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1 Integrins Mediate Enhancement of Human Airway Smooth Muscle Cytokine Secretion by Fibronectin and Type I Collagen1
Department of Asthma, Allergy and Respiratory Science, The Guy’s, King’s and St. Thomas’ School of Medicine, King’s College London, Guy’s Hospital Campus, London, United Kingdom
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Abstract |
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-dependent secretory responses of human ASM cells, and characterized the involvement of specific integrins. Cytokine production (eotaxin, RANTES, and GM-CSF) was evaluated by ELISA, RT-PCR, and flow cytometry. Function-blocking integrin mAbs and RGD (Arg-Gly-Asp)-blocking peptides were used to identify integrin involvement. IL-1-dependent release of eotaxin, RANTES, and GM-CSF was enhanced by fibronectin and by fibrillar and monomeric type I collagen, with similar changes in mRNA abundance. Collagen types III and V had no effect on eotaxin or RANTES release but did modulate GM-CSF. Analogous changes in intracellular cytokine accumulation were found, but in <25% of the total ASM cell population. Function-blocking Ab and RGD peptide studies revealed that 
21, 51, v1, and v3 integrins were required for up-regulation of IL-1-dependent ASM secretory responses by fibronectin, while 21 was an important transducer for type I collagen. Thus, fibronectin and type I collagen enhance IL-1-dependent ASM secretory responses through a 1 integrin-dependent mechanism. Enhancement of cytokine release from ASM by these ECM components may contribute to airway wall inflammation and remodeling in asthma. |
Introduction |
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ECM proteins ligating via cell surface integrin ECM receptors regulate diverse cellular functions including growth, migration, survival, and the maintenance of differentiated status (14). Histologic studies of asthmatic airways have revealed abnormalities in both the quantity and composition of ECM proteins. Collagen types I, III, and V; fibronectin; tenascin; hyaluronan; versican; and laminin 2/2 chains are increased, whereas other major ECM components including collagen type IV and elastin are decreased (5, 6, 7, 8, 9). Increased type I collagen, hyaluronan, and versican have been found localized within and surrounding ASM bundles from asthmatics (9, 15). In the bronchoalveolar lavage fluid of asthmatics, increased levels of fibronectin, hyaluronan, and laminin products are found (reflecting increased ECM turnover), which correlate with asthma severity (16). These studies support marked ECM changes in the airway wall in asthma. However, little is known of the impact of enrichment of the airway wall with collagen and fibronectin on airway cell function.
Previously, we have reported that both fibronectin and type I collagen enhance responses of cultured human ASM cells to mitogens such as PDGF-BB and -thrombin (17). Bonacci et al. (18) report similar findings with fibroblast growth factor-2-dependent proliferation of bovine tracheal smooth muscle cultured on type I collagen; and Freyer et al. (19) have shown that both type I collagen and fibronectin increase survival of human ASM cells. Moreover, ASM cells cultured from asthmatic individuals or grown on homologous ECM from asthmatic ASM cells proliferate more rapidly (20). Collectively, these observations affirm that ASM in situ likely is subjected to the influences of signaling through ECM/integrin interactions, and have led to the suggestion that alterations in airway wall ECM microenvironment in remodeling in asthma may favor enhanced survival and growth responses of ASM during inflammation (17, 18, 19). However, it is unknown whether the ECM has analogous effects on ASM secretory responses.
In the present study, we hypothesized that enrichment of the airway wall ECM environment with fibronectin and type I collagen, as occurs in asthma, confers increased secretory properties of ASM. We compared IL-1/TNF--dependent eosinophil-activating cytokine release from human ASM cells that were cultured either on plastic (Pl) or fibronectin or on varying forms of collagen. Moreover, we characterized the integrin heterodimer subunits expressed by ASM and their involvement in modulating secretory responses.
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Materials and Methods |
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Human ASM cells were obtained in accordance with procedures approved by the Guy’s and St. Thomas’ Hospitals’ Research Ethics Committee from the lobar or main bronchus of 45 nonasthmatic patients (mean age, 64 ± 4 years; range, 28–78 years; 24 male, 21 female) undergoing lung resection for carcinoma of the bronchus using methods described previously (13). Fluorescent immunocytochemical and flow cytometric techniques confirmed that near-confluent, FBS-deprived human ASM cells (passage 2) stained (>95%) for smooth muscle-specific -actin and calponin (17). Cells at passages 3–5 were used in all experiments.
