help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on September 7, 2006, doi:10.1164/rccm.200603-394OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200603-394OCv1
174/11/1179    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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kaur, D.
Right arrow Articles by Brightling, C. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kaur, D.
Right arrow Articles by Brightling, C. E.
American Journal of Respiratory and Critical Care Medicine Vol 174. pp. 1179-1188, (2006)
© 2006 American Thoracic Society
doi: 10.1164/rccm.200603-394OC

Original Article

Airway Smooth Muscle and Mast Cell–derived CC Chemokine Ligand 19 Mediate Airway Smooth Muscle Migration in Asthma

Davinder Kaur*, Ruth Saunders*, Patrick Berger, Salman Siddiqui, Lucy Woodman, Andrew Wardlaw, Peter Bradding and Christopher E. Brightling

Institute for Lung Health, and Department of Infection, Inflammation and Immunity, University of Leicester, Leicester, United Kingdom; Laboratoire de Physiologie Cellulaire Respiratoire, INSERM E356; and Université Victor Segalen, Bordeaux, France

Correspondence and requests for reprints should be addressed to Dr. C.E. Brightling, M.R.C.P., Ph.D., Department of Respiratory Medicine, University Hospitals of Leicester, Groby Road, Leicester LE3 9QP, UK. E-mail: ceb17{at}le.ac.uk


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Airway smooth muscle (ASM) hyperplasia is a feature of asthma, and increases with disease severity. We hypothesized that this results from migration of ASM or progenitors in response to chemokines derived from ASM or mast cells within the ASM bundle.

Objectives: To examine expression of the chemokine receptor, CC chemokine receptor (CCR) 7, in vivo by ASM in patients with asthma and healthy control subjects, and by primary cultures of ASM and fibroblasts; to define expression of its ligands, CC chemokine ligand (CCL) 19 and CCL21, in bronchial biopsies, and primary cultures of ASM and mast cells; and to investigate CCR7's role in ASM migration and repair.

Methods: ASM was isolated from bronchoscopy and resection tissue. Receptor and chemokine expression was examined by immunohistochemistry, immunofluorescence, flow cytometry, ELISA, and reverse transcriptase–polymerase chain reaction. CCR7 function was examined by intracellular calcium measurements, chemotaxis, wound healing assays, and measurement of cell proliferation.

Measurements and Main Results: ASM, myofibroblasts, and fibroblasts expressed CCR7. CCL19, but not CCL21, was highly expressed in bronchial biopsies by mast cells and vessels in asthma of all severities, ASM in severe disease, and ex vivo ASM and mast cells. ASM CCR7 activation by CCL19-mediated intracellular calcium elevation and concentration-dependent migration, but not proliferation. Importantly, mast cell and ASM-derived CCL19 mediated ASM migration and repair.

Conclusions: The CCL19/CCR7 axis may play an important role in the development of ASM hyperplasia in asthma.

Key Words: airway smooth muscle • asthma • CC chemokine ligand 19 • CC chemokine receptor 7 • mast cells



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Both an increase in mast cell numbers in the airway smooth muscle (ASM) bundles and smooth muscle hyperplasia are features of asthma. The cause of this smooth muscle hyperplasia is unknown; emerging evidence suggests that ASM progenitors may migrate to the smooth muscle bundle in asthma to contribute to this hyperplasia.

What This Study Adds to the Field
We report for the first time that ASM and fibroblasts express functional CCR7, and that both ASM and mast cell-derived CCL19 mediate ASM migration in asthma via activation of CCR7, providing a novel chemotactic pathway for the recruitment of ASM or ASM progenitors to the asthmatic ASM compartment.

 
Asthma is a common disease and remains a significant cause of morbidity and mortality worldwide. It affects 10% of children and 5% of adults, and its prevalence continues to rise (1). The immunopathology of asthma is characterized by the presence of airway inflammation together with features of tissue repair known as remodeling (2). The airway inflammation in asthma is typically eosinophilic, with increased expression of T helper (Th) type 2 cytokines (3). In addition, the number of mast cells localized within the airway smooth muscle (ASM) bundle is increased in asthma, and is related to the degree of airway hyperresponsiveness (4, 5). Airway remodeling encompasses several structural changes in the airway wall, including reticular lamina and basement membrane thickening, an increased number of subepithelial myofibroblasts, and increased ASM mass. This latter feature stems from a combination of both ASM hyperplasia (6) and hypertrophy, which increases with disease severity (7).

