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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 738-742, (2002)
© 2002 American Thoracic Society

Original Article

Cysteinyl Leukotrienes Promote Human Airway Smooth Muscle Migration

Krishnan Parameswaran, Gerard Cox, Katherine Radford, Luke J. Janssen, Roma Sehmi and Paul M. O'Byrne

Asthma Research Group, Firestone Institute for Respiratory Health, St. Joseph's Healthcare, and Department of Medicine, McMaster University, Hamilton, Ontario, Canada

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cysteinyl leukotrienes promote airway smooth muscle (ASM) contraction and proliferation. Little is known about their role in ASM migration. We investigated this using cultured human ASMs (between the second and fifth passages) obtained from the large airways of resected nonasthmatic lung. Platelet-derived growth factor-BB (1 ng/ml) promoted significant (3.5-fold) ASM migration of myocytes across collagen-coated 8-µm polycarbonate membranes in Transwell culture plates. Leukotriene E4 (10-7, 10-8, 10-9 M) did not demonstrate a chemotactic effect; it did promote chemokinesis. Priming by leukotriene E4 (10-7 M) significantly augmented the directional migratory response to platelet-derived growth factor (1.5-fold, p < 0.05). This was blocked by montelukast (10-6 M), demonstrating the effect to be mediated by the cysteinyl leukotriene receptor. The "priming effect" was also partially attenuated by prostaglandin E2 (10-7 M). Whereas both the chemokinetic and the chemotactic "primed" responses were equally attenuated by a p38 mitogen-activated protein kinase inhibitor (SB203580, 25 µM) and by a Rho-kinase inhibitor (Y27632, 10 µM), the chemotactic response showed greater inhibition than chemokinesis by a phosphatidylinositol-3 kinase inhibitor (LY294002, 50 µM). These experiments suggest that cysteinyl leukotrienes play an augmentary role in human ASM migration. The phosphatidylinositol-3 kinase pathway is a key signaling mechanism in the chemotactic migration of ASM cells in response to cysteinyl leukotrienes.

Key Words: cysteinyl leukotrienes • airway smooth muscle migration • chemotaxis • montelukast • phosphatidylinositol-3 kinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The inflammatory process that characterizes the pathophysiology of asthma involves a number of cells and mediators. The cysteinyl leukotrienes (Cys-LTs) C4, D4, and E4 have a number of inflammatory effects relevant to the pathophysiology of asthma (1). Inhalation of leukotriene (LT) E4 causes eosinophil influx into the airway wall (2). In vitro, both LTD4 and LTE4 by themselves are weak chemoattractants for eosinophils, but they augment the chemoattractant effects of other factors such as eotaxin (3). The effects of Cys-LTs on the migrational response of other inflammatory cells, in particular airway smooth muscle (ASM), are less well known. Over the past 10 years, the role of ASM in asthma has been recognized to extend beyond bronchoconstriction (4). They participate in the inflammatory and remodeling processes by producing an array of cytokines, chemokines, and matrix proteins (5). An increase in the size and number of ASMs has been reported in asthma (6). Cys-LTs facilitate this process by promoting ASM proliferation (7). Electron microscopic studies of biopsies obtained during the late response to allergen challenge indicate that muscle cells may dedifferentiate in response to allergen to form motile, contractile cells with a myofibroblast phenotype (8). It is likely that these cells migrate away from the original blocks of deep ASMs to sites close to the disrupted reticular basement membrane, similar to the process of ASM migration and remodeling described in atheromatous plaques and postangioplasty stenosis (9). The migrational property of ASM cells in response to platelet-derived growth factor (PDGF) has recently been demonstrated (10). No information is available on whether Cys-LTs promote ASM migration.

We hypothesized that Cys-LTs are also chemoattractants for ASM and promote the migration of ASM. This effect is likely to be mediated through the G-protein–coupled Cys-LT1 receptor. The objectives of this study were to investigate whether human ASM cells migrate in response to Cys-LTs and to investigate the signaling mechanism of this migratory response.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Roswell Park Memorial Institute (RPMI)-1640 culture medium, fetal calf serum, bovine serum albumin, and PDGF-BB were purchased from Invitrogen Canada Inc. (Burlington, ON, Canada). Vitrogen collagen solution (3.2 mg/ml) was purchased from Cohesion Technologies Inc. (Palo Alto, CA) and was diluted to 1.5 mg/ml with sterile deionized water. LTE4 and prostaglandin E2 (PGE2) were purchased from Cayman Chemicals (Ann Arbor, MI). Inhibitors of p38 mitogen-activated protein (MAP) kinase (SB203580), Rho-kinase (Y27632), and phosphatidylinositol-3 (PI-3) kinase (LY294002) were purchased from Sigma (Oakville, ON, Canada). Diff-Quik Wright Giemsa solution was purchased from VWR International (Mississauga, ON, Canada). Montelukast was a generous gift from Merck Frosst Canada (Kirkland, PQ, Canada).