Surface coating with ECM proteins
Lyophilized human plasma fibronectin (Sigma-Aldrich), rat monomeric type I collagen (ICN Chemicals), and human fibrillar collagen types I, III, and V (Chemicon International) were reconstituted in sterile PBS. ECM proteins (0.1–10 µg/ml) diluted in PBS were adsorbed to tissue culture plasticware overnight at 37°C as previously described (17). Excess unbound ECM protein was removed by aspiration and washing with PBS. Unoccupied protein-binding sites were blocked with 0.1% BSA for 30 min.
Cell stimulation and application of blocking mAbs
Near-confluent, FBS-deprived cells from flasks were seeded (5000 cells/cm2) in DMEM containing 1% FBS on to Pl or ECM substrate (0.1–10 µg/ml)-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 mAbs (1 µg/ml) or isotype-matched control Abs with continuous roller mixing before seeding. After attachment, cells were washed twice with FBS-free RPMI 1640 (containing 25 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin/100 µg/ml streptomycin) and then stimulated for 24 h in RPMI 1640 with 1 ng/ml recombinant human IL-1 or 10 ng/ml recombinant human TNF-. Soluble RGD integrin-blocking peptides (10 µM) or a negative control peptide were added directly to adherent cells 30 min before stimulation with cytokines. Integrin function-blocking anti-human mouse mAbs (Chemicon International, unless stated otherwise) were anti-1 (clone FB12), anti-2 (clone P1E6), anti-3 (clone P1B5), anti-5 (clone P1D6), anti-v (clone L230, gift from Dr. J. F. Marshall (John Vane Science Center, London, U.K.)(21)), anti-1 (clone 6S6), anti-v3 (clone LM609), and anti-3 (clone 25E11). Mouse-purified or isotype-matched IgG (Chemicon International) was used as a nonimmune control.
Flow cytometric labeling of surface integrins and intracellular cytokines
Cell surface integrins were localized by binding of the above anti-human integrin subunit mAbs. Near-confluent growth-arrested cells on Pl were harvested using trypsin/EDTA, washed twice in PBS (200 x g for 5 min), resuspended, and fixed with 200 µl of 4% formaldehyde (methanol-free, EM-grade; Polysciences) for 30 min on ice in round-bottom FACS tubes. Fixed cells (20,000/tube) were resuspended in PBS containing 3% FBS for 30 min to prevent nonspecific mAb binding. After centrifugation, cells were incubated on ice with the anti- or anti- integrin subunit unconjugated Abs (1 µg/ml for 30 min), washed in PBS, then incubated in the dark with an anti-mouse PE-conjugated secondary Ab (Sigma-Aldrich) for 30 min and washed twice in PBS.
Cells on Pl or ECM substrates (10 µg/ml) for 24 h in the absence or combined presence of cytokine stimulation were harvested and fixed as above. The protein transport inhibitor, brefeldin A, was not used in these studies. Cells (20,000/tube) were permeablized with 100 µl of 0.5% saponin (catalog no. S-4521; Sigma-Aldrich)/0.1% BSA in PBS containing Abs (1 µg/ml). Abs used (BD Pharmingen, unless stated otherwise): PE-conjugated anti-GM-CSF (clone no. BVD2-21C11), PE-conjugated anti-RANTES (clone no. 2D5), PE-conjugated anti-eotaxin (clone no. 43915; R&D Systems), or PE-conjugated isotype-matched control Abs (IgG1/IgG2a). Numbers of PE-labeled cells were determined on a FACSCalibur flow cytometer (BD Biosciences) using CellQuest Pro software (BD Biosciences) (22). After analysis, cells were confirmed microscopically to be intact.
Measurement of cytokine levels by ELISA
Levels of eotaxin, RANTES, and GM-CSF were determined in duplicate in the same samples of cell-conditioned medium by specific sandwich ELISA as described previously (13). Manual cell counts were repeated after collection of cell-conditioned medium to confirm 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 Pl to correct for small variations in release between cell lines.
