help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on April 17, 2008, doi:10.1165/rcmb.2007-0091OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2007-0091OCv1
39/4/475    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nair, P.
Right arrow Articles by Cox, P. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nair, P.
Right arrow Articles by Cox, P. G.
American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 475-481, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0091OC

The Effects of Leptin on Airway Smooth Muscle Responses

Parameswaran Nair1, Katherine Radford1, Adrian Fanat1, Luke J. Janssen1, Marc Peters-Golden2 and P. Gerard Cox1

1 Firestone Institute for Respiratory Health, St. Joseph's Healthcare, and Department of Medicine, McMaster University, Hamilton, Ontario, Canada; and 2 Division of Pulmonary & Critical Care Medicine, University of Michigan, Ann Arbor, Michigan

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


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Obesity is associated with asthma and airway hyperresponsiveness. Leptin modulates some of the proinflammatory effects observed in obesity. The objective of this study was to determine the effects of leptin on airway smooth muscle responses. The effect of leptin (0.1–100 ng/ml) on migration (toward platelet-derived growth factor [PDGF], 10 ng/ml, across collagen-coated membrane in Transwell culture plates), proliferation (by BrDU incorporation), and cytokine production (by Bioplex bead assay) of cultured human airway smooth muscle cells from nine nonasthmatic donors was assessed. Effects of leptin on the contractile responses were studied in bovine tracheal smooth muscle rings. Leptin receptor expression and activation of STAT-3, Src kinase, Suppressor of Cytokine Signaling-3 (SOCS-3), and COX were evaluated by Western blotting and PCR. PGE2 levels in supernatant were assessed by enzyme immunoassay. Human airway smooth muscle cells express leptin receptor, which, when engaged, phosphorylated STAT-3. Leptin inhibited PDGF-induced human airway smooth muscle migration and proliferation and IL-13–induced eotaxin production. Leptin did not stimulate cytokine synthesis and did not evoke contractile responses or inhibit isoproterenol-induced relaxation of carbachol-induced contraction of bovine tracheal rings. The inhibitory effects on migration and eotaxin production are not due to activation of SOCS-3 but are partly due to increased production of PGE2 because they were attenuated by indomethacin. In conclusion, leptin inhibited human airway smooth muscle proliferation, migration toward PDGF, and IL-13–induced eotaxin production. This is partly mediated by PGE2 secretion from smooth muscle cells induced by leptin. The association between obesity and asthma is unlikely to be due to a direct effect of leptin on airway smooth muscle.

Key Words: leptin • airway smooth muscle • migration • proliferation • cytokines • PGE2



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The proinflammatory effects of obesity in asthma are unlikely to be due to a direct effect of leptin on airway smooth muscle.

 
There has been a global increase in the prevalence of obesity and of asthma over the past decade. Several large cross-sectional studies and a few prospective studies have demonstrated a strong positive relationship between obesity and asthma prevalence and severity (1–5). However, a causal relationship has not been established, and more recent studies have failed to find an association between obesity and airflow obstruction (6) or airway hyperresponsiveness (7) in patients with self-reported asthma.

Possible mechanisms for the apparent relationship between obesity and asthma include the direct mechanical effects of obesity on the airway, the absence of the bronchoprotective effect of deep inhalations in obese individuals (8), and an increase in airway hyperresponsiveness associated with airway narrowing (9). Obesity may also induce airway hyperresponsiveness by immunological mechanisms (10). Obesity is recognized to be a proinflammatory state (11). It is associated with an increase in macrophage numbers within adipose tissue that secrete TNF-{alpha}, inducible nitric oxide synthase, and IL-6 (12), which may lead to airway inflammation. Thus, in obese individuals, the biological activity of adipose tissue may increase the risk for developing asthma and airway inflammation.

Some of the proinflammatory effects of adipose tissue have been attributed to the hormone leptin, which is produced by adipose tissue (13). The leptin receptor (OB-R) is a member of the class I cytokine receptor family and has a single transmembrane-spanning domain. Six isoforms (OB-Ra through OB-Rf), resulting from alternative splicing, differ in the length of their intracellular tails but share identical extracellular binding domains. Thus, the isoforms can be classified as short, long, and secreted. The long full-length isoform (OB-Rb) is responsible for most of the known effects of leptin.

