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Published ahead of print on April 12, 2007, doi:10.1165/rcmb.2006-0172OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 240-247, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0172OC

Modulation of Human Airway Smooth Muscle Migration by Lipid Mediators and Th-2 Cytokines

Krishnan Parameswaran, Katherine Radford, Adrian Fanat, Jancy Stephen, Caroline Bonnans, Bruce D. Levy, Luke J. Janssen and P. Gerard Cox

Firestone Institute for Respiratory Health, St. Joseph's Healthcare and Department of Medicine, McMaster University, Hamilton, Ontario, Canada; and Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts

Correspondence and requests for reprints should be addressed to Dr. K. Parameswaran, Firestone Institute for Respiratory Health, St. Joseph's Healthcare, 50 Charlton Avenue East, Hamilton, ON, L8N 4A6 Canada. E-mail: parames{at}mcmaster.ca


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Cysteinyl leukotrienes and the T helper (Th)-2 cytokines IL-5 and IL-13 directly modulate human airway smooth muscle functions such as contraction and proliferation. We studied the effects of other lipid mediators involved in asthma pathophysiology such as prostaglandin D2 (PGD2), lipoxin, and isoprostanes, and the cytokines, IL-5, IL-4, and IL-13 on human airway smooth muscle cell migration. Chemotaxis and chemokinesis of cultured airway smooth muscle cells from humans without asthma (second to fifth passages, n = 6) were studied using collagen-I–coated polycarbonate membranes in Transwell culture plates. Receptor expression and kinase activation were studied by flow cytometry, polymerase chain reaction, and Western blotting techniques. In contrast to LTE4- stimulated (10–6 M) chemokinesis and LTE4-primed migration toward platelet-derived growth factor (PDGF), isoprostane 15-F2t-IsoP, and IL-5 were neither chemotactic nor chemokinetic. PGD2 (10–10–10–6 M) was a chemoattractant and primed migration toward PDGF through the DP2/CRTh2 receptor. Although airway smooth muscle cells did not express the lipoxin A4 cognate receptor, LTE4-primed migration toward PDGF was blocked by lipoxin A4 (10–6 M), suggesting that this is mediated through CysLT1R antagonism. IL-13 (10 ng/ml), but not IL-4 (0.1–100 ng/ml), augmented migration toward PDGF. This was associated with increased Src-kinase phosphorylation and up-regulation of PDGF-{alpha} and -beta receptors, and was attenuated by IL-13R{alpha}– and IL-4R{alpha}–neutralizing antibodies, an Src-kinase antagonist (PP1, 3 µM), a CysLT1R antagonist, montelukast (10–6 M), and by lipoxin A4 (10–6 M). PGD2 and IL-13 promote human airway smooth muscle migration. IL-13 can promote airway smooth muscle migration through Src-kinase and leukotriene-dependent pathways. This may contribute to the accumulation of smooth muscle cells in remodeled airway submucosa.

Key Words: airway smooth muscle migration • IL-13 • PGD2 • cysteinyl leukotriene • CRTh2 receptor



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We demonstrate novel biological effects of prostaglandin D2 and IL-13 on human airway smooth muscle cells. Both of these can promote human airway smooth muscle migration, and this may contribute to the accumulation of smooth muscle cells in remodeled airway submucosa.

 
Airway smooth muscle, in addition to causing bronchoconstriction, is a major component of the remodeled airway in patients with long-standing asthma (1). In addition to the increased smooth muscle mass, shorter smooth muscle–to-epithelium distances are selectively seen in airway tissue of subjects with severe asthma, and this correlates with the degree of airflow obstruction (2). One mechanism of airway smooth muscle accumulation in the subepithelial region may be by migration of myocytes from the deeper layer, a process analogous to the remodeling observed in atheromatous blood vessels. Although there is no in vivo evidence to support this notion, airway smooth muscle cells from patients with asthma show more migratory capabilities compared with cells from normal subjects (3).