RT-PCR
Total RNA was isolated using the RNeasy mini kit (Qiagen), and its concentration was determined using the RiboGreen RNA quantification kit (Molecular Probes). Full-length first-strand cDNA was synthesized using 2 µg of total RNA in a 33-µl reverse transcriptase reaction mixture with Ready-To-Go You-Prime beads and random hexamer primers (Amersham Biosciences) as previously described (22). To confirm PCR were performed within the linear for amplification, the number of cycles was initially varied from 20 to 28. The size of the amplicons corresponded to predicted sizes (eotaxin, 182 bp; RANTES, 332 bp; GM-CSF, 277 bp), confirmed by sequencing. Primers used in RT-PCR were: eotaxin (24 cycles), 5'-CCC AAC CAC CTC CTG CTT TAA C-3' as sense and 5'-CCA GAT ACT TCA TGG AAT CCT GCA C-3' as antisense; RANTES (26 cycles), 5'-TGC TTT GCC TAC ATT GCC CG-3' as sense and 5'-CTG GGG AAG GTT TTG TAA CTG C-3' as antisense; GM-CSF (22 cycles), 5'-TGA GTA GAG ACA CTG CTG CTG A-3' as sense and 5'-TCA AAG GGG ATG ACA AGC AGA A-3' as antisense; 18S rRNA, 5'-TGA CTC AAC ACG GGA AAC CTC AC-3' as sense and 5'-GGA CAT CTA AGG GCS TCA CAG ACC-3' as antisense. cDNA samples were amplified as previously described (22). All primers were purchased from MWG Biotech.
Statistical analysis
Data are mean ± SEM from cells cultured from n patient donors. Data were compared using one- or two-way ANOVA, where appropriate, followed by Bonferroni’s t test post hoc to evaluate statistical differences between treatment groups (SigmaStat; SPSS). A p value of <0.05 was considered significant.
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Results |
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IL-1 (1 ng/ml) induced marked release of eotaxin, RANTES, or GM-CSF from human ASM cells when cultured on Pl (Fig. 1), confirming previous observations (13). After culture in plates precoated with fibronectin or fibrillar type I collagen, IL-1-stimulated cells were significantly more secretory compared with culture on Pl (p < 0.05–0.01; Figs. 1 and 2). A similar concentration-dependent increase in IL-1-dependent secretory capacity was found with monomeric type I collagen (p < 0.05–0.01; Fig. 2), which did not differ from the up-regulation found with fibrillar type I collagen (p > 0.05, two-way ANOVA; Fig. 2). Up-regulation of IL-1-dependent eotaxin or RANTES release was not found with culture on either type III or V collagen (p > 0.05, n = 5; data not shown), although in the same samples there was a concentration-dependent decrease (52 ± 3% at 10 µg/ml) in GM-CSF with type V collagen and an increase in GM-CSF release (195 ± 13% at 10 µg/ml) with type III collagen compared with IL-1-stimulated cells on Pl (p < 0.05–0.01, n = 5–6; data not shown). Additionally, fibronectin and type I collagen (10 µg/ml) each potentiated GM-CSF release by 156 ± 5% and 137 ± 7%, respectively (p < 0.05–0.01 compared with Pl) when cells were costimulated with maximally effective concentrations of IL-1 (1 ng/ml) and TNF- (10 ng/ml). In all experiments, release of eotaxin, RANTES, or GM-CSF from cells cultured on these ECM substrates (1 or 10 µg/ml) in the absence of IL-1/TNF- did not differ from release by unstimulated cells on Pl (p > 0.05, n = 6–7 for each ECM; Figs. 1 and 2).
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To examine whether up-regulation of IL-1-dependent secretory responses by ECM substrates involved increased gene activation, cytokine mRNA levels were examined in human ASM cells cultured on fibronectin. Consistent with previous reports, treatment of cells growing on Pl with IL-1 (1 ng/ml) for 16 h increased the mRNA abundance for eotaxin, RANTES, and GM-CSF (p < 0.05–0.01; Fig. 3) (10, 11). A further increase in eotaxin and GM-CSF mRNA occurred in IL-1-stimulated cells after culture on 10 µg/ml fibronectin (p < 0.05; Fig. 3). An apparent increase in IL-1-dependent RANTES mRNA was also found, but this failed to reach significance. No change in 18S rRNA between the treatments was found (Fig. 3). In two separate experiments, a similar enhancement of IL-1-dependent eotaxin, RANTES, and GM-CSF mRNA abundance at 16 h was found after culture on fibrillar type I collagen (10 µg/ml), compared with stimulation on Pl (not shown).