Leptin concentrations are increased in obese individuals (14) and more so in patients with asthma who are obese (15). Although leptin has a proliferative effect on CD4+ T cells, the effect of leptin on asthma is unlikely to be on the Th2 allergic phenomenon because leptin increased Th1 cytokine production and decreased Th2 cytokines (16). Leptin has been reported to increase airway responsiveness induced by ovalbumin (OVA) aerosol challenge in OVA-sensitized mice (17). The increase in OVA-induced airway responsiveness occurred in the absence of any effects of leptin on inflammatory cell influx or Th2 cytokine expression. This suggests that leptin can directly modulate airway smooth muscle function. In this study, we examined the effects of leptin on human airway smooth muscle proliferation, migration, and cytokine synthesis and the effects of leptin on contraction and relaxation of bovine airway smooth muscle. Some of the data have been presented in the form of an abstract (18).


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Human Airway Smooth Muscle Culture
Portions of human lungs that were resected for cancer at St. Joseph's Healthcare, Hamilton, Ontario, Canada were obtained after obtaining informed consent from nine patients (mean age 54 yr, mean FEV1 74% predicted, mean body mass index 29 kg/m2) and approval from the hospital Research Ethics Board. Smooth muscle tissue was isolated from macroscopically disease-free areas of human bronchi. Airway smooth muscle cells were grown to confluence as previously described (19). The cells were passaged two to five times and used for the experiments. Human aortic vascular smooth muscle cells were obtained from PromoCell (Heidelberg, Germany).

Western Blotting
Leptin receptor expression on human airway smooth muscle cells was examined by Western blotting. Protein extracted from smooth muscle cells was subjected to 10% SDS-PAGE, and the separated proteins were electrophoretically transferred to Hybond ECL nitrocellulose membranes (Amersham Life Sciences, Oakville, ON, Canada). Blots were incubated with the anti–OB-R rabbit polyclonal antibody (Santa Cruz Biotechnologies Inc., Santa Cruz, CA) against a recombinant protein corresponding to aminoacids 541 through 840 of the internal domain of human OB-R (detects short and long forms of OB-R) followed by incubation with donkey anti-rabbit IgG and then incubated with horseradish peroxidase complex. Peroxidase-labeled proteins were visualized by enhanced chemiluminescence reagents (Amersham Life Sciences). Phospho and total Signal Transducer and Activator of Transcription-3 (STAT-3) were examined after treating the cells with 100 ng/ml leptin using 1/1,000 rabbit anti–STAT-3 antibody (Cell Signaling, Boston, MA) and secondary donkey anti-rabbit antibody (GE Healthcare Bio-Sciences Inc., Baie d'Urfé, PQ, Canada). Src-kinase phosphorylation was assessed using a monoclonal antibody that recognized activated Src (pY418 rabbit polyclonal anti-phospho-Src; BioSource International, Camarillo, CA). Cyclooxygenase (COX)-1 and -2 activation on smooth muscle cells were examined by using monoclonal antibodies (1/1,000) against both proteins (R&D, Minneapolis, MN). Specific proteins were recognized using a secondary antibody conjugated to horseradish peroxidase and enhanced chemiluminescence (Amersham Life Sciences).

Migration Assay
Migration experiments were performed using a 6.5-mm Transwell culture plate with an 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 (19). Assays were done in duplicate using tissues from six different lung specimens. Chemotaxis was studied by adding the chemoattractant only to the outer well. The chemoattractants studied were platelet-derived growth factor (PDGF)-BB (10 ng/ml) (Invitrogen Canada Inc., Burlington, ON, Canada) and leptin (1–100 ng/ml) (Reprotech Inc., Rocky Hill, NJ). The effect of leptin on migration toward PDGF was studied by treating the cells with 100 ng/ml leptin for 30 minutes before adding PDGF.