The cause of ASM hyperplasia in asthma is unknown, and is often attributed to increased proliferation. Indeed, ASM proliferation is increased in ex vivo asthmatic ASM (8), but several reports have been unable to demonstrate increased ASM proliferation in vivo (6, 7). An alternative explanation is that ASM progenitors, either located within the airway wall or derived from peripheral blood fibroblast progenitors (fibrocytes) (9), migrate to the ASM bundle and differentiate into ASM. In support of this view, myofibroblasts expressing fibrocyte markers have been identified after ovalbumin challenge in a mouse model of asthma and after allergen challenge in human disease (10). Importantly, human fibrocytes express CCR3, CCR5, CCR7, and CXC receptor (CXCR) 4 (9), but only CCR7 (9) and CXCR4 (11) are functional. We have shown that the ligand for CXCR4, CXCL12, is constitutively released by the bronchial epithelium, but not by ASM (12). We therefore hypothesized that CCR7 is expressed by ASM and that ASM itself, or mast cells, are important sources of the CCR7 ligands CC chemokine ligand (CCL) 19 and CCL21.

In this study, we have demonstrated, using a number of approaches, that ASM and fibroblasts express functional CCR7 and that both ASM and mast cell–derived CCL19 mediate ASM migration and repair. Thus, the CCR7/CCL19 axis provides a novel chemotactic pathway for the recruitment of ASM or ASM progenitors to the asthmatic ASM compartment. Some of the results of these studies have been previously reported in the form of abstracts (13, 14).


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
A detailed description of these methods can be found in the online supplement.

Subjects
Subjects were recruited from Leicester, United Kingdom. Those with asthma gave an appropriate history and had objective evidence of variable airflow obstruction and/or airway hyperresponsiveness, as described previously (12). Severity was defined using GINA (Global Initiative for Asthma) guidelines (15). Subjects were nonsmokers with less than a 5 pack-year history. Subjects underwent bronchoscopy (16), and mucosal biopsies were embedded into glycolmethacrylate resin (17) or used for ASM isolation. Clinical characteristics are described in Table 1. The study was approved by the Leicestershire Ethics Committees. All patients gave their written, informed consent.


View this table:
[in this window]
[in a new window]
  		TABLE 1. SUBJECTS' CLINICAL CHARACTERISTICS

 
Cell Isolation and Culture
Pure ASM bundles were isolated from bronchial biopsies (n = 4 subjects with asthma) and large airway tissue from subjects undergoing surgery (n = 9). Primary ASM was cultured in Dulbecco's modified Eagle's medium (DMEM) Glutamax-1 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin, 100 µM nonessential amino acids, and 1 mM sodium pyruvate, and characterized as described previously (12). Primary fibroblasts were cultured from bronchial biopsies (n = 4) after removal of the ASM bundles and cultured in the same conditions as the ASM cells. Fibroblast cells were characterized by immunofluorescence as positive for the fibroblast marker anti–prolyl-4-hydroxylase (Dako, Ely, Cambridgeshire, UK) and negative for {alpha}-smooth muscle actin ({alpha}-SMA; Sigma, Gillingham, Dorset, UK).

Human lung mast cells (HLMCs) were obtained from normal lung (n = 7) obtained at surgery using immunomagnetic affinity purification (18). Cells were cultured in DMEM, 10% FBS supplemented with stem cell factor (SCF) (100 ng/ml), interleukin (IL)-10 (10 ng/ml), and IL-6 (50 ng/ml; R&D Systems, Abingdon, Oxfordshire, UK). The human mast cell line-1 (HMC-1) cell line was a generous gift from Dr. J. Butterfield (Mayo Clinic, Rochester, MN). HMC-1 cells were maintained in Iscove's modified DMEM (19). Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood from healthy donors by centrifugation on Ficoll.