Smooth Muscle Culture
Portions of human lungs that were resected at St. Joseph's Healthcare (Hamilton, ON, Canada) were obtained with the cooperation of the Division of Thoracic Surgery, after obtaining approval from the institutional review board. Smooth muscle tissue was isolated from macroscopically disease-free areas of human bronchi. ASM cells were grown to confluence, as described before (11), in RPMI medium containing 10% fetal calf serum and penicillin-streptomycin (100 U/ml–100 µg/ml). The confluent cell growth exhibited the typical "hill and valley" appearance under light microscopy and also had caveolae and gap junctions by electron microscopy (data not shown). The cells were passaged between two to five times and were used for the migration assay.

Smooth Muscle Migration Assay
Migration experiments were performed using a 6.5-mm Transwell culture plate with an 8.0-µM pore polycarbonate membrane separating the inner and the outer chambers (Fisher Scientific Limited, Nepean, ON, Canada). Both chambers of the culture plate were treated overnight (at 4°C) with Type 1 collagen solution, which was then aspirated, and the chambers dried for 4 hours in a laminar flow hood. The inserts were then washed thoroughly with sterile deionized water before using them for the experiments. Bovine serum albumin-RPMI medium was added to both chambers 30 minutes before treatments and was aspirated immediately before the experiment. Confluent cells were maintained in growth-factor free medium for 24 hours before the experiments. The cells were then harvested with trypsin (0.05% and 0.53 mM ethylenediaminetetraacetic acid), counted, centrifuged (1,000 r.p.m. for 10 minutes), and resuspended at 8.0 x 105 cell/ml in 0.3% bovine serum albumin-RPMI medium. The cells (vol 100 µl) were then plated on the upper side the membrane. The chemoattractants (vol 600 µl) were added to the lower wells. After 5 hours of incubation at 37°C, the membranes were peeled off, and the cells on the upper face of the membranes were scraped using a cotton swab. Cells that migrated to the lower face of the membrane were fixed with 3.7% formaldehyde and were stained with Diff-Quik. The number of migrated cells on the lower face of the filter was counted in four random fields under x20 magnification (microscope, Olympus BX40; camera, Sony 3CCD Power HAD video camera, Japan; software, Northern Eclipse, Empix Imaging, Mississauga, ON, Canada). Assays were done in duplicate using tissues from six to eight different lung specimens except for the experiment investigating the effect of PGE2, where only four different specimens were used.

Migration Experiments
The migratory response to different doses of PDGF-BB (0.1, 1, and 10 ng/ml) was initially studied by adding PDGF to the lower wells to identify the optimal dose to be used as a positive control. Based on our previous studies, which demonstrated a greater effect of inhaled LTE4 compared with LTD4 on human bronchial mucosal eosinophil infiltration (2), all of the LT experiments were performed with LTE4. Chemokinesis (nondirectional or random migration) to LTE4 (10-7, 10-8, 10-9 M) was studied by adding it to both the upper and lower wells. Chemotaxis (directional migration) was studied by adding the doses only to the lower wells. To study whether LTE4 could augment or "prime" the migratory response to PDGF, the smooth muscle cells were incubated with LTE4 (10-7 M) for 30 minutes before the migration assay. To investigate whether the priming effect of LTE4 was mediated through the Cys-LT receptor, montelukast, a specific Cys-LT receptor antagonist (10-6, 10-8, 10-10 M), was added to the upper well along with the smooth muscle cells and LTE4 during the "priming" experiments. Similarly, the effect of PGE2 on the chemotactic response to LTE4 and PDGF was studied by adding it (10-7 M) to the upper well along with the smooth muscle cells and LTE4. All the chemicals were diluted in 0.3% bovine serum albumin-RPMI buffer. LTE4 and PGE2 were extracted from solutions of methanol using compressed nitrogen gas. The RPMI-bovine serum albumin buffer solution was used as the control for all of the assays.