Proliferation Assay
Cell proliferation was also determined using a colorimetric assay based on the measurement of BrdU incorporation during DNA synthesis according to the manufacturer's instructions (Roche Diagnostics, Laval, PQ, Canada). Briefly, confluent cells were detached by trypsinization, counted, and plated onto 96-well microtiter plates at a density of 104 cells/well in RPMI supplemented with 10% FBS for 48 hours. The growth medium was then switched to RPMI only, and the cells were exposed to this medium for 24 hours to induce quiescence and synchronize the cell cycle. The vehicle and test substances were then added in triplicate or sextuplicate wells (10 µl/well). Six hours later, the thymidine analog BrdU was added (10 µl/well), and the cells were incubated for a further 18 hours, during which the BrdU was incorporated into the newly synthesized DNA of proliferating cells. Total exposure time of the cells to test substances was 24 hours. The culture medium was then removed, and the cells were denatured with 70% ethanol and 0.5 M HCl, treated with nucleases for 30 minutes at 37°C, and incubated with 1:100 diluted mouse anti-BrdU mAbs conjugated to peroxidase for 30 minutes at 37°C. After removing the antibody conjugate, substrate solution was added for 20 minutes. The absorbance (optical density) was measured at 405 nm with a reference wavelength at 590 nm using an ELISA plate reader (Model 550; Bio-Rad, Oakville, ON, Canada).

Cytokine and Mediator Synthetic Assay
Confluent cells were transferred to 0.3% BSA in RPMI for 48 hours and treated with IL-13 (10 ng/ml) or Cytomix (IL-1β 5 ng/ml, TNF-{alpha} 30 ng/ml, and IFN-{gamma} 100 ng/ml) (all from PeproTech Inc., Rocky Hill, NJ) and/or leptin (0.1–100 ng/ml) overnight. To assess the role of prostanoids and leukotrienes, the cyclooxygenase inhibitor indomethacin (10–6 M) (Sigma Aldrich, Oakville, ON, Canada) or the cysteinyl leukotriene (CysLT) antagonist montelukast (Cayman Chemical Co., Ann Arbor, MI) was added 30 minutes before IL-13, PDGF, or Cytomix or leptin. IL-8, IFN-{gamma}, TNF-{alpha}, RANTES, and Eotaxin in smooth muscle supernatant were measured by ELISA using the Bioplex bead assay (Bio-Rad Laboratories, Mississauga, ON, Canada). The limit of detection was 10 pg/ml. Prostaglandin (PG)E2 levels in smooth muscle culture supernatants were measured by a sensitive enzyme immunoassay according to the manufacturer's instructions (Assay Designs, Ann Arbor, MI). The limit of detection for PGE2 was 13.4 pg/ml.

STAT-3 activation was assessed using a DNA-binding transcription factor ELISA assay (TransAm) according to the manufacturer's instructions (Active Motif, Carlsbad, CA). Briefly, nuclear extracts from untreated and from smooth muscle cells treated with 100 ng/ml leptin were incubated with anti-STAT3 primary antibody (1/500) in 96-well plates that had immobilized oligonucleotides containing the STAT-3 consensus binding site (5'-TTCCCGGAA-3') and were then probed with secondary antibody (1/1,000) conjugated to horseradish peroxidase. After the antibody conjugate was removed, substrate solution was added, and the absorbance (optical density) was measured at 450 nm with a reference wavelength at 655 nm using an ELISA plate reader (Model 550; Bio-Rad).

PCR
Suppressor of cytokine signaling–3 (SOCS-3) m-RNA expression on human airway smooth muscle cells was assessed by PCR. RNA (0.2 µg) was reversed transcribed. Briefly, the RNA was incubated with Super Script II reverse transcriptase (Invitrogen Canada) and mixed with oligo (dT) in a 20-µl reaction at 37°C for 30 minutes. The reaction was inactivated by incubation at 94°C for 5 minutes. The PCR was performed as previously described (20). Briefly, a semiquantitative form of PCR was used with β-actin as a standard. The following primer sequences were used (5'->3'): SOCS-3 sense (GGA CCA GCG CCA CTT CTT CAC) and SOCS-3 antisense (TAC TGG TCC AGG AAC TCC CGA). The primers were made by MOBIX Lab (McMaster University, Hamilton, ON). The reverse transcription reaction was mixed with 2.5 units of Taq DNA polymerase (Invitrogen Canada) and 60 µM of each primer in final 50-µl volume. β-actin amplifications were done as separate but parallel reactions using the identical cycling conditions. SOCS-3 amplifications were performed in a Perkin Elmer Gene Amp PCR system (Applied Biosystems, Streetsville, ON, Canada) with an initial denaturing step of 94°C for 2 minutes, followed by 30 cycles of 45 seconds at 94°C, 45 seconds at 58°C, and 2 minutes at 72°C. The final cycle ended with 10 minutes at 72°C. C2C12 mouse myoblasts (an immortalized mouse myoblast cell line; ATCC, Rockville, MD) served as the positive control.