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To investigate whether enhancement of cytokine production by ECM substrates occurred in all ASM cells or in a subset, intracellular cytokine levels were examined by flow cytometry in fixed saponin-permeablized cells. In preliminary studies, positive labeling for eotaxin, RANTES, or GM-CSF was detected in <2% of the total population after stimulation with IL-1 for 24 h, which increased to 4–5% after culture on 10 µg/ml fibronectin or fibrillar type I collagen (n = 2). Given these low levels of labeling, cells in subsequent studies were costimulated with maximally effective concentrations of IL-1 (1 ng/ml) and TNF- (10 ng/ml), shown previously in ELISA studies to act synergistically to induce release of GM-CSF and RANTES (10, 13). Optimal numbers of GM-CSF (12.2 ± 3.7%)-, RANTES (19.03 ± 3.72%)-, or eotaxin (11.4 ± 3.41%)-labeled cells occurred with stimulation for 24 h (data not shown). Kinetics for GM-SCF and RANTES accumulation were similar, consistent with our previous ELISA findings in IL-1-stimulated cells (13). In keeping with the current ELISA (Figs. 1 and 2) and mRNA findings (Fig. 3), culture on fibronectin or fibrillar type I collagen further increased the number of IL-1/TNF--stimulated eotaxin-, RANTES-, or GM-CSF-positive cells by 
1.5-fold (p < 0.05–0.01 compared with stimulated cells on Pl; Fig. 4). Likewise, culture on type III collagen increased IL-1/TNF--stimulated GM-CSF (p < 0.05) but not RANTES labeling, which overall resembled closely the ELISA findings with collagen type III (described above).
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Having defined a probable role for ECM substrates in the regulation of cytokine production from human ASM cells, subsequent experiments examined possible integrin receptors that could mediate this response. Flow cytometry of cells on Pl confirmed that 5 and 1 integrin subunits were universally expressed and that 50–60% of cells had detectable surface 1, 2, 3, and v integrin subunits and 50% expressed the v3 heterodimer (Fig. 5A). To exclude possible underestimation of the number of labeled cells due to cleavage of Ab recognition sites by the trypsin used to remove the cells from the culture flasks, we compared this profile of integrin subunit expression in cells harvested using PBS-containing EDTA (0.5 mM for 20 min) and found no differences, which was further supported by examination of cultured human ASM cells in situ by reflected UV light immunofluorescence microscopy (T. T.-B. Nguyen and S. J. Hirst, unpublished observations).
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/TNF--dependent intracellular eotaxin and RANTES labeling was reduced by 1 µg/ml 1 integrin subunit-blocking mAb (p < 0.05, Table I). Likewise, in ELISA studies, blocking the binding of matrix factors to 2 or 1 integrin subunits abolished enhancement of IL-1-dependent eotaxin release by either fibronectin or fibrillar type I collagen (p < 0.05 compared with control IgG1, Fig. 5, B and C). Function-blocking mAbs (1 µg/ml) to 5 or v subunits had a similar effect, but only against enhancement by fibronectin (p < 0.05 compared with control IgG1). Abs against 3 or 3 subunits were without effect (p > 0.05 compared with control IgG1). However, the v3 heterodimer-specific blocking mAb (1 µg/ml) abolished enhancement of eotaxin release by fibronectin (p < 0.05 compared with control IgG1). A similar profile of neutralization was obtained when IL-1-dependent RANTES release by either fibronectin or type I collagen was examined in supernatants in place of eotaxin (data not shown, n = 4).
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-dependent eotaxin release by fibronectin (p < 0.05, Fig. 5D). This result is in keeping with a role for 51, v1, and v3 integrin-mediated secretory signals, though 31, 81, IIb3, v5, and v6 are also known to be RGD sensitive (23). |
Discussion |
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-dependent cytokine secretory signal. ELISA studies indicated that enhancement by fibronectin was mediated through multiple 1 integrins expressed by ASM including 21, 51, and v1, but also by v3, as function-blocking mAbs directed against these integrin subunits or to the v3 heterodimer prevented enhancement of IL-1-dependent eotaxin release by fibronectin, as did blocking a fibronectin recognition site with soluble RGD peptides. Similarly, enhancement by fibrillar collagen type I appeared to involve the 21 integrin. Furthermore, flow cytometric analysis indicated these events may involve a minority of cultured ASM cells because intracellular cytokine expression elicited by IL-1/TNF- and its potentiation by the ECM could only be detected in <25% of the total population; even under conditions considered to induce maximal cytokine release when measured by ELISA (10, 13).