Signal Transduction Pathways
The influence of the p38 MAP kinase pathway, the Rho-kinase pathway, and the PI-3 kinase pathway on smooth muscle migration was studied using selective pharmacologic inhibitors to the kinases. The p38 MAP kinase inhibitor (SB203580, 25 µM), the Rho-kinase inhibitor (Y27632, 10 µM), and the PI-3 kinase inhibitor (LY294002, 50 µM) were added to the upper wells along with the smooth muscle cells with and without LTE4 (depending on whether the experiments were to study chemokinesis or chemotaxis) 30 minutes before the migration assay and remained in contact with the cells for the entire 5 hours of the migration assay.

Statistical Analysis
Data were summarized using means and SDs. The means of migrated smooth muscle cells were compared for each of the experiments by factorial analysis of variance statistics using the Statistical Package for Social Sciences (SPSS for Windows, version 10.0, Chicago, IL). The source of significant variation was identified by Student–Newman–Keuls test. Statistical significance was accepted for p values of less than 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ASM cells showed significant migratory response to 1 and 10 ng/ml doses of PDGF-BB (Figure 1) . A 1-ng/ml dose (an approximate 3.5-fold response compared with the negative control) was selected as the optimal dose for all further experiments. In comparison, ASM cells did not show any chemotactic response to any of the doses of LTE4. However, they showed a dose-dependent weak chemokinetic response to LTE4 (Figure 2) .



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  		Figure 1. Dose-dependent migratory response of cultured human ASM cells to PDGF-BB. *p < 0.05 compared with control.

 


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  		Figure 2. Chemotactic and chemokinetic responses of cultured human ASM cells to LTE4. LTE4 caused significant chemokinesis, but not chemotaxis. *p < 0.05 compared with control; p < 0.05 compared with corresponding chemotactic response.

 
LTE4 augmented the directional migratory response of ASM cells to PDGF by approximately 1.5-fold. This was almost completely inhibited by montelukast 10-6 M, but not by doses of 10-8 and 10-10 M (Figure 3) . PGE2 (10-7 M) also attenuated the migratory response to PDGF alone (p < 0.05), and the "LTE4-primed" response to PDGF (p < 0.05). The magnitude of inhibitory effect was greater on the primed response (Figure 4) .



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  		Figure 3. Priming effect of LTE4 on PDGF-induced migratory response. The augmented response of LTE4 pretreated smooth muscle cells to PDGF was inhibited by montelukast. *p < 0.05 compared with control; p < 0.05 compared with unprimed response to PDGF; {ddagger}p < 0.05 compared with a primed response to PDGF.

 


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  		Figure 4. The effect of PGE2 on human ASM migration. PGE2 attenuated PDGF, and LTE4-induced ASM migration. p < 0.05 compared with PDGF-induced migration; #p < 0.05 compared with the LTE4-primed migratory response to PDGF.

 
All of the three kinase inhibitors significantly attenuated the directional migratory response to PDGF, without any significant differences between their effects (Figure 5) . In contrast, the PI-3 kinase inhibitor caused a greater inhibitory response on the LTE4-primed directional migration than the inhibitory effects of the MAP kinase inhibitor and the Rho-kinase inhibitor. The PI-3 kinase inhibitor had little effect on the chemokinetic response to LTE4, which was significantly attenuated by the MAP kinase inhibitor and the Rho-kinase inhibitor.



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  		Figure 5. The effect of protein kinases involved in the signal transduction pathways of human ASM chemotaxis and chemokinesis to PDGF and LTE4. Inhibitors of MAP kinase (MAPKI), Rho-kinase (RhoKI), and the phosphatidylinositol-3 kinase (PI3KI) attenuated PDGF-induced migration of primed and unprimed smooth muscle cells. PI3KI did not attenuate the chemokinetic response to LTE4, whereas MAPKI and RhoKI did. *p < 0.05 compared with the respective control experiments; p < 0.05 compared with the inhibitory effect of MAPKI and RhoKI.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study show that LTE4 does not promote directional migration of nonasthmatic human ASM cells, but it causes nondirectional movement or chemokinesis. However, LTE4 augments the migratory response to a growth factor, an effect that is mediated through the Cys-LT1 receptor. The PI-3 kinase signaling pathway plays an important role in this chemotactic response, which may be relevant in the remodeling process in airways that are asthmatic.