Preparation of Bovine Tracheal Rings
Lobes of lung and tracheae were obtained from cows (136–454 kg) killed at a local abattoir and immediately put in ice-cold physiologic solution for transport to the laboratory. Lobes of lung were pinned out, the overlying parenchyma and pulmonary vasculature were removed, and strips of trachealis were excised (4–5 mm long and 1 mm thick).

Muscle Bath Techniques
Tracheal strips were mounted into 3-ml muscle baths using stainless steel hooks inserted into the lumen. One hook was tied with silk suture (Ethicon 4-O) to a Grass FT.03 force transducer; the other was attached to a Plexiglas rod that served as an anchor. Tissues were bathed in Krebs-Ringer's buffer and maintained at 37°C. Tissues were passively stretched to impose a preload tension of 1 g (determined to allow maximal responses). Isometric changes in tension were amplified, digitized (two samples per second), and recorded online (DigiMed System Integrator; MicroMed, Louisville, KY) for plotting on the computer. Tissues were equilibrated for 1 hour before starting the experiments, during which time the tissues were challenged with 60 mM KCl three times to assess the functional state of each tissue. Tissues were then washed, and the preload was readjusted just before onset of the study (i.e., at the end of the equilibrium period). Tissues were treated with carbachol (10–7 M) and isoproterenol (10–8 to 10–5 M) in the presence and absence of leptin (1 µg/ml).

Statistical Analysis
Statistical analysis was performed with ANOVA using the different time points or experimental conditions as within-subject factors. The source of significant variation was identified by predefined contrasts. P < 0.05 was considered significant. All analyses were performed using SPSS-version 13.0 (Statistical Package for Social Sciences, Chicago, IL).


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Leptin Receptor Expression and Signaling
Human airway smooth muscle cells express the leptin receptor OB-R (Figure 1A), which when engaged causes STAT-3 phosphorylation (Figure 1B). The receptor expression was not up-regulated by PDGF (Figure 1A). This was confirmed by a TransAm Assay. Compared with untreated cells, leptin resulted in a mean 153% (SD 45%) increase in STAT-3 activity (optical density). The corresponding value for PDGF was 123% (SD 36%). Leptin did not cause Src-kinase phosphorylation; neither did leptin have any effect on PDGF-induced Src-kinase phosphorylation (data not shown). The anti-inflammatory effect was not due to activation of SOCS-3 because airway smooth muscle cells do not express SOCS-3 (Figure 1C).


Figure 1
Figure 1
Figure 1
View larger version (110K):
[in this window]
[in a new window]

  		Figure 1. (A) Leptin receptor (OB-R) expression on human airway smooth muscle cells. Western blots show that human airway and vascular (aortic) smooth muscle cells express the 140-kD leptin receptor. This is unaffected by treatment with platelet-derived growth factor (PDGF) (10 ng/ml for 30 min to 12 h). The data shown are a summary of four experiments. (B) Western blots demonstrating that leptin (100 ng/ml, 30 min) increases phospho-STAT-3 levels, but not total STAT-3, compared with untreated control. The data shown are a summary of four experiments. (C) Polymerase chain reaction amplification and immunoblots did not show SOCS-3 expression on human airway smooth muscle cells. C2C12 cell lines acted as positive controls.

 
Effects on Human Airway Smooth Muscle Proliferation, Migration, and Cytokine Production
Leptin caused a concentration-dependent inhibition of smooth muscle proliferation (promoted by 10 ng/ml PDGF) (Figure 2), migration toward PDGF (10 ng/ml) (Figure 3), and eotaxin production (not IL-8, IFN-{gamma}, TNF-{alpha}, and RANTES) stimulated by IL-13 (Table 1 and Figure 4). Leptin did not promote muscle proliferation, migration (data not shown), or cytokine synthesis (Table 1).


