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Published ahead of print on May 18, 2006, doi:10.1164/rccm.200509-1420OC
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American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 379-385, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200509-1420OC

Original Article

Extracellular Matrix Regulates Enhanced Eotaxin Expression in Asthmatic Airway Smooth Muscle Cells

Vivien Chan, Janette K. Burgess, Jonathan C. Ratoff, Brian J. O'Connor, Anne Greenough, Tak H. Lee and Stuart J. Hirst

King's College London School of Medicine, Medical Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma, and Division of Asthma, Allergy, and Lung Biology, London, United Kingdom; Department of Pharmacology, University of Sydney; and Woolcock Institute of Medical Research, Sydney, Australia

Correspondence and requests for reprints should be addressed to Stuart J. Hirst, Ph.D., King's College London School of Medicine, MRC & Asthma UK Centre in Allergic Mechanisms of Asthma, Thomas Guy House, Guy's Hospital Campus, London SE1 9RT, UK. E-mail: stuart.hirst{at}kcl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with fibronectin and collagen are key features of asthma. Previously, we have reported these ECM proteins enhance ASM synthetic function.

Objective: We compared ASM cultured from endobronchial biopsies from subjects with and without asthma to assess if asthmatic cells were hypersecretory and determined whether the underlying mechanism involved autocrine ECM production.

Methods and Measurements: Cells from subjects with and without asthma were cultured on plastic or in plates precoated with ECM proteins. Cytokine production was evaluated by enzyme-linked immunosorbent assay and by reverse transcriptase–polymerase chain reaction. Function-blocking integrin antibodies were used to identify integrin involvement.

Results: Baseline eotaxin and its production after stimulation with interleukin (IL)-13, IL-1beta, or tumor necrosis factor-{alpha} was increased (2.5- to 6.0-fold) in ASM cells cultured from subjects with asthma compared with healthy subjects. When seeded on ECM from asthmatic ASM, IL-13–dependent eotaxin release from healthy or asthmatic ASM was enhanced compared with culture on healthy ECM. The ECM substrates fibronectin and type I collagen each enhanced IL-13–dependent eotaxin release, and Western immunoblot indicated that fibronectin expression was higher in asthmatic ASM cells. Integrin-blocking antibodies revealed that {alpha}5beta1 was required for more than 50% of the enhanced IL-13–dependent eotaxin release by ASM cells from subjects with asthma, whereas {alpha}2beta1 or {alpha}vbeta3 neutralization lacked effect.

Conclusion: The data indicate that ASM cells cultured from subjects with asthma are hypersecretory compared with cells from healthy donors and that autocrine fibronectin secretion acting via {alpha}5beta1 in part underlies this effect.

Key Words: airway remodeling • airway smooth muscle • asthma • eotaxin • extracellular matrix • integrin

Tissue remodeling and persistent inflammation are key features of the airway wall in asthma (1, 2). Two prominent components of the remodeling process in the causation of airway narrowing and hyperresponsiveness in patients with asthma include accumulation of airway smooth muscle (ASM) and alterations in the amount and composition of extracellular matrix (ECM) proteins (3–7). In addition to ASM accumulation, evidence suggests that ASM may contribute to airway wall inflammatory events in asthma by expressing cell adhesion receptors and costimulatory molecules, and by releasing multiple cytokines and chemokines, including those that activate eosinophils such as eotaxin (CCL 11) (8, 9).

ECM proteins provide not only mechanical support for airway structure and function but also regulate the function of cells embedded therein, including migration, survival, proliferation, and differentiation (10). In the airway wall of patients with asthma, collagen types I, III, and V, fibronectin, tenascin C, hyaluronan, versican, and laminin {alpha}2/beta2 chains are increased, whereas other major ECM components, including collagen type IV and elastin, are decreased (3–7). Increased type I collagen, hyaluronan, and versican have been found localized within and surrounding ASM bundles from patients with asthma (7, 11). In the bronchoalveolar lavage (BAL) fluid of patients with asthma, there are increased levels of fibronectin, hyaluronate, and laminin products (reflecting increased ECM turnover), which correlate with asthma severity (12).