Products of arachidonic acid metabolism such as prostaglandins, leukotrienes, lipoxins, and isoprostanes have important biological effects in asthma. We have demonstrated that cysteinyl leukotrienes augment the chemotaxis of airway smooth muscle cells toward platelet-derived growth factor (PGDF), and that this process is attenuated by prostaglandin E2 (4). Very little is known about the effect of the other prostanoids on airway smooth muscle migration. Mast cells, which are a rich source of prostaglandin D2 (PGD2) and leukotrienes, are often found in close proximity to smooth muscle bundles (5). It is possible that PGD2 may promote airway smooth muscle migration toward the subepithelial basement membrane. Lipoxins, which are endogenous products of the 12- and 15-lipoxygenase pathways, are "braking" signals for the proinflammatory actions of cysteinyl leukotrienes (6), and are capable of inhibiting endothelial and inflammatory cell migration (7, 8). Isoprostanes, produced by nonenzymatic peroxidation of prostaglandins by free radicals and reactive oxygen species, are potent constrictors of airway and vascular smooth muscle cells via the Rho and Rho-kinase pathways (9). Since these enzymes are critically involved in the organization of smooth muscle cytoskeleton, it is likely that their regulation could modulate cell movement.

T helper (Th)-2 cytokines such as IL-5 and IL-13 have direct contractile and regulatory effects on human airway smooth muscle. IL-5 has been reported to increase the contractile response of human bronchial rings to acetylcholine (10). IL-13 has been reported to reduce isoproterenol-induced relaxation in cultured human airway smooth muscle cells (11), augment allergen-induced airway hyperresponsiveness in mice (12), and enhance carbachol and potassium chloride–induced contraction and calcium signals in murine tracheal rings (13). Further, it is speculated that cysteinyl leukotrienes can modulate some effects of IL-13 on human airway smooth muscle, particularly on its synthetic functions (14). It is not known whether these cytokines have any direct effects on human airway smooth muscle migration.

In this study, we examined the effects of PGD2, lipoxin, an isoprostane 15-F2t-IsoP, and the cytokines IL-4, IL-5, and IL-13 on human airway smooth muscle migration. We specifically studied their effects on human airway smooth muscle cell chemokinesis and chemotaxis. Some of the results of these studies have been previously reported in the form of abstracts (15, 16).


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Human Airway Smooth Muscle and Fibroblast Culture
Portions of human lungs that were resected at St. Joseph's Healthcare (Hamilton, ON, Canada) were obtained after obtaining informed consent from the patients (Table 1) and approval from the hospital Research Ethics Board. Smooth muscle tissue was isolated from macroscopically disease-free areas of human bronchi. Fibroblasts were isolated from portions of lung parenchyma. The cells were grown to confluence, as described before (4). The smooth muscle cells were passaged between 2 and 5 times and used for the migration assay. Smooth muscle cells were distinguished from fibroblasts based on light and electron microscopy, caveolae, and gap junction.


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  		TABLE 1. SUBJECT CHARACTERISTICS

 
Migration Assay
Migration experiments were performed using a 6.5-mm Transwell culture plate with a 8.0-µM pore, collagen-I–coated, polycarbonate membrane separating the inner and the outer chambers (Fisher Scientific Limited, Nepean, ON, Canada) as previously described (4). Assays were done in duplicate using tissues from up to six different lung specimens. Additional detail on the method for making these measurements is provided in the online supplement.

Migration Experiments
Chemotaxis was studied by adding the chemoattractant only to the outer well. Chemokinesis was studied by adding the chemoattractant to both the inner and outer wells. The chemoattractants studied were PDGF-BB (1 ng/ml; Invitrogen Canada Inc., Burlington, ON, Canada), PGD2 (10–9–10–7 M), isoprostane 15-F2t-IsoP (10–9–10–7 M) (both from Cayman Chemical, Ann Arbor, MI), lipoxin A4 (10–9–10–7 M) (Calbiochem-EMD Biosciences Inc, San Diego, CA), IL-4 (0.1–100 ng/ml), IL-5 (0.1–10 ng/ml), and IL-13 (0.1–10 ng/ml) (all from Pepro Tech Inc., Rocky Hill, NJ). The priming effect of the chemoattractants was studied by treating smooth muscle cells with the chemoattractants for 30 min before setting up the migration experiments toward PDGF. The effects of cysteinyl leukotrienes and lipoxin A4 on migration toward IL-13 were studied by treating the smooth muscle cells with montelukast (10–6 M; kind gift from MerckFrosst, Kirkland, PQ, Canada) and with lipoxin A4 (10–6 M) for 30 min before the migration experiments. The signaling mechanism of chemotaxis toward PGD2 was studied by using antagonists against thromboxane receptor (GR32191B), DP1 receptor (BWA868C), and DP2/CRTh2/thromboxane receptor (BAYu3405) (all 10–10–10–6 M; Cayman Chemical). The mechanisms of migration toward IL-13 were studied by treating the smooth muscle cells with IL-13R{alpha}– and IL-4R{alpha}–neutralizing antibodies (1 µM; R&D Systems, Minneapolis, MN) and with an Src-kinase antagonist PP1 (3 µM; Calbiochem-EMD Biosciences) for 30 min before the experiments.