Figure 2
View larger version (8K):
[in this window]
[in a new window]

  		Figure 2. Leptin caused a concentration-dependent inhibition of PDGF (10 ng/ml)-stimulated smooth muscle proliferation. This was attenuated by indomethacin (10–6 M) but not by montelukast (10–6 M). *P < 0.05 compared with PDGF alone. Data are shown as mean and SD of six experiments.

 

Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		Figure 3. Leptin caused a concentration-dependent inhibition of PDGF (10 ng/ml)-stimulated smooth muscle chemotaxis. This was abolished by indomethacin (10–6 M) but not by montelukast (10–6 M). *P < 0.05 compared with PDGF alone. Data are shown as mean and SD of six experiments.

 

Figure 4
View larger version (8K):
[in this window]
[in a new window]

  		Figure 4. Leptin caused a concentration-dependent inhibition of IL-13–stimulated eotaxin production from smooth muscle cells. This was attenuated by indomethacin but not by montelukast. *P < 0.05 compared with PDGF alone. Data are shown as mean and SD of nine experiments.

 

View this table:
[in this window]
[in a new window]

  		TABLE 1. CYTOKINE SYNTHESIS FROM HUMAN AIRWAY SMOOTH CELLS STIMULATED OVERNIGHT WITH IL-13 (10 ng/ml) OR CYTOMIX (IL-1β 5 ng/ml, TNF-{alpha} 30 ng/ml, IFN-{gamma} 100 ng/ml)

 
Effects on Bovine Airway Smooth Muscle Contraction and Relaxation
Leptin did not cause contraction of bovine tracheal strips or enhance carbachol-induced contraction. Leptin had no effect on the relaxant effects of isoproterenol on carbachol-induced contraction (Figures 5A and 5B).


Figure 5
View larger version (10K):
[in this window]
[in a new window]

  		Figure 5. (A) Leptin (1,000 ng/ml) did not enhance carbachol-induced contraction (expressed as percentage of KC- induced contraction) of bovine tracheal smooth muscle strips. Data are shown as mean of four separate experiments. (B) Leptin (1,000 ng/ml) had no effect on isoprotenerol-induced relaxation of carbachol-induced contraction of bovine tracheal smooth muscle strips. Data are shown as mean of four separate experiments.

 
Modulation by Prostanoids
The inhibitory effect of leptin on proliferation and migration toward PDGF and eotaxin production stimulated by IL-13 were partially abolished by indomethacin but not by montelukast (Figures 2–4GoGo and Table 1). Leptin increased COX-2 expression on airway smooth muscle cells (Figure 6A) and caused a 1.92-fold increase in PGE2 stimulated by PDGF (Figure 6B). Neither indomethacin nor monteukast had an effect on the other cytokines.


Figure 6
Figure 6
View larger version (58K):
[in this window]
[in a new window]

  		Figure 6. (A) Prostaglandin (PG)E2 synthesis from airway smooth muscle cells stimulated by leptin (100 ng/ml). PGE2 levels increased 1.9-fold compared with baseline (*P < 0.05). This was attenuated by indomethacin (10–6 M) (**P < 0.05). PDGF (10 ng/ml) and indomethacin alone did not have significant effects. (B) Leptin induced COX-1 and COX-2 on airway smooth muscle cells. Incubation with leptin (100 ng/ml) caused COX-1 (at 2 h, maximal 24 h) and COX-2 induction (at 2 h, maximal 8 h). A representative blot from three experiments (in duplicate) is shown.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
This study reports a number of novel observations. Human airway smooth muscle cells express leptin receptor, which, when engaged, phosphorylated STAT-3. Leptin inhibited PDGF-induced human airway smooth muscle migration and proliferation and IL-13–induced eotaxin production. Leptin did not evoke contractile responses or inhibit isoproterenol-induced relaxation of carbachol-induced contraction of bovine tracheal rings or stimulate cytokine synthesis in human airway smooth muscle cells. The inhibitory effects are not due to activation of SOCS-3 or inhibition of Src-kinase but are likely due in part to COX induction and production of PGE2 because they were abolished by indomethacin. The results suggest that the proinflammatory effects of obesity in asthma are unlikely to be due to a direct effect of leptin on airway smooth muscle.