Enrichment of the airway wall with collagen and fibronectin in asthma is potentially important for the regulation of ASM synthetic function. Previously, we have reported that both type I collagen and fibronectin enhance thrombin- or platelet-derived growth factor-BB–stimulated proliferation of human ASM cells cultured from subjects without asthma (13) and that this is mediated by multiple ECM binding beta1 integrins (14). Bonacci and coworkers (15) reported similar findings with fibroblast growth factor-2 (FGF-2)–dependent proliferation of bovine tracheal smooth muscle cultured on type I collagen, and Freyer and coworkers (16) have shown that several ECM substrates, including type I collagen and fibronectin, increase the survival of human ASM cells from nonasthmatic subjects. Moreover, ASM cells cultured from individuals with asthma and grown on a homologous ECM substrate from asthmatic ASM cells proliferate more rapidly (17). In keeping with this observation, the composition of ECM proteins secreted by ASM cells from patients with asthma differs from that of nonasthmatic individuals. ECM from asthmatic ASM cells comprises an increased content of growth-promoting type I collagen (13, 14), with a concomitant reduction in ASM growth–attenuating substrates such as laminin and chrondroitin sulfate (13, 18). Aside from effects on proliferation, we have recently reported that the ECM regulates ASM cell secretory function. We found that induction of release of multiple eosinophil-activating cytokines, including eotaxin from healthy human ASM cells by interleukin (IL)-1beta or tumor necrosis factor (TNF)-{alpha}, was potentiated by culture on fibronectin or type I collagen (19).

In this study, we cultured ASM cells from endobronchial biopsies from subjects with and without asthma to assess whether asthmatic cells exhibit amplified secretory responses. Furthermore, we hypothesized that autocrine production of ECM proteins known to be up-regulated in asthma contributed to the amplified secretory response in ASM cells from individuals with asthma. Some of the results of this study have been previously reported in the form of an abstract (20).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Culture of Human ASM Cells
ASM cells from healthy subjects and patients with glucocorticoid naive atopic asthma were obtained in accordance with procedures approved by the Research Ethics Committees of King's College Hospital (Study 11-03-209) and Guy's and St. Thomas' Hospitals (Study 05/Q0704/72) by deep endobronchial biopsy (see Table 1 for patient details). See the online supplement for full details of patient recruitment and endobronchial biopsy. ASM bundles were visualized using a dissecting microscope and dissected free of surrounding tissue using fine needles. Cells were grown by explant culture from ASM bundle fragments in 12-cm2 flasks using methods described previously (13, 21). The presence of ASM bundles in endobronchial biopsies was confirmed histologically (Figure E1 of the online supplement). Fluorescent immunocytochemistry routinely confirmed that near-confluent, fetal bovine serum (FBS)–deprived ASM cells (passage 2) stained (> 95%) for smooth muscle–specific {alpha}-actin, desmin, and calponin (13, 21) (Figure E2). Cell passages 3–7 were used in all experiments.


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  		TABLE 1. DETAILS OF PATIENTS WITH ASTHMA AND HEALTHY SUBJECTS

 
Surface Coating with ECM Proteins
Lyophilized human plasma fibronectin (Sigma-Aldrich, Poole, UK), rat tail monomeric type I collagen (ICN Chemicals, Thame, UK), and human fibrillar collagen type I (Chemicon, Chandler's Ford, UK) were adsorbed to tissue culture plasticware overnight at 37°C as previously described (13, 14, 19). Native ECM from healthy or asthmatic ASM cells was used as a substrate on which subsequent ASM cells were seeded. ECM free of cells was prepared by treatment of ASM cells (20,000 cells/cm2) in 24-well plates with hypotonic ammonium hydroxide (20 mM) as described previously (17). Unoccupied protein-binding sites were blocked by incubation with 0.1% bovine serum albumin for 30 min.

Cell Stimulation and Application of Blocking Antibodies
Near-confluent, FBS-deprived cells from flasks were seeded (5,000 cells/cm2) in Dulbecco's modified Eagle's medium containing 1% FBS onto plastic or ECM substrate precoated plasticware and left overnight at 37°C. In some experiments, cells in suspension were pretreated for 30 min at room temperature with integrin function–blocking monoclonal antibodies (1 µg/ml) or isotype-matched control antibodies with continuous roller mixing before seeding, described previously (19). After attachment, cells were washed twice with FBS-free Roswell Park Memorial Institute (RPMI)–1640 and then stimulated for 24 h in RPMI-1640 (0.5 ml) containing human recombinant cytokines (R&D Systems, Abingdon, UK). Blocking anti-human monoclonal antibodies (Chemicon) were anti-{alpha}2 (clone P1E6), anti-{alpha}5 (clone P1D6), anti-beta1 (clone 6S6), and anti-{alpha}vbeta3 (clone LM609). Mouse purified isotype-matched IgG (Chemicon) was used as a nonimmune control. Eotaxin levels were determined in duplicate by specific sandwich ELISA as described previously (19). Manual counts were performed after collection of cell-conditioned media to confirm that cell numbers were unchanged after stimulation and to allow cytokine levels to be expressed initially in nanograms per milliliter per million cells before normalization for stimulation on plastic to correct for small variations in release between cell lines.