CCR7 and Ligand Expression
CCR7 protein expression in biopsies was analyzed by immunohistochemistry (4) using a CCR7 monoclonal antibody (mAb; a gift from Millennium, Cambridge, MA). In primary ASM cells, CCR7 mRNA expression was analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR), and primary ASM and fibroblast protein expression were analyzed by flow cytometry (CCR7 mAb CD197; eBioscience, Wembley, UK) and immunofluorescence (CCR7 mAb; Millennium).

CCL19 and CCL21 protein expression was analyzed in biopsies by immunohistochemistry (4) with mAb to CCL19 and CCL21 (R&D Systems), in primary ASM cells, PBMC, HLMC, and HMC-1 cells by flow cytometry and immunofluorescence with mAb to CCL19 and CCL21 (R&D Systems). CCL19 expression in primary ASM cell supernatants and lysates was measured by ELISA (R&D Systems). Isotype controls were used where appropriate (Dako).

Functional Assays
Calcium imaging.
Changes in cytosolic Ca2+ concentration ([Ca2+]i) in ASM cells in response to CCL19 (100 ng/ml) were measured by ratiometric imaging on FURA-2–loaded cells using Openlab software (Improvision, Coventry, UK) (20).

Chemotaxis Assays.
We found that chemotaxis assays using standard Transwells with fibronectin or collagen-coated 8-µm inserts did not give us reproducible results for ASM migration toward platelet-derived growth factor (PDGF; R&D Systems) as a positive control (data not shown). Therefore, we developed a novel two-dimensional chemotaxis assay. ASM cells and fibroblasts were seeded onto 8–rectangular-well plates coated with 10 µg/ml fibronectin at a density of 0.25 x 106 cells/well and allowed to adhere overnight. Cells were then serum deprived in insulin/transferrin/sodium selenite (ITS) (ITS+3; Sigma, St. Louis, MO) medium for 24 h before experimentation. Cells were scratched along a line predrawn across the width of the well, on the underside of the 8-well plate, 22 mm from the bottom of the well. Any cells remaining between this line and the upper edge of the well were removed by scraping. The cells were then washed four times with ITS media to remove any debris. A piece of blotting paper (25 mm x 6 mm; Sigma) was then placed along the upper edge of the well, secured in place using silicon grease. The blotting paper was then impregnated with 200 µl of control media, recombinant CCL19 (25–200 ng; R&D Systems), or HMC-1 lysates (in a proportion of 1:2–20 HMC-1:ASM cells) before addition of 1.5 ml ITS media to the well in the presence or absence of CCL19 neutralizing antibody (5 µg/ml; R&D Systems) or isotype control (5 µg/ml; R&D Systems). Photomicrographs were taken at baseline and after 6 h using a Nikon Coolpix 5400 (Nikon, Japan) camera and an Olympus CK2 microscope (Olympus, Tokyo, Japan), at 200x magnification, across the width of the cell edge. The cells were then incubated at 37°C in 5% CO2. Using Powerpoint (Microsoft, Redmond, WA), the position of the cells at 0 h was traced around and transferred to the corresponding photograph at 6 h. A blinded observer counted the number of cells that had moved across the line at 6 h, under the different conditions.

To validate our novel chemotaxis assay, we assessed the within- and between-observer repeatability. Two blinded observers enumerated the number of ASM cells in 22 high-powered fields (from 4 donors) that had migrated after 6 h either toward PDGF (40 ng) or in ITS controls. One of these observers repeated the measurements. The within-observer intraclass correlation coefficient was 0.97 and the between-observer coefficient was 0.91, confirming that our assay is highly repeatable.

Wound healing assay.
As with the chemotaxis assays, ASM cells were seeded onto 8-well fibronectin-coated plates, serum deprived for 24 h in ITS medium, and then wounded using a sterile 200-µl pipette tip in a predetermined grid pattern. After wounding ASM cells were washed four times with ITS media before addition of ITS media plus vehicle/CCL19 (50–200 ng/ml) in the presence or absence of CCL19 neutralizing antibody or isotype control. Wounds were then photographed at baseline and after 6 h. The number of cells that had moved into the wound was counted by a blind observer.