Although leptin receptor expression has been demonstrated in human airways (21), the effects of leptin on human airway smooth muscle functions have not been reported. The effects of leptin on human vascular smooth muscle cells are conflicting. Bohlen and colleagues (22) demonstrated an inhibition of cultured aortic smooth muscle cell proliferation by increasing leptin concentrations of 50 and 100 ng/ml. They observed that the short isoforms of OB-R were expressed in a 10- to 27-fold excess compared with the long isoform in these cells and that leptin down-regulated the short isoforms significantly, whereas the long isoform was not influenced. The authors suggested that leptin inhibits vascular smooth muscle cell growth by modulating leptin receptor isoforms. In contrast, Oda and colleagues (23) have reported that leptin (100 ng/ml) stimulates human vascular smooth muscle proliferation and chemotaxis and increases phosphorylation of MAP-kinases and phosphoinositol 3-kinases. Although the effects of leptin on human airway smooth muscle contraction have not been examined, leptin has been described to inhibit spontaneous and oxytocin-induced contraction of human uterine smooth muscle (24). The effects of leptin on the synthetic functions of human airway or vascular smooth muscle cells are not known.

We examined various signaling pathways to explore the mechanism behind our observations. Cultured airway smooth muscle cells expressed leptin OB-R receptor. Although we did not characterize the receptor isoforms (25), the monoclonal antibody directed against aminoacids in the intracytoplasmic domain of the receptor is likely to have detected the long-isoform (OB-Rb) because OB-Rb is the only leptin receptor isoform that contains the STAT-3 binding site (26). Because we did not observe any stimulatory effect of leptin on airway smooth muscle function, it is unlikely that activation of Janus kinases (JAK)-2 by leptin led to phosphorylation of Akt or Src-kinase. It is likely, therefore, that these pathways are not activated by leptin in human airway smooth muscle cells. To explain the inhibitory effect of leptin on smooth muscle proliferation, migration, and cytokine synthesis, we speculated that leptin, through the Tyr1138 residue of the internal portion of the OB-R, activates STAT-3, which in turn would be substrates of JAK. Phospho-STAT-3 then translocates to the nucleus, ultimately leading to transcription of suppressor of cytokine signaling-3 (SOCS-3) (27). SOCS-3 expression is known to inhibit tyrosine phosphorylation of OB-R, thus providing an important feedback mechanism for receptor signaling at the transcriptional level. However, we were unable to identify SOCS-3 mRNA or protein on human airway smooth muscle cells.

We therefore examined another potential mechanism to explain our observations. Leptin is reported to induce cyclo-oxygenase 2 in murine macrophages (28) and to increase cytosolic phospholipase activity in murine and rat alveolar macrophages (29), leading to synthesis of CysLT and prostanoids through the lipooxygenase and cyclooxygenase pathways, respectively. The CysLTs are described to augment airway smooth muscle contraction, proliferation (30), migration (19), and cytokine synthesis, and prostaglandin E2 can inhibit these processes by increasing intracellular cyclic AMP (19, 31). The inhibitory effect of leptin on PDGF-stimulated migration and IL-13–stimulated eotaxin synthesis was partly dependent on cyclooxygenase activity, as evidenced by the loss of inhibition by indomethacin, but not by montelukast. Consistent with this, leptin induced COX-1 and COX-2 and increased PGE2 production from the airway smooth muscle cells. We have not identified all the mechanisms by which leptin attenuated PDGF-induced smooth muscle proliferation. It may be possible that leptin, through yet undescribed pathways, inhibits STAT-6 or MAP-kinases, leading to decreased cyclin-D expression on smooth muscle cells. This needs further investigation. We are also unable to explain the lack of effect of leptin on bovine airway smooth muscle. Although the long form of the leptin receptor is reported to be widely expressed in bovine tissues (32), the lack of effect may be related to the relative lack of functional receptors on tracheal smooth muscle tissues.

In summary, leptin inhibited human airway smooth muscle proliferation, migration toward PDGF, and IL-13–induced eotaxin production. This is partly mediated by PGE2 secretion from smooth muscle cells induced by leptin. Leptin did not stimulate proinflammatory cytokine release from airway smooth muscle cells. The proinflammatory effects of obesity in asthma are unlikely to be due to a direct effect of leptin on airway smooth muscle.