Reverse Transcriptase–Polymerase Chain Reaction
Total RNA was isolated using the RNeasy mini kit (Qiagen, Hilden, Germany), and its concentration determined using the RiboGreen RNA quantification kit (Molecular Probes, Leiden, The Netherlands). Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed for detection of eotaxin mRNA (24 cycles; eotaxin sense primer 5'-CCC AAC CAC CTC CTG CTT TAA C-3'; antisense 5'-CCA GAT ACT TCA TGG AAT CCT GCA C-3') as previously described (19, 22). Cycle number was initially varied from 20 to 28 to confirm RT-PCR reactions were within the linear range for amplification. The size of the eotaxin amplicon corresponded to its predicted size of 182 bp. Amounts of eotaxin mRNA were compared with 18S ribosomal RNA (rRNA; 12 cycles; 5'-TGA CTC AAC ACG GGA AAC CTC AC-3' sense primer and 5'-GGA CAT CTA AGG GCS TCA CAG ACC-3' antisense) present in each sample. All primers were purchased from MWG Biotech (Ebersberg, Germany).

Western Immunoblot
Total cell protein extracts from ASM cells in suspension were prepared and separated (7.5 µg/lane) by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, described previously (22). Lysates were prepared from cells in suspension to limit the contribution of fibronectin adsorbed to tissue culture plasticware. Fibronectin was detected with a rabbit polyclonal antibody (H-300; Santa Cruz Biotechnology, Santa Cruz, CA). The primary antibody was detected with a goat anti-rabbit IgG horseradish peroxidase–conjugated secondary (Santa Cruz Biotechnology). Blots were stripped and reprobed with a mouse anti-GAPDH monoclonal antibody (clone 6G5; Biogenesis, Poole, UK) to control for loading differences. Signals were visualized by enhanced chemiluminescence (Amersham-Pharmacia, Amersham, UK) and quantified (ImageQuant, Molecular Dynamics, Sunnyvale, CA) on autoradiographs that depicted bands within a linear range of exposure.

Statistical Analysis
Data are expressed as mean ± SEM of duplicate or triplicate observations obtained from cells cultured from "n" patient subjects. Data were compared using one- or two-way analysis of variance (ANOVA), where appropriate, followed by Bonferroni's t test post hoc to evaluate statistical differences between treatment groups (SigmaStat; SPSS, Inc., Chicago, IL). A probability value (p) of less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Up-regulation of Eotaxin in Asthmatic ASM Cells
The cytokines IL-1beta (10 ng/ml) and TNF-{alpha} (10 ng/ml), either alone or in combination with IL-13 (10 ng/ml), induced release of eotaxin from ASM cells cultured from healthy subjects (p < 0.01 by two-way ANOVA, n = 6), confirming previous observations (22, 23). Cells from patients with asthma also released eotaxin when stimulated with these cytokines, but levels were increased compared with release from healthy subjects (p < 0.001 by two-way ANOVA, n = 6) ranging from 2.5- to 6.0-fold depending on the stimulus (Figure 1A). Of note, constitutive levels of eotaxin release from ASM cells from patients with asthma exceeded those from healthy control subjects when stimulated with IL-1beta, TNF-{alpha}, or IL-13 alone (p < 0.05). In keeping with enhanced release of eotaxin protein from ASM cells from patients with asthma, IL-13–dependent eotaxin mRNA levels were examined and found to be greater in ASM cells cultured from patients with asthma compared with healthy cells (Figure 1B).