The mitogenic and contractile response of cultured human ASM cells has been studied more than their migratory response. This study demonstrated the ability of the human ASM cell to migrate in response to a chemoattractant gradient, as has been previously shown with nonhuman (10) and non-ASM cells (12). The methods employed to culture the cells were similar to previously published reports (13). It is possible that the cells may have included some fibroblasts and myofibroblasts and that the culture may have represented a composite of "airway structural cells." The clinical or biologic relevance of this, however, is not known. The study confirmed previous observations that PDGF is a chemoattractant for tracheal smooth muscle cells of dogs (10); however, the magnitude of response was less than previously reported. This may represent differences in the species studied or study conditions.

Cys-LTs have various biologic effects on smooth muscles mediated through the G-protein–coupled Cys-LT1 receptor (14) and perhaps through an uncharacterized receptor (7). Although LTD4 and LTE4 are believed to act through the same Cys-LT receptor, we observed greater eosinophil cell infiltration into airway mucosa after LTE4 inhalation than after LTD4 inhalation in mildly atopic patients with asthma (2). Therefore, in this series of experiments, we employed LTE4 to study the migratory response of smooth muscle cells. The Cys-LT1 receptor-mediated signal transduction is mostly coupled through the G{alpha}i (inhibition of cAMP) and the G{alpha}q (phospholipase C, protein kinase C, and calcium-mediated) pathways (15) to cause smooth muscle contraction (16). LTD4 can cause actin cytoskeleton reorganization, a critical component of smooth muscle adhesion, spreading, and movement by coupling with a pertussin toxin–sensitive G protein, Rho-guanosine triphosphatases, and tyrosine phosphorylation pathways (17). In keeping with this observation, we observed that LTE4 causes a significant chemokinetic response in ASM cells (Figure 2), which was attenuated by inhibitors of the Rhogr-kinase and the p38 MAP kinase pathways. The PI-3 kinase pathway (17), unlike the Rho-kinase pathway (18), was not critically important in this nondirectional movement, although LTD4 has been observed to induce a translocation of PI-3 kinase to a membrane fraction in human intestinal epithelial cells (19). However, the Class 1b isoform of PI-3 kinase {gamma}, which is activated by the ß{gamma} subunits of heterotrimeric G proteins, has not been demonstrated in ASMs (20).

Unlike the significant chemokinetic response, ASM did not show a chemotactic response to LTE4. Directed movement along a concentration gradient of chemical attractants is a complex event mediated by chemoattractant receptors belonging to the seven-transmembrane helix receptor family (21). After binding to their ligands, these receptors transmit their signals to heterotrimeric G proteins, which then dissociate into {alpha} and ß{gamma} subunits. The latter bind and activate target enzymes such as phospholipase C, PI-3 kinase, and adenylyl cyclase. The PI-3 kinase is critically involved in maintaining a balance of migration-promoting and migration-suppressing activities of Rho-guanosine triphosphate (GTP)-ase activating protein (GAP), particularly to PDGF, to drive chemotaxis (22, 23). Indeed, we observed that the "LT-primed" chemotactic response to PDGF showed greater inhibition by the PI-3 kinase inhibitor than the inhibitors of the p38 MAP kinase pathway and the Rho-kinase pathway. LY294002 is a competitive inhibitor of the ATP site of PI-3 kinase (24). In most biologic systems, evidence for an involvement of PI-3 kinase is obtained by treating cells with 5 to 20 µM of LY294002 (25). The dose that we used (50 µM) may have been high enough to cause some nonspecific effects but has been previously reported to inhibit PI-3 kinase–mediated human smooth muscle migration (26) and proliferation (27). We would have expected to see a similar response on PDGF-induced cell migration, which is also predominantly chemotactic and regulated by PI-3 kinase (28). Although the PI-3 kinase inhibitor caused a greater inhibition on PDGF-induced cell migration than the MAP kinase and the Rho-kinase inhibitors, this was not statistically significant. Perhaps the migration of human smooth muscle cells (29) and their signal transduction pathways is different from that in Chinese hamster ovary 602 cell lines (21) and rat ELT3 cell lines (23).