   Acknowledgments
 
The authors thank the Departments of Thoracic Surgery and Pathology for their cooperation in obtaining resected lung specimens and T. Tazzeo for her help with the muscle bath experiments.


   Footnotes
 
The study was funded by an Ontario Thoracic Society Block-Term Grant. Dr Nair was supported by a clinician-scientist award from the Canadian Institutes of Health Research.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0091OC on April 17, 2008

Conflict of Interest Statement: N.P. has received honoraria for scientific lectures (Merck $2,000, Atlanta $2,000, Novartis $1,000). These were not drug promotion talks, but the sessions were supported by unrestricted grants. N.P. also holds two investigator-initiated industry-supported grants. 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. L.J.J. has received $70,000 from Astra-Zeneca to study the mechanisms underlying B-adrenoceptor desensitization in airway smooth muscle and $75,000 from Glaxo-SmithKline to study isoprostane-induced airway hyperresponsiveness. M.P.-G. has served as a consultant and speakers' bureau member for Merck and has recently received honoraria for those services, but none is related to the subject of this manuscript. P.G.C. has received honoraria from various organizations and pharmaceutical companies for academic lectures, but none were related to the subject of this manuscript.

Received in original form March 18, 2007

Accepted in final form February 18, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Tantisiri KG, Weiss ST. Complex interactions in complex traits: asthma and obesity. Thorax 2001;56:64–74.
  2. Chinn S. Obesity and asthma: evidence for and against a causal relation. J Asthma 2003;40:1–16.[CrossRef][Medline]
  3. Camargo CA, Weiss ST, Zhang S, Willett WC, Speizer FE. Prospective study of body mass index, weight change, and risk of adult-onset asthma in women. Arch Intern Med 1999;159:2582–2588.[Abstract/Free Full Text]
  4. Chinn S, Downs SH, Anto JM, Gerbase MW, Leynaert B, de Marco R, Janson C, Jarvis D, Kunzli N, Sunyer J, et al. Incidence of asthma and net change in symptoms in relation to changes in obesity. Eur Respir J 2006;28:763–771.[Abstract/Free Full Text]
  5. Shore SA, Johnston RA. Obesity and asthma. Pharmacol Ther 2006;110:83–102.[CrossRef][Medline]
  6. Sin DD, Jones RL, Man SF. Obesity is a risk factor for dyspnea but not for airflow obstruction. Arch Intern Med 2002;162:1477–1481.[Abstract/Free Full Text]
  7. Schachter LM, Salome CM, Peat JK, Woolcock AJ. Obesity is a risk for asthma and wheeze but not airway hyperresponsiveness. Thorax 2001;56:4–8.[Abstract/Free Full Text]
  8. Boulet LP, Turcotte H, Boulet G, Simard B, Robichaud P. Deep inspiration avoidance and airway response to methacholine: influence of body mass index. Can Respir J 2005;12:371–376.[Medline]
  9. Litonjua AA, Sparrow D, Weiss ST. The FEF25–75/FVC ratio is associated with methacholine airway hyperresponsiveness. Am J Respir Crit Care Med 1999;159:1574–1579.[Abstract/Free Full Text]
  10. Shore SA. Obesity and asthma: lessons from animal models. J Appl Physiol 2007;102:516–528.[Abstract/Free Full Text]
  11. Visser M, Bouter LM, McQuillan GM, Wener MH, Harris TB. Elevated C-reactive protein levels in overweight and obese adults. JAMA 1999;282:2131–2135.[Abstract/Free Full Text]
  12. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Rerrante AW Jr. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest 2003;112:1796–1808.[CrossRef][Medline]
  13. Fantuzzi G. Adipose tissue, adipokines, and inflammation. J Allergy Clin Immunol 2005;115:911–919.[CrossRef][Medline]
  14. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL, et al. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 1996;334:292–295.[Abstract/Free Full Text]