Figure 1
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  		Figure 1. Eotaxin production is increased in airway smooth muscle (ASM) cells cultured from patients with asthma compared with healthy control subjects. (A) Eotaxin release from human ASM cells after stimulation for 24 h with interleukin (IL)-1beta (1 ng/ml), tumor necrosis factor (TNF)-{alpha} (10 ng/ml), or IL-13 (10 ng/ml). Data are mean ± SEM of duplicate values from independent experiments using cells cultured from six different healthy subjects or patients with asthma. Mean cell counts were 6,563 ± 530 and 6,255 ± 318 for healthy subjects and patients with asthma, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, compared with healthy control subjects. (B) Increased eotaxin mRNA (182 bp) in asthmatic ASM cells after stimulation with IL-13 (10 ng/ml) for 6 h. Bands are representative of two experiments using cells cultured from two subjects with asthma and two healthy control subjects. Unstim = unstimulated.

 
ECM from Asthmatic ASM Enhances Eotaxin Release
We have previously reported that ECM components found localized within and surrounding ASM bundles from patients with asthma up-regulate IL-1beta/TNF-{alpha}–stimulated chemokine production from ASM cells cultured from healthy subjects (19). In keeping with these findings, levels of mRNA encoding eotaxin in healthy ASM cells and released eotaxin protein induced by IL-13 (10 ng/ml) were approximately twofold increased on fibronectin compared with culture on plastic (p < 0.01–0.001 by two-way ANOVA; Figure 2). An IL-13 concentration–dependent increase in eotaxin release from healthy ASM cells was also found with monomeric type I collagen (p < 0.05–0.01; Figure 2C), which did not differ from the up-regulation found with fibrillar type I collagen (p > 0.05 by two-way ANOVA). The same profile of enhanced eotaxin release with these ECM substrates occurred when IL-4 was used in place of IL-13 (data not shown), which up-regulates eotaxin in these cells by the IL-13 receptor complex (23). In all experiments, release of eotaxin from cells cultured on these ECM substrates in the absence of IL-13 (or IL-4) did not differ from release by unstimulated cells on plastic (p > 0.05; Figures 2B and 2C).


Figure 2
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  		Figure 2. Enhancement of IL-13 (10 ng/ml)–dependent eotaxin by defined extracellular matrix substrates. (A) IL-13–dependent eotaxin mRNA (182 bp) was increased after culture on 10 µg/ml fibronectin. Bands are representative of separate experiments using cells cultured from two healthy subjects. Eotaxin levels determined by ELISA after culture on (B) fibronectin or (C) type I monomeric or fibrillar collagen. Data are mean ± SEM of duplicate values from independent experiments using cells cultured from 6–7 subjects. *p < 0.05, **p < 0.01, ***p <0.001, compared with stimulated cells on plastic.

 
Because these ECM components are also secreted by asthmatic ASM cells either in the intact airway or in culture (17), we next examined the effects of native ECM on IL-13–dependent secretory responses in ASM cells from patients with and without asthma. ECM from asthmatic ASM (20,000 cells/cm2) enhanced IL-13–dependent eotaxin release from either healthy or asthmatic cells by around eightfold compared with fivefold by ECM from similar numbers of healthy cells (p < 0.05 by two-way ANOVA; Figure 3).


Figure 3
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  		Figure 3. Native extracellular matrix (ECM) derived from asthmatic ASM cells enhances IL-13–dependent eotaxin release at 24 h from ASM cells cultured from either healthy control subjects or subjects with asthma. Data are shown as the fold increase above unstimulated and are mean ± SEM of duplicate values from independent experiments using cells cultured from six subjects, which were seeded on ECM substrates from at least three subjects. Baseline values for eotaxin (ng/ml/million cells) were 16.35 ± 12.24 (open squares), 38.07 ± 18.22 (closed squares), 20.89 ± 14.27 (open circles), and 38.35 ± 20.89 (closed circles). *p < 0.05 compared with ECM from healthy subjects.

 
To account for up-regulation of IL-13–dependent eotaxin release from asthmatic or healthy ASM by ECM from asthmatic ASM cells, differences in total cell fibronectin content were examined by Western immunoblot in ASM cells cultured from individuals with and without asthma. Constitutive levels of fibronectin (intense band at 250 kD) were increased by 1.90- ± 0.03-fold in ASM cells cultured from patients with asthma compared with healthy control subjects (p < 0.05); stimulation with IL-13 (10 ng/ml) for 24 h had no detectable effect on fibronectin content (Figure 4).


Figure 4
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  		Figure 4. Western immunoblot analysis showing increased fibronectin content in lysates from ASM cells cultured from an individual with asthma. Lysates were prepared from cells in suspension to limit the contribution of fibronectin adsorbed to tissue culture plasticware. Equal loading of proteins was confirmed by reblotting membranes for GAPDH. Blots are representative of three experiments using cells cultured from three subjects with asthma and three healthy control subjects.