Receptor Expression
Expression of lipoxin A4 receptor (ALX) and cysteinyl leukotriene receptor-1 (CysLT1R) gene expression on human airway smooth muscle cells were examined by reverse transcriptase polymerase chain reaction using previously described methods (17). CysLT1R expression was also examined by flow cytometry using a polyclonal anti-CysLT1R antibody directed against the carboxyl-terminal portion of the receptor as previously described (18). The measurements were made after incubating the cells overnight with varying concentrations of IL-13. PDGF receptor {alpha} and beta expression on airway smooth muscle cells were examined by Western blot analysis. Serum-starved smooth muscle cells were treated with IL-13 for 30 min and for 5 h, and protein was extracted, resolved by SDS-PAGE, transferred to nitrocellulose filters, and probed with monoclonal antibodies that recognized PDGFR-{alpha} and beta (Santa Cruz Biotechnology Inc, Santa Cruz, CA). CRTh2/DP2 receptor expression on airway smooth muscle cells was examined by Western blot analysis using a polyclonal antibody directed against the N-terminal of the CRTh2/DP2 receptor (Cayman Chemical). This was also confirmed by Western blot using a rat monoclonal antibody against human CRTh2 receptor (clone BM16, catalog number 558412; BD Biosciences, Mississauga, ON, Canada). Peripheral blood mononuclear cells were isolated by percoll gradient separation (19) from normal healthy donors and used as positive control. Additional details on the method for making these measurements is provided in the online supplement.

Kinase Activation
Airway smooth muscle cells, starved for 24 h in serum-free medium, were treated with various concentrations of IL-13. After 30 s, total and activated Src kinase expression was examined by Western blot analysis as previously described (20). Additional detail on the method for making these measurements is provided in the online supplement.

Cysteinyl Leukotriene Synthesis
Cysteinyl leukotriene synthesis by human airway smooth muscle cells was assessed by measuring their levels by a sensitive enzyme immunoassay (limit of detection 26.6 pg/ml; Assay Designs, Ann Arbor, MI) in the supernatant of growth-arrested muscle cells treated overnight with IL-13 (10 ng/ml).

Analysis
Statistical analysis was performed using the repeated-measures ANOVA test using the various experimental conditions as within-subject factors. Sources of significant variations were identified by predefined contrasts. P < 0.05 was considered statistically significant. All analyses were performed using SPSS version 13.0 (Statistical Software for Social Sciences, Chicago, IL).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effects of PGD2, Lipoxin, and Isoprostane 15-F2t-IsoP
PGD2 was a chemoattractant for human airway smooth muscle cells. At the highest concentration (10–7 M), a 1.5-fold increase in the number of migrating cells was observed over control condition (P < 0.05). PGD2 did not have any chemokinetic effect when it was added to both sides of the polycarbonate membrane. The chemotactic effect was inhibited in a concentration-dependent manner by the DP2/CRTh2 receptor antagonist (BAYu3405), but not by the DP1 (BWA868C) and thromboxane (GR32191B) receptor antagonists (Figure 1A). Consistent with this, we observed that human airway smooth muscle cells express the CRTh2/DP2 receptor, suggesting that the chemotactic effect of PGD2 is mediated through the DP2 receptor (Figure 1B).