  15. Sood A, Ford ES, Camargo CA Jr. Association between leptin and asthma in adults. Thorax 2006;61:300–305.[Abstract/Free Full Text]
  16. Lord GM, Matarese G, Howard JK, Baker RJ, Bloom SR, Lechler RI. Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression. Nature 1998;394:897–901.[CrossRef][Medline]
  17. Shore SA, Schwartzman IN, Mellema MS, Flynt L, Imrich A, Johnston RA. Effect of leptin on allergic airway responses in mice. J Allergy Clin Immunol 2005;115:103–109.[CrossRef][Medline]
  18. Parameswaran K, Fanat A, Radford K, Sharma AM, Cox P. The effects of leptin and adiponectin on human airway smooth muscle cell migration and proliferation. Eur Respir J 2004;24:97s.
  19. Parameswaran K, Cox G, Radford K, Janssen LJ, Sehmi R, O'Byrne PM. Cysteinyl leukotrienes promote human airway smooth muscle migration. Am J Respir Crit Care Med 2002;166:738–742.[Abstract/Free Full Text]
  20. Spangenburg EE. SOCS-3 induces myoblast differentiation. J Biol Chem 2005;280:10749–10758.[Abstract/Free Full Text]
  21. Bruno A, Chanez P, Chiappara G, Siena L, Giammanco S, Gjomarkaj M, Bonsignore G, Bousquet J, Vignola AM. Does leptin play a cytokine-like role within the airways of COPD patients? Eur Respir J 2005;26:398–405.[Abstract/Free Full Text]
  22. Bohlen F, Kratzsch J, Mueller M, Seidel B, Friedman-Einat M, Witzigmann H, Teupser D, Koerner A, Storck M, Thiery J. Leptin inhibits cell growth of human vascular smooth muscle cells. Vascul Pharmacol 2007;46:67–71.[CrossRef][Medline]
  23. Oda A, Taniguchi T, Yokoyama M. Leptin stimulates rat aortic smooth muscle cell proliferation and migration. Kobe J Med Sci 2001;47:141–150.[Medline]
  24. Moynihan AT, Hehir MP, Glavey SV, Smith TJ, Morrison JJ. Inhibitory effect of leptin on human uterine contractility in vitro. Am J Obstet Gynecol 2006;195:504–509.[CrossRef][Medline]
  25. Tartaglia LA. The leptin receptor. J Biol Chem 1997;272:6093–6096.[Free Full Text]
  26. Ghilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH, Skoda RC. Defective STAT signaling by the leptin receptor in diabetic mice. Proc Natl Acad Sci USA 1996;93:6231–6235.[Abstract/Free Full Text]
  27. Frühbeck G. Intracellular signalling pathways activated by leptin. Biochem J 2006;393:7–20.[CrossRef][Medline]
  28. Raso GM, Pacilio M, Esposito E, Coppola A, Di Carlo R, Meli R. Leptin potentiates IFN-gamma-induced expression of nitric oxide synthase and cyclo-oxygenase-2 in murine macrophage J774A.1. Br J Pharmacol 2002;137:799–804.[CrossRef][Medline]
  29. Mancuso P, Canetti C, Gottschalk A, Tithof PK, Peters-Golden M. Leptin augments alveolar macrophage leukotriene synthesis by increasing phospholipase activity and enhancing group IVC iPLA2 (cPLA2gamma) protein expression. Am J Physiol Lung Cell Mol Physiol 2004;287:L497–L502.[Abstract/Free Full Text]
  30. Panettieri RA, Tan EM, Ciocca V, Luttmann MA, Leonard TB, Hay DW. Effects of LTD4 on human airway smooth muscle cell proliferation, matrix expression, and contraction in vitro: differential sensitivity to cysteinyl leukotriene receptor antagonists. Am J Respir Cell Mol Biol 1998;19:453–461.[Abstract/Free Full Text]
  31. Goncharova EA, Billington CK, Irani C, Vorotnikov AV, Tkachuk VA, Penn RB, Krymskaya VP, Panettieri RA Jr. Cyclic AMP mobilizing agents and glucocorticoids modulate human smooth muscle cell migration. Am J Respir Cell Mol Biol 2003;29:19–27.[Abstract/Free Full Text]
  32. Chelikani PK, Glimm DR, Kennelly JJ. Tissue distribution of leptin and leptin receptor mRNA in the bovine. J Dairy Sci 2003;86:2369–2372.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2007-0091OCv1
39/4/475    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nair, P.
Right arrow Articles by Cox, P. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nair, P.
Right arrow Articles by Cox, P. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2008 American Thoracic Society.