 
Attenuation of Eotaxin Release from Asthmatic ASM Cells by Integrin-specific Blocking Antibodies
Having defined a probable role for the ECM underlying amplified IL-13–dependent eotaxin production from asthmatic ASM cells, subsequent experiments examined possible integrin receptors that could mediate this response using maximally effective concentrations of integrin-blocking monoclonal antibodies (1 µg/ml for each) (14, 19).

To confirm antibody selectivity, the panel of blocking antibodies was examined first on enhancement of IL-13–dependent eotaxin release from healthy ASM cells that were cultured on the defined ECM substrates, fibronectin or type I collagen (10 µg/ml for each ECM). Blocking the binding of matrix factors to {alpha}2beta1 integrin subunits abolished enhancement of IL-13–dependent eotaxin release by monomeric type I collagen (p < 0.01 compared with control IgG1) but not fibronectin. In contrast, blocking of {alpha}5beta1 was effective against enhancement by fibronectin (p < 0.01 compared with control IgG1) but not type I collagen (Figures 5A and 5B). Neutralization of {alpha}vbeta3 with a heterodimer-specific blocking antibody did not prevent enhancement of IL-13–dependent eotaxin release by either fibronectin or type I fibrillar collagen (p > 0.05 compared with control IgG1).


Figure 5
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  		Figure 5. Enhanced IL-13–dependent eotaxin release from asthmatic ASM cells is partially integrin dependent. Integrin-blocking antibody (1 µg/ml) selectivity was confirmed in control experiments with IL-13 (10 ng/ml)–stimulated ASM cells from healthy subjects cultured on defined ECM substrates comprising either (A) type I fibrillar collagen (n = 4) or (B) fibronectin (n = 7, 10 µg/ml). In C, integrin-blocking antibodies were examined on IL-13–stimulated ASM cells on plastic from healthy subjects or subjects with asthma. Data are mean ± SEM of duplicate values from independent experiments using cells cultured from 4–7 subjects. *p < 0.05, **p < 0.01, compared with IgG1 treatment.

 
When cells from healthy subjects were plated directly on plastic, IL-13–dependent eotaxin release was suppressed by the combination of {alpha}2 with beta1 integrin blocking and by {alpha}5 and beta1 integrin block (p < 0.05 compared with control IgG1). In contrast, IL-13–dependent eotaxin release from asthmatic ASM cells, although much increased compared with healthy subjects (p < 0.001 by two-way ANOVA), was blocked by antibodies against {alpha}5 and beta1 integrin subunits (p < 0.01 compared with control IgG1) but not by neutralization of {alpha}2 and beta1 (Figure 5C). Blocking of {alpha}vbeta3 was without effect regardless of whether ASM cells on plastic were derived from patients with asthma or healthy control subjects (p > 0.05 compared with control IgG1).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we report that ASM cells from subjects with asthma express higher levels of eotaxin both constitutively and in response to multiple cytokines implicated in airway inflammation in asthma, when compared with ASM cells cultured from healthy control subjects. IL-13 was selected as a stimulus with which to address possible mechanisms underlying enhanced eotaxin production from ASM cells from patients with asthma because of its proposed central role in eosinophilic inflammation and remodeling in asthma (24, 25). Both constitutive and IL-13–inducible eotaxin mRNA and protein were increased in ASM cells cultured from patients with asthma, suggesting the enhancement was likely transcriptionally regulated, although post-transcriptional mechanisms could contribute to the difference. Elsewhere, we have demonstrated selected airway wall ECM components that are increased in asthma, such as collagen and fibronectin (3–7), up-regulate ASM cell secretory responses (19). Although absolute amounts of ECM substrates in contact with ASM cells in the asthmatic airway are unknown, we hypothesized that the prevailing asthmatic ECM environment in part mediated the enhanced secretory response of ASM cells from patients with asthma. In keeping with this possibility, fibronectin, at concentrations found to enhance ASM cell proliferative and survival responses (13, 15, 16), provided a transcriptionally regulated enhancement of the IL-13–dependent eotaxin secretory signal in cells from healthy subjects. This is compatible with our recent findings with IL-1beta and TNF-{alpha} (19). Human ASM cells in culture secrete an ECM network of mixed composition (17). ECM from asthmatic ASM cells was found to enhance IL-13–dependent eotaxin release from ASM cells cultured from either healthy subjects or subjects with asthma and Western immunoblot revealed that cells from subjects with asthma expressed higher constitutive levels of fibronectin. ELISA studies also indicated that at least 50% of the enhanced eotaxin response in asthmatic ASM cells was blocked after treatment with blocking antibodies to {alpha}5beta1 (major fibronectin receptor) integrin but not {alpha}2beta1 (collagen type I receptor) or {alpha}vbeta3 (vitronectin receptor) integrins.