Figure 1
Figure 1
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  		Figure 1. (A) PGD2 was a chemoattractant for human airway smooth muscle cells. At the highest concentration, a 1.5-fold increase in the number of migrating cells was observed over control condition. This was inhibited in a concentration-dependent manner by the DP2/CRTh2 receptor antagonist (BAYu3405), but not by the DP1 (BWA868C) and thromboxane (GR32191B) receptor antagonists. The doses shown in the figure are 10–6 M. Data are shown as mean and SD of five experiments (*P < 0.05 compared with control, {dagger}P < 0.05 compared with PGD2). (B) CRTh2/DP2 receptor expression on human airway smooth muscle cells by Western blotting using a polyclonal antibody (Cayman Chemicals) (left panel). Peripheral blood mononuclear cells (PBMNC) were used as positive control. Human CRTh2 has three glycosylation sites. The mean and SD of the optical density units of the Western blots from five experiments are shown (*P < 0.05 compared with negative control). The panel on the right shows Western blots on smooth muscle cells from three additional subjects using a monoclonal antibody (clone BM16; BD Biosciences), confirming the expression of the 46-kD protein.

 
Lipoxin was also neither chemotactic nor chemokinetic for human ASM. However, it attenuated LTE4-primed migration toward PDGF (Figure 2A). Human airway smooth muscle cells do not express ALX (Figure 2B), and since the attenuating effect was similar to that found with a CysLT1 receptor antagonist, it is likely this effect is mediated through the CysLT1 receptor that is expressed on human airway smooth muscle cells.


Figure 2
Figure 2
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  		Figure 2. (A) Lipoxin A4 was not chemotactic and did not have any effect on PDGF-induced migration. However, it attenuated LTE4-primed migration toward PDGF. This effect was comparable to the effect of montelukast in attenuating LTE4-primed migration toward PDGF. Data are shown as mean and standard deviations of six experiments. *P < 0.05 compared with control, {dagger}P < 0.05 compared with untreated cells migrating toward PDGF, {ddagger}P < 0.05 compared with LTE4-primed migration toward PDGF. (B) PCR and immunoblotting demonstrates CysLT1 receptor mRNA expression, but not lipoxin A4 receptor (ALX) expression, on human airway smooth muscle cells (hASM). cDNA from human peripheral blood is used a positive control.

 
Isoprostane 15-F2t-IsoP was neither chemotactic nor chemokinetic at the concentrations that were studied (see Figure E1 in the online supplement).

Effects of IL-5 and IL-13
IL-4, IL-5. IL-4 and IL-5 were neither chemotactic nor chemokinetic, and they did not prime migration toward PDGF at any of the doses that were studied (Figures E2 and E3). IL-13 was also not chemotactic or chemokinetic; however, it primed migration toward PDGF at the highest dose that was studied (P < 0.05) (Figure 3A).


Figure 3
Figure 3
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  		Figure 3. (A) IL-13 was not a chemoattractant. However, smooth muscle cells treated with 10 ng/ml of IL-13 showed augmented migration toward PDGF. Data are shown as mean and SD of five experiments. *P < 0.05 compared with control, {dagger}P < 0.05 compared with migration toward PDGF. (B) IL-13–primed migration toward PDGF was attenuated by an IL-13 receptor neutralizing antibody, an IL-4 receptor antibody, an Src-kinase inhibitor (PP1), montelukast, and lipoxin A4. Data are shown as mean and SD of six experiments. *P < 0.05 compared with control, {dagger}P < 0.05 compared with migration toward PDGF, {ddagger}P < 0.05 compared with IL-13–primed migration toward PDGF.

