-instilled airways (as determined by 3,3'-diaminobenzidine staining, counterstained with hematoxylin). Pretreatment with cyclophosphamide prevented IL-13-induced TNF-
expression (Fig. 5).
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Effect of IL-13 on leukocyte recruitment and mucin production.
The airway epithelium of control rats contained few leukocytes, but
instillation of IL-13 into the airways caused time-dependent neutrophil
and eosinophil recruitment (Fig. 6,
top). In cyclophosphamide-treated rats, blood neutrophils
and eosinophils were depleted (after cyclophosphamide, venous blood
neutrophil count = 1.8 ± 0.5%; eosinophil count = 0%;
n = 5 rats). Pretreatment with cyclophosphamide
inhibited recruitment of neutrophils and eosinophils into the airways
(Fig. 6, top) and prevented IL-13-induced mucin production
(Fig. 6, bottom).
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DISCUSSION |
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In the present studies, we examined the role of EGFR activation in IL-13-induced GC metaplasia. We used IL-13 because it shares a receptor component and signaling pathways with IL-4, and it was demonstrated to be necessary and sufficient for the asthma phenotype, including GC metaplasia with mucin overproduction, in murine experimental asthma (15, 32). The present studies are based on previous observations that EGFR activation causes GC metaplasia in airways (28), an effect that was prevented by pretreatment with a selective EGFR tyrosine kinase inhibitor. The GC metaplasia occurred by conversion of nongranulated secretory cells to GC (20).
First, we examined the effect of IL-13 on GC metaplasia in tracheal epithelium of pathogen-free rats. GC metaplasia was determined by the volume density of AB-PAS-stained mucous glycoconjugates in the airway epithelium. Rat airway GC are reported to express MUC5AC mucin (20, 28). The expression of MUC5AC protein also was determined immunohistochemically in IL-13-induced GC metaplasia. In control rats, the airway epithelium contained only sparse AB-PAS and MUC5AC staining. IL-13 increased AB-PAS and MUC5AC staining dose dependently. These results indicate that IL-13 induces GC metaplasia and MUC5AC mucin production in rat airway epithelium.
Next, we examined the relationship between IL-13-induced GC metaplasia
and the EGFR system. Control rats showed little EGFR expression in
airway epithelium, but instillation of IL-13 increased EGFR expression.
Pretreatment with the selective EGFR tyrosine kinase inhibitor BIBX1522
prevented IL-13-induced AB-PAS and MUC5AC staining dose dependently and
completely. These findings indicate that EGFR activation is involved in
IL-13-induced mucin production. EGFR was expressed in IL-13-stimulated
airway epithelium, and TNF- has been shown to induce EGFR expression
in airway epithelium (20, 28). Several cell types are
known to produce TNF-
, including epithelial cells (28),
mast cells (4), neutrophils (33), eosinophils
(12), and macrophages (22). Therefore, we
examined the effect of instillation of IL-13 on TNF-
protein
expression. In control animals, there was little TNF-
protein
expression, but IL-13 increased TNF-
expression significantly in the
epithelium and especially in infiltrating neutrophils.
EGFR expression must be followed by EGFR activation to induce mucin production. This activation may occur via two different pathways. The binding of EGFR ligands to EGFR activates the intrinsic receptor tyrosine kinase and induces tyrosine phosphorylation (17, 28). Alternatively, activation of EGFR tyrosine phosphorylation may occur via a ligand-independent transactivation mechanism such as stimulation by oxidative stress (14, 16, 27). Takeyama et al. (27) showed that oxygen-free radicals released from neutrophils when they are activated by the chemoattractant IL-8 cause mucin synthesis in human airway epithelial cells, an effect that is prevented by a selective EGFR tyrosine kinase inhibitor (BIBX1522) and by antioxidants. We hypothesized that leukocyte recruitment by IL-13 could provide oxygen-free radicals, which then could cause EGFR activation. IL-4 is reported to recruit leukocytes into airways (8, 29). We confirmed this with IL-13 instillation, which caused time-dependent leukocyte recruitment, which started after ~8 h and which was maximal within 24 h. Pretreatment with cyclophosphamide inhibited leukocyte recruitment into airways and prevented IL-13-induced mucin production.
Next, we examined the effect of IL-13 on neutrophil chemotaxis in
vitro. IL-13 decreased neutrophil movement dose dependently. Because
IL-13 did not cause neutrophil chemotaxis, we hypothesized that IL-13
stimulates the production of a neutrophil chemoattractant in the
epithelium. In control animals, the airway epithelium did not stain for
IL-8-like chemoattractant, but instillation of IL-13 resulted in
positive staining with an anti-human IL-8 antibody. Pretreatment with
an IL-8 blocking antibody inhibited IL-13-induced leukocyte recruitment
and mucin production. These findings indicate that IL-13 induces an
airway epithelial IL-8-like chemoattractant, which causes neutrophil
recruitment. Blocking antibody to human IL-8 has been shown previously
to inhibit tissue edema in rat carrageenan-induced footpad swelling
(9), to inhibit neutrophil accumulation in the rat skin
sites injected with human IL-8 (21), and to inhibit
glycogen-induced neutrophil accumulation in the rat peritoneum; it is
also protective against lung and dermal vascular injury in rats after
the deposition of IgG immune complexes (21). In the
present studies, we used both anti-human IL-8 and anti-rat macrophage
inflammatory protein (MIP)-1 antibodies (polyclonal anti-rat
MIP-1
; Biosource) for immunohistochemical staining and neutralization of IL-8-like chemoattractant activity in the airway. Both antibodies had similar effects. There was no significant difference between anti-human IL-8 antibody and anti-rat MIP-1
antibody on neutrophil recruitment and GC metaplasia in IL-13-instilled airways (data not shown).
Previous studies showed that human neutrophils incubated with IL-8
cause the release of oxygen-free radicals, resulting in EGFR activation
and mucin production (27). We suggest the following sequence of events by which IL-13 causes mucin production in the airway
epithelium: 1) IL-13 induces the production of a
chemoattractant in the airway epithelium (rat homolog of IL-8), which
results in neutrophil recruitment into airways; 2) the
recruited neutrophils release TNF-, which induces EGFR in the airway
epithelium; 3) recruited neutrophils also release oxygen
free radicals, causing EGFR tyrosine kinase phosphorylation, resulting
in mucin production. In the present studies, we cannot rule out a role
for eosinophils in IL-13-induced GC production. However, the number of
recruited neutrophils greatly exceeded those of eosinophils after IL-13 instillation. Furthermore, Cohn et al. (7) showed that
eosinophils are not essential for Th2-induced airway mucus production.
The novel pathways involved in Th2 cytokine (IL-13)-mediated GC production reported here suggest new methods for therapy of allergic hypersecretion, which is an important cause of death in acute asthma.
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ACKNOWLEDGEMENTS |
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This study was supported in part by private funds.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. A. Nadel, Cardiovascular Research Institute and Depts. of Medicine and Physiology, Univ. of California, San Francisco, CA 94143-0130 (E-mail: janadel{at}itsa.ucsf.edu).
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. Section 1734 solely to indicate this fact.
Received 24 April 2000; accepted in final form 11 August 2000.
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