Although the involvement of specific ECM components in up-regulation of airway and vascular smooth muscle responses such as attachment, proliferation, migration, and survival is well established (17, 18, 24, 25), up-regulation of cytokine secretory capacity by fibronectin or collagen ECM substrates to our knowledge has not previously been reported in mesenchymal cells from the lung or elsewhere. In ELISA studies, we found that overnight culture of human ASM cells in plates precoated with fibronectin, fibrillar, or monomeric denatured type I collagen increased IL-1-dependent cytokine release compared with similarly stimulated cells cultured on Pl. Collagen types III and V had no effect on IL-1-dependent eotaxin or RANTES release, but differentially affected GM-CSF release. Thus, different ECM proteins could elaborate varying patterns of cytokine secretion compared with Pl, suggesting these differences when present did not result from a nonspecific protein interaction, rather they reflected a specific response to the ECM. Enhancement by both fibrillar and monomeric type I collagen suggests both the native helical configuration (fibrillar) and proteolytic denatured (monomeric) forms of type I collagen, occurring after cleavage of fibrillar collagen in inflammation by matrix metalloproteinases (26), are sufficient to elicit secretory signals in ASM. Moreover, the addition of soluble forms of fibronectin or type I collagen directly to the cell cultures does not result in potentiation of IL-1-dependent eotaxin or RANTES release, suggesting enhancement of cytokine release requires these ECM substrates to be in the polymerized form present after coating (Q. Peng and J. S. Hirst, unpublished observations).
The extent of enhancement of cytokine release was consistent across each of the substrates examined with GM-CSF being the most increased (200%) followed by eotaxin (150%) and RANTES being the least susceptible (130%). In keeping with this enhancement of eosinophil-activating cytokine protein release by ECM substrates, analogous increases in these cytokines were found with techniques examining intracellular cytokine expression (flow cytometry) and mRNA abundance (RT-PCR), though the increase for RANTES mRNA failed significance, which may reflect either insufficient powering or saturation of the PCR. Enhancement of cytokine mRNA levels by fibronectin or type I collagen implies the increase in eotaxin, RANTES, and GM-CSF protein detected by the ELISA and flow cytometry studies involves increased cytokine gene transcription or stabilization of mRNA transcripts. Although not examined in the present study, it was recently shown that GM-CSF release from TNF--stimulated eosinophils in the presence of fibronectin involved mainly GM-CSF mRNA stabilization, which was dependent on ERK phosphorylation and the RNA binding proteins, YB-1 and HuR (27). Elsewhere, evidence suggests that growth factors can bind ECM components to form complexes that enhance subsequent growth factor activity through integrin collaboration with growth factor receptors. In the case of VEGF interacting with fibronectin to enhance endothelial cell migration, the amplified response was attributed to sustained ERK activation requiring activation of both the Flk-1 (VEGF receptor) and 51 (fibronectin receptor) (28). Whether similar mechanisms operate in human ASM cells remains an open question.
This study is among the first with ASM cells to demonstrate intracellular cytokine expression by flow cytometric techniques. The synthesis and release of proinflammatory mediators by ASM is a relatively new finding (29) and has almost without exception been estimated by ELISA- or RT-PCR-based methods in experiments that have not been designed to distinguish between signals generated from a subset of cells rather than the total population. Although the single labeling flow cytometry method used here demonstrated that culture on fibronectin or type I collagen potentiated intracellular cytokine expression (also type III collagen in the case of GM-CSF elaboration) in human ASM cells stimulated for 24 h with maximally effective concentrations of IL-1 and TNF- in combination, overall numbers of positive-labeled cells generally did not exceed 25% of the total population indicating that only a subpopulation of human ASM cells could be induced to express eosinophil-activating cytokines under the current experimental conditions. It is possible that further increases in the frequency of positive cells may have been detected at time points beyond 24 h, though inclusion of the protein transport inhibitor brefeldin A either reduced labeling or had no effect and was therefore omitted (D. Lai and S. J. Hirst, unpublished observations). Technical difficulties associated with the high autofluorescence of human cultured ASM cells (Fig. 4A) precluded use of double- or triple-labeling strategies to examine whether those cells expressing eotaxin also expressed RANTES or GM-CSF, and so it remains possible that distinct multiple populations of cells exist with each capable of expressing one or more cytokine. Likewise, the current study did not ascertain whether culture on various ECM substrates increased expression levels in a single subpopulation of cells already expressing or recruited additional cells. Nevertheless, the data raise an important question concerning the possible heterogeneity of ASM cell cytokine expression, which has not previously been addressed.