 
IL-13 signaling. We further characterized the mechanisms involved in IL-13–primed migration toward PDGF. It was attenuated by both an IL-13–neutralizing antibody and an IL-4R{alpha} antibody, suggesting that the effects of IL-13 are mediated through the common IL-4R{alpha} subunit (Figure 3B). IL-13 up-regulated PDGF-{alpha} and beta receptors (Figure 4A) and activated Src-kinase (Figure 4B). The effect on PDGF-{alpha} receptor was less modest (1.7-fold at 30 min and 0.7-fold at 5 h) compared with the PDGF-beta receptor (3.2-fold at 30 min and 3.4-fold at 5 h over control). However, IL-4 did not increase Src-kinase phosphorylation (Figure 4B). IL-13–primed migration toward PDGF was also attenuated by an Src-kinase inhibitor (PP1) (Figure 3B). These experiments suggest that up-regulation of PGDF receptor and activation of Src-kinase may be two mechanisms by which IL-13 facilitates human airway smooth muscle migration. Finally, IL-13–primed migration was attenuated by a CysLT1 receptor antagonist (montelukast) and by lipoxin A4 (Figure 3B), suggesting that cysteinyl-leukotrienes are involved in this process. Although IL-13 did not increase CysLT secretion from human airway smooth muscles (Figure E4), there was a modest increase in CysLT1R expression when airway smooth muscle cells were treated with IL-13 (Figure E5).


Figure 4
Figure 4
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  		Figure 4. (A) IL-13 up-regulated PDGF-beta receptors on airway smooth muscle cells. The bar graphs show mean and SD of the optical density arbitrary units at 30 min and at 5 h from four experiments. *P < 0.05 compared with control. (B) Western blotting showing both PDGF and IL-13, but not IL-4, increased Src-kinase phosphorylation. IL-13–induced Src-kinase phosphorylation was attenuated by an IL-13 receptor neutralizing antibody. The bar graphs show mean and SD of the optical density arbitrary units from four experiments. *P < 0.05 compared with control, {dagger}P < 0.05 compared with effect of IL-13.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We report a number of novel observations. First, PGD2 is a chemoattractant for human airway smooth muscle cells acting through the CRTh2/DP2 receptor. Second, IL-13 facilitates human airway smooth muscle migration through an Src-kinase–dependent pathway. Third, leukotriene and IL-13–primed human airway smooth muscle migration is attenuated by lipoxin A4, demonstrating a novel aspect of lipoxin biology. Finally, we demonstrate that IL-5 and isoprostane 15-F2t-IsoP are not chemoattractants for airway smooth muscle cells. Human airway smooth muscle chemotaxis promoted by PGD2 and by IL-13 may contribute to the accumulation of smooth muscle in the submucosa and to the remodeling process in patients with asthma.

PGD2 has long been known to be a potent bronchoconstrictor. This is believed to be mediated through the thromboxane receptor (21) because the classic DP1 receptor activation increases intracellular cAMP, which leads to airway smooth muscle relaxation. The proximity of smooth muscle cells to mast cells in the submucosa of patients with asthma led us to examine whether PGD2, which is a mast cell product, could be a chemoattractant for smooth muscle cells. Unlike cysteinyl leukotrienes that are not chemoattractants, we observed that PGD2 was a weak chemoattractant for airway smooth muscle cells. This is unlikely through the DP1 receptor because its activation increases intracellular cAMP, which would inhibit migration (22). A DP1 receptor antagonist, as expected, did not have any effect on smooth muscle chemotaxis. The effect was not mediated through the thromboxane receptor either, because it was unaffected by the thromoboxane inhibitor, GR32191B. Since cells that express the newly described DP2/CRTh2 receptor (23) show chemotaxis toward PGD2 (24), we examined and observed DP2/CRTh2 receptor expression on human airway smooth muscle cells. Chemotaxis was attenuated in a concentration-dependent manner by BAYu3405. We therefore conclude that PGD2 promotes human airway smooth muscle chemotaxis through this novel receptor. We did not examine the exact mechanism, but speculate that it is by coupling through G{alpha}i proteins. This needs further investigation. The effect of PGD2 on airway smooth muscle chemotaxis that we observed was opposite to those described by Kohyama and colleagues (25), who reported that PGD2 inhibited migration of human fibroblasts toward fibronectin. It is possible that this due to differences in expression of DP1 and DP2/CRTh2 receptors on the two cell types and activation in response to various chemoattractant stimuli. Mast cell products such as PGD2 and CC chemokine ligand 19 (26) may thus contribute to the smooth muscle hyperplasia in chronic asthma.