Eotaxin (CCL 11), a CC family chemokine, is an important eosinophil chemoattractant in both mice and humans (26, 27). Eotaxin was originally believed to be selective for eosinophils but is also chemotactic for basophils, Th2 lymphocytes, tryptase–chymase mast cells (28), and ASM (29). Its physiologic properties extend beyond chemotaxis to include induction of eosinophil degranulation and IgE-independent degranulation of basophils (30, 31). We and others have identified cultured ASM cells as a source of eotaxin mRNA and protein (8, 22). It is unlikely that the differences in eotaxin production we observed between the asthmatic and healthy cells reflect a tissue culture artifact. Other reports show increased connective tissue growth factor (CTGF) mRNA and protein in ASM cultured from patients with asthma compared with similar cells from nonasthmatic individuals (32). The same study confirmed increased CTGF mRNA in ASM cells from asthmatic intact airway sections. Likewise, other secreted factors, including vascular endothelial growth factor and basic FGF (FGF-2) are elevated in ASM bundles in patients with chronic obstructive pulmonary disease (33, 34), and elevated eotaxin has previously been reported in intact ASM bundles from a patient with severe asthma (8). The level of immunoreactive eotaxin released from nonasthmatic human ASM after stimulation with IL-13 in combination with IL-1beta or TNF-{alpha} is approximately 20,000 times greater than that induced by cytokine-stimulated airway epithelial cells, as reported by Lilly and coworkers (35). Thus, our finding that ASM cells from patients with asthma exhibit a further two- to fourfold enhancement in eotaxin release suggests that, under specific conditions, ASM could be a major source of eotaxin in the asthmatic airway, although the activity of eotaxin on infiltrating cells may be tightly regulated by mast cell tryptase (36). A consensus of new data indicates the presence of an inherent abnormality of ASM in asthma that not only encompasses its amplified contractile (37) and proliferative (17, 21) function but now includes enhanced secretory responses involving the release of eotaxin. The exact nature of the abnormality producing enhanced IL-13–dependent eotaxin release by ASM cells from subjects with asthma and its consequences have not been fully elucidated. One possibility is that autocrine eotaxin production acting via activation of cell surface CCR3 enhances ASM migration (29), thereby contributing to the increased content of ASM in asthmatic airways.

The source of ECM underlying enhanced eotaxin release may include the ASM itself. Recent cell culture–based studies indicate proliferation of ASM from patients with asthma is increased (21) and that autocrine ECM production from asthmatic ASM cells enhances FBS-dependent proliferation of ASM cultured from either healthy subjects or subjects with asthma (17). Here, a similar experiment was performed to examine chemokine production in place of cell proliferation. We found that IL-13–dependent eotaxin release by ASM cells from either healthy individuals or individuals with asthma was similarly potentiated by subsequently seeding cells onto an ECM from asthmatic ASM cells compared with that from healthy subjects. Potentially, this could involve variation in the overall secreted quantity of ECM, having an otherwise identical composition, due to varying growth rates between ASM cells from healthy individuals and individuals with asthma. Alternatively, it might reflect overall differences in ECM composition secreted from asthmatic ASM cells compared with healthy control cells. Elsewhere, and in the present study (Figure 4), we have shown that enhancement of inducible eotaxin release is related to the concentration of ECM coating (19). Thus, to exclude a simple concentration effect, ASM cells from healthy subjects or subjects with asthma were seeded at similar densities (20,000 cells/cm2), and cell-free ECM was prepared 48 h later, before significant growth had occurred. Intrinsic differences in ECM substrate composition between healthy and asthmatic ASM cells are supported by the Western immunoblot finding that fibronectin levels were higher in ASM cell suspension lysates from two subjects with asthma compared with two healthy subjects. In each case, the magnitude of the increased fibronectin content in asthmatic cells was 1.9-fold, and similar to the 2.5-fold difference found in the enhancement of IL-13–dependent eotaxin release between ASM cells cultured on ECM from subjects with asthma and from healthy control subjects (Figure 3). That fibronectin is increased in the asthmatic airway (3–7) and that it enhanced IL-13–dependent eotaxin release lend further credibility to its possible involvement in the enhanced secretory capacity of ASM cells from individuals with asthma, although other ECM substrates up-regulated in asthma also up-regulate inducible eotaxin release from ASM, including type I collagen (Figure 2C). Consistent with earlier findings (19), both fibrillar and monomeric type I collagen enhanced IL-13–stimulated eotaxin release from healthy ASM cells. This suggests both the native helical configuration (fibrillar) and proteolytic denatured (monomeric) forms of type I collagen, which occur after cleavage of fibrillar collagen in inflammation by matrix metalloproteinases (38), are sufficient to amplify secretory signals in ASM. That the effect of type I collagen was less marked than fibronectin is perhaps a reflection of normal airway wall homeostasis given that collagen is one of the predominant ECM components in the healthy airway. Elsewhere, we have recently reported that collagen type I and fibronectin can act synergistically to enhance growth factor–induced proliferation of nonasthmatic human ASM cells (14). A similar synergistic interaction may occur for eotaxin release, but this was not examined in the present study.