Similar to the chemotactic effect on peripheral blood eosinophils (3) and the proliferative effects on cultured ASMs (7), we observed that LTE4 itself, although not having a chemotactic effect on ASM cells, augmented the chemotactic effects of a growth factor, that is, PDGF. This effect was mediated through the Cys-LT receptor as it was abolished by montelukast, a specific Cys-LT receptor antagonist. However, we do not know the precise signal transduction pathway linking the G-protein–coupled receptor pathway and the receptor tyrosine kinase pathway. Because the priming effect was attenuated by the Rho-kinase, p38 MAP kinase, and the PI-3 kinase inhibitors, the interaction is likely to be upstream of these enzymes. Moreover, given that activated G{alpha}q (through phospholipase C and protein kinase C) and activated Gß{gamma} and receptor tyrosine kinases (through PI-3 kinase {gamma}, Src-family kinases signaling to Shc homology proteins) can stimulate Ras and in turn activate the MAP kinase pathway (30), it is possible that the interaction may be at the level of Ras or Src kinases. Indeed Src-family kinases are involved in migration of cultured vascular smooth muscle cells (12), and they regulate PI-3 kinase and protein kinase B activation to modulate neutrophil chemotaxis (31). It is unlikely that Cys-LTs directly stimulate PI-3 kinase in human ASM to augment PDGF-stimulated chemotaxis, but rather via activation of Src kinases. Alternatively, the priming effect may simply reflect the increased motility of cells triggered by LT-induced cytoskeletal reorganization. This needs further investigation.

The inhibitory effect of PGE2 on growth factor–induced ASM cell migration is probably similar to the E-prostanoid 1 and 2 (EP1 and EP2) receptor–coupled, cAMP-dependent, protein kinase A–mediated, inhibitory effect on fibroblast chemotaxis (32) and human ASM relaxation (33). The inhibitory effect on LTE4-primed chemotactic response may represent an interaction between the prostanoid (EP) receptor signaling cascade and the Cys-LT1 receptor signaling cascades, by an inhibitory effect on the Rho-kinase pathway. This needs further investigation. PGE2 may provide an endogenous mechanism to check the uninhibited facilitatory effects of Cys-LTs in promoting airway inflammation and airway hyperresponsiveness in patients with asthma. Indeed, inhaled PGE2 has been shown to mitigate human ASM proliferation (34) and allergen-induced airway responses and airway inflammation (35).

ASM plays an important role in the process of airway remodeling in patients with asthma (5). Cys-LTs, albeit causing a modest inflammatory effect when inhaled, play important roles in promoting airway remodeling in murine models of asthma (36). They may do so by augmenting the proinflammatory and mitogenic effects of other cytokines and chemokines relevant to the pathophysiology of asthma. Pretreatment with LT receptor antagonists attenuates an allergen-induced increase in airway hyperresponsiveness (37). This experiment, despite the relatively small magnitude of the migratory response, provides another potential mechanism for how Cys-LTs may contribute to the accumulation of smooth muscle in the airway and airway remodeling. It needs to be proven whether the migratory responses observed in this study are enhanced in smooth muscle from patients with asthma. Drawing on the analogy of the migration of vascular smooth muscles and the inhibitory effects of statins in preventing vascular remodeling in atherosclerosis (38), prevention of ASM migration provides an attractive therapeutic intervention for preventing airway remodeling in asthma.

In conclusion, this study provides evidence that ASM cells show a chemotactic response to PDGF. LTE4, although it is not directly a chemoattractant, augments this response, which is inhibited by montelukast. The PI-3 kinase pathway is a key signaling mechanism of ASM chemotaxis.


   Acknowledgments
 
The authors thank Dr. W. Gerthoffer, University of Nevada, for his helpful advice; Dr. J. Miller, Dr. J. E. M. Young, and Dr. J. Urschel, St. Joseph's Healthcare, Hamilton, for providing us with resected lung specimens; and J. Otis, McMaster University, for her help with preparing the figures. Dr. Parameswaran is a postdoctoral fellow of the Canadian Institutes of Health Research. Montelukast was a generous gift from Merck Frosst Canada.


   FOOTNOTES
 
Supported by a Block-Term Grant Award from the Ontario Thoracic Society.

Received in original form April 6, 2002; accepted in final form June 3, 2002


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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
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