The second novel observation was the ability of IL-13 to prime migration of smooth muscle cells toward PDGF. This was only observed at the highest concentration of IL-13 that was studied. This could be through a number of possible mechanisms. IL-13 signals through the IL-13 receptor that has two components: an IL-13R{alpha}1 and IL-13R{alpha}2 chain. IL-4 signals through the IL-4 receptor, which has two dimers: an IL-4R{alpha} and IL-4R{gamma}c. IL-13R{alpha}1 binds IL-13 with low affinity, but a heterodimer of IL-13R{alpha}1 and IL-4R{alpha} acts as a high-affinity functional receptor for both human IL-13 and IL-4. IL-13R{alpha}1 does not form dimers with {gamma}c, and IL-13R{alpha}2 is not known to signal (27). Human airway smooth muscle cells are reported to express both IL-13 and IL-4 receptors (28). Since IL-13–primed migration was inhibited by both IL-13 and IL-4 receptor neutralizing antibodies, it appears to be mediated through the IL-13R{alpha}1/ IL-4R{alpha} heterodimer. Therefore it is not clear why IL-4, which signals through the same receptor complex, did not have the same chemotactic effect on airway smooth muscle cells. Recently, there is some evidence that IL-13 might have effects that are independent of IL-4R{alpha} (29). This mechanism is unlikely to be the explanation for our observation, because IL-13–primed migration was inhibited by the IL-4 receptor antibody.

Since Src-kinase activity is critical for chemotaxis (20, 30), and IL-13 has been reported to activate Lsk, a protein tyrosine kinase with homology to the COOH-terminal Src kinase (31), we examined and demonstrated that treatment of airway smooth muscle cells with IL-13, but not IL-4, increased the levels of phosphorylated Src-kinase. Further evidence for the involvement of Src-kinase was provided by the inhibition of IL-13–primed migration by an Src-kinase inhibitor. Although IL-4 has been reported to activate Src in murine cell lymphoma cell lines (32), this has not been reported in human airway smooth muscle cells. This might be one of the reasons for the lack of chemotactic effect of IL-4 on airway smooth muscle cells. Further experiments are necessary to clarify the signaling pathways. IL-5 also did not have any effect on human airway smooth muscle chemotaxis. In animal models of allergen sensitization, IL-4 and IL-13, but not IL-5, are critical for the development of sustained airway hyperreactivity and airway remodeling after allergen exposure (33). It is thus possible that IL-5, while necessary for the recruitment and survival of airway eosinophils, may not be necessary for the accumulation of smooth muscles in the submucosa.

Next, we examined another potential mechanism by which IL-13 could augment migration toward PDGF. IL-13, via a STAT-6–dependent mechanism, is reported to up-regulate PDGF-AA and PDGF receptor-{alpha} proteins in human fibroblasts (34). In keeping with this, we observed that treatment of smooth muscle cells with concentrations of IL-13 that primed migration toward PDGF, increased PDGF receptor {alpha} and beta expression. There was greater up-regulation of the beta than the {alpha} receptor at both 30 min and 5 h. PDGF-BB activates both {alpha}- and beta-receptor subunits, and promotes cell migration in many cell types, including fibroblasts and smooth muscle cells. PDGF-AA activates {alpha}-receptor subunit only, and its role for cell migration is still debatable (35). Activation of the {alpha}-subunit has been reported to antagonize beta-subunit–stimulated smooth muscle cell migration (36). Human airway smooth muscle cells in culture express 5–6 times for PDGF receptor beta-subunits compared with the {alpha}-subunits (37). It is possible that greater expression of the beta-receptor than the {alpha}-receptor by IL-13 may contribute to the increased chemotaxis toward PDGF-BB. However, we cannot be certain about the kinetics of receptor expression because we examined only total protein and not the phosphorylated receptor.