Integrins comprising {alpha}beta heterodimers are the principal receptors mediating multiple cell responses to ECM substrates (39). Previously, we and others have reported that approximately 50 to 60% of healthy human ASM cells express the promiscuous vitronectin receptor {alpha}vbeta3, 65 to 70% of cells express {alpha}2 integrin required for type I collagen binding, and {alpha}5 (major fibronectin receptor) and beta1 subunits are universally expressed (14, 16, 19). Here, our ELISA experiments with integrin function–blocking monoclonal antibodies showed that enhancement of IL-13–stimulated eotaxin release by fibronectin required binding of {alpha}5beta1 but not {alpha}2beta1 or {alpha}vbeta3 and that enhancement of type I collagen involved {alpha}2beta1. To implicate autocrine ECM production in the enhanced eotaxin release from asthmatic ASM cells, the same panel of integrin-blocking antibodies was examined on IL-13–stimulated eotaxin release from ASM cells from subjects with and without asthma cultured on plastic. Eotaxin release from healthy ASM cells could be reduced by combinations of antibodies that prevent both fibronectin and type I collagen binding. In contrast, the marked increase in IL-13–stimulated eotaxin release found with asthmatic cells was reduced only by blocking antibodies to fibronectin binding. Although the concentrations of blocking antibodies used in the present study were maximal (14, 19), the combination of anti-{alpha}5 and anti-beta1 was only partially effective (~ 50%) in preventing enhanced IL-13–stimulated eotaxin release from asthmatic cells. This suggests additional mechanisms independent of ECM binding to these beta1 integrins contribute to increased secretory responses in ASM from individuals with asthma. Collectively, however, the data suggest that autocrine ECM does contribute in part to IL-13–stimulated eotaxin release. Observed differences in integrin requirements between ASM cells from individuals with asthma and healthy subjects may reflect different integrin expression patterns and studies are underway to test this hypothesis.

In summary, we have shown that ASM cells cultured from subjects with asthma express increased amounts of eotaxin, and enhanced autocrine fibronectin secretion acting via {alpha}5beta1 integrin appears to underlie a major portion of the amplified response. Our findings further support the hypothesis that the ECM environment surrounding ASM cells favors enhanced synthetic responses during inflammation and remodeling in asthma.


   Acknowledgments
 
The authors thank the research nursing and technical staff in the department for recruitment and screening of volunteers, and for assistance with endobronchial biopsy, and Dr. Trang Nguyen for histologic characterization of smooth muscle bundles in endobronchial biopsy samples.


   FOOTNOTES
 
Supported by grants from Asthma UK (Nos. 00/44, 05/027). J.K.B. is supported by a National Health and Medical Research Council Australia Peter Doherty Fellowship (No. 165722).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200509-1420OC on May 18, 2006

Conflict of Interest Statement: V.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.K.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.C.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.J.O. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.H.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.J.H. received $3,000 for speaking at a conference (October 2003) sponsored by GlaxoSmithKline (GSK), and was a discussant at a scientific meeting organized and financed by GSK in January 2004.

Received in original form September 12, 2005; accepted in final form May 16, 2006


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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