Integrins, which comprise heterodimers are the principal receptors mediating multiple cell responses to ECM substrates (30). Their extracellular domains recognize short peptide sequences (e.g., Arg-Gly-Asp (RGD) found on some ECM proteins (fibronectin and vitronectin)), while the intracellular domains are involved in the formation of focal adhesion complexes and downstream signaling events. Our data provide new information that 50% of human ASM cells express the promiscuous vitronectin receptor, v3, and 65–70% of cells express 2 integrin required for type I collagen binding. These data confirm an earlier report showing similar expression patterns of multiple 1 integrin family subunits including 1, 3, and v (19). In keeping with this earlier report, 5 and 1 subunits were found to be universally expressed. ELISA studies with integrin function-blocking mAbs suggest that enhancement of IL-1-stimulated eotaxin release by fibronectin involved multiple integrins including 21, 51, v1, and v3; while enhancement on type I collagen involved 21. The findings with fibronectin support the consensus that a single ECM substrate may ligate several integrin heterodimers. For example, 31, 51, v1, and v3, which are expressed on ASM cells are all known to bind fibronectin with varying affinities (31). Furthermore, the finding that in each case blocking Abs abolished enhancement of cytokine release by fibronectin or type I collagen, irrespective whether the target integrins were universally (5 and 1) or partially expressed (2 and v), supports our hypothesis from the intracellular cytokine flow cytometry findings that the enhancement of eotaxin by fibronectin or type I collagen in the ELISA study likely involved only a subpopulation of human ASM cells that also express 2 and v integrin subunits.
Block of fibronectin-induced potentiation of IL-1-dependent eotaxin release, as well as that by type I collagen, by 2 subunit neutralization was an unexpected finding as 21 is the major type I collagen receptor. The response to fibronectin may depend on autocrine type I collagen production, possibly via an IL-1-dependent GM-CSF-mediated amplification loop, and GM-CSF has recently been shown to induce the production of type I collagen by ASM cells (32). Alternatively, collagen-binding regions are present in purified fibronectin (33) and other recent data in a cell-free system has revealed that reconstituted 21 heterodimers ligate both type I collagen and fibronectin (34).
In conclusion, we have demonstrated that interstitial ECM proteins such as fibronectin and type I collagen, among the most widely expressed proteins in the lung, enhance release of IL-1-stimulated eotaxin, RANTES, or GM-CSF from human ASM cells. The underlying mechanism involves increased cytokine mRNA levels and appears to require multiple 1 integrins as well as v3 in the case of fibronectin. Although the relevance of these events has not been demonstrated in vivo, their significance is unlikely to be restricted to a culture environment. The fact that fibronectin and collagen are increased in asthmatic airways (5, 6, 7, 8, 9) adds weight to the hypothesis that such changes in the ECM environment surrounding ASM cell may favor not only enhanced survival (19) and growth (17, 18) responses, but also enhanced secretion during inflammation and remodeling.
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Acknowledgments |
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Footnotes |
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1 This study was supported by Asthma U.K. (no. 00/44), the Special Trustees of Guy’s and St. Thomas’ Hospitals, and the Wellcome Trust (no. 051435). 
2 Q.P. and D.L. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Stuart J. Hirst, Department of Asthma, Allergy and Respiratory Science, The Guy’s, King’s and St. Thomas’ School of Medicine, Thomas Guy House, Guy’s Hospital Campus, London, U.K. SE1 9RT. E-mail address: stuart.hirst{at}kcl.ac.uk
4 Abbreviations used in this paper: ASM, airway smooth muscle; ECM, extracellular matrix; Pl, plastic.
Received for publication August 18, 2004. Accepted for publication November 16, 2004.
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1-integrin-ligand interactions. Anal. Biochem. 306:305.[Medline]This article has been cited by other articles:
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) or combined presence of 1 ng/ml IL-1
). *, p < 0.05 compared with stimulated cells on Pl. 
, p < 0.05; 