Cysteinyl leukotrienes augment migration of airway smooth muscle cells toward PDGF (4). Since they are reported to mediate some of the effects of IL-13 in murine models of allergen-induced hyperresponsiveness and remodeling (14, 38), we examined their involvement in IL-13–primed smooth muscle migration toward PDGF. Montelukast, a CysLT1 receptor antagonist, attenuated IL-13–primed migration toward PDGF, suggesting a role for cysteinyl leukotrienes in this process. It is unlikely to be due to synthesis of leukotrienes because we were unable to demonstrate increased levels in smooth muscle cells treated with IL-13. However, consistent with a previous report of CysLT1R upregulation by IL-13 (18), we observed a modest up-regulation of CysLT1R expression by IL-13. CysLT1R has also been reported to exhibit inverse agonist signaling that can be inhibited by montelukast (39). It is likely that this is one of the mechanisms by which montelukast attenuated IL-13–primed migration toward PDGF. Since lipoxins are natural braking signals for the proinflammatory effects of cysteinyl leukotrienes (40) and can attenuate allergen-induced airway inflammation including IL-13–mediated effects (41), we also examined the effects of lipoxin A4 in this process. Although lipoxins by themselves had no effect on chemotaxis toward PDGF, they attenuated both leukotriene-primed and IL-13–primed chemotaxis toward PDGF. Since human airway smooth muscle cells did not express ALX, we assume that lipoxin A4 competed with cysteinyl leukotrienes at CysLT1 receptors for inhibition. This needs further investigation.

Thus IL-13 may have augmented PDGF-induced chemotaxis by a combination of the three mechanisms described above (i.e., increasing Src-kinase phosphorylation, increasing PDGF receptor expression, and increasing CysLT1 receptor expression). It is unlikely to be by any one single mechanism, as the augmentation was observed only at the highest dose of IL-13 that was studied and the effects on the individual mechanisms studied were modest.

Finally, since isoprostanes play a fundamental role in actin cytoskeleton rearrangement via the Rho–rho kinase pathway, and since they are reported to have chemotactic activity for inflammatory cells such as the neutrophils (42), we examined the effect of one isoprostane on airway smooth muscle chemotaxis. 15-F2t-IsoP, which acts mainly through the FP and TP receptors at the concentrations that we studied, was neither chemotactic nor chemokinetic. It is likely that TP receptors are not involved in airway smooth muscle migration. As previously described, TP receptor antagonists did not have any effect on migration toward PDGF. Further, a thromboxane A2 analog (U46619) did not have any effect on airway smooth muscle chemotaxis or chemokinesis (data not shown). Although we did not observe 15-F2t-IsoP to be chemoattractant for airway smooth muscle cells, we cannot generalize this to all the other isoprostanes because they have a myriad of structure and have species and tissue specificities (9).

In summary, we demonstrate novel biological effects of PGD2 and IL-13 on human airway smooth muscle cells. PGD2 is a chemoattractant for human airway smooth muscle cells acting through the CRTh2/DP2 receptor. IL-13 can promote airway smooth muscle migration through Src-kinase and leukotriene-dependent pathways that can be regulated by lipoxin A4. This novel mechanism may contribute to the accumulation of smooth muscle cells in remodeled airway submucosa.


   Acknowledgments
 
The authors acknowledge the help of the division of Thoracic Surgery and the department of Pathology of St. Joseph's Healthcare Hamilton in obtaining airways, and Professor Sven-Erik Dahlen, Karolinska Institute, Sweden for helpful discussions regarding leukotriene biology.


   Footnotes
 
K.P. was supported by a Clinician-Scientist Award from the Canadian Institutes of Health Research. This study was funded, in part, by a Firestone Institute-AstraZeneca Research Grant Award.

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: 10.1165/rcmb.2006-0172OC on April 12, 2007

Conflict of Interest Statement: K.P. has received honoraria for lectures, $4,000 (GlaxoSmithKline [GSK], $1,500 from AstraZeneca [AZ], and $2000 from Merck) in the past 2 years, and investigator-initiated unrestricted study grants (GSK $500,000 and Sepracor $160,000). K.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. B.D.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.J.J. has received, as research grants, $186,040 from GSK for 2002–2006, and $250,000 from AZ for 2003–2007. P.G.C. has received speaker's fees from AZ, GSK, Altans, Boehringer-Ingelheim (B-I), and Pfizer of approximately $11,500 in 2005. He has participated in Advisory Board meetings on an ad hoc basis, typically 1 per year for each of B-I and GSK. He carries out clinical research in asthma sponsored by Asthmatx Inc., Mountain View, CA and basic science research with sponsorship from AZ and GSK.

Received in original form May 15, 2006

Accepted in final form February 26, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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