Proliferation.
ASM proliferation was assessed by cell counts and using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, according to the manufacturer's instructions (Promega, Southampton, UK).

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 4 (GraphPad, San Diego, CA). Data are presented as mean (± SEM), except cell numbers in bronchial biopsies, which are presented as median (interquartile range [IQR]). Nonparametric data were analyzed using the Mann-Whitney test and parametric data by using t tests or analysis of variance. Differences were considered significant when p values were less than 0.05.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CCR7 Expression by ASM and Myofibroblasts
Immunohistochemistry.
ASM bundles expressed strong immunoreactivity for CCR7 in all subjects, both in those with asthma (n = 6) and those without (n = 5; Figures 1A–1C). The number of {alpha}-SMA+ cells in the bronchial submucosa distinct from the ASM bundle was increased in asthma 6 (IQR, 7) cells/mm2 compared with normal controls 0 (IQR, 3) cells/mm2 (p = 0.014). All of the {alpha}-SMA+ cells colocalized to CCR7+ cells (Figures 1D–1F).


Figure 1
Figure 1
View larger version (70K):
[in this window]
[in a new window]
  		Figure 1. CC chemokine receptor (CCR) 7 expressed by airway smooth muscle (ASM) and myofibroblasts in vivo. Example photomicrographs of a bronchial biopsy illustrating: (A) isotype control (original magnification: x100); (B) {alpha}-smooth muscle actin ({alpha}-SMA) expression (original magnification: x100); (C) sequential section demonstrating colocalization of CCR7 with the ASM bundle (original magnification: x100); (D) {alpha}-SMA staining of cells located between a vessel and the ASM bundle (arrows; original magnification: x200); (E) sequential section demonstrating colocalization of {alpha}-SMA with CCR7 (arrows; original magnification: x200). (F) The number of {alpha}-SMA+ cells in the submucosa was increased in asthma compared with healthy control subjects. Horizontal bars represent medians.

 
mRNA expression.
A PCR product of a size consistent with CCR7 was generated by RT-PCR from RNA purified from primary cultured ASM cells (n = 3; Figure 2A). DNA sequencing confirmed that this PCR product encoded the CCR7 sequence.


Figure 2
View larger version (75K):
[in this window]
[in a new window]
  		Figure 2. CCR7 expressed by ex vivo ASM and fibroblast cells. (A) Polymerase chain reaction (PCR) products generated by reverse transcriptase–PCR using CCR7-specific primers from purified primary cultured ASM RNA. The lower panel shows the positive control beta-actin (350 bp), and the upper panel shows CCR7 (530 bp). The example fluorescent histograms illustrate (B) cell surface CCR7 expression and (C) CCR7 expression by permeabilized ASM assessed by flow cytometry. (D) CCR7 expression by ASM and fibroblasts was confirmed by immunofluorescence. Fibroblast cells also stained strongly for the fibroblast marker, but were negative for {alpha}-SMA, which was highly positive in the ASM cells (nuclei stained blue and fluorescence in green).

 
Flow cytometry and immunofluorescence.
The proportion of primary cultured ASM cells that expressed CCR7 on their cell surface was 42 ± 3% (n = 7; Figure 2B). There was no difference in expression between ASM derived from patients with asthma and from normal control subjects, and the expression of CCR7 was not up-regulated by activation by CCL19 (data not shown). The CCR7 expression by permeabilized ASM was 53 ± 10% (n = 5; Figure 2C). Fibroblasts also expressed CCR7 on their surface (30 ± 10%; n = 3) and intracellularly (71 ± 14%; n = 3). The expression of CCR7 by ASM and fibroblasts was also confirmed by immunofluorescence (Figure 2D).

ASM CCR7 Functional Response to CCL19 Activation
Calcium imaging.
After ASM activation by CCL19, 81% of cells responded with a transient rise in [Ca2+]i (n = 72 individual cells, 3 donors). The mean increase in [Ca2+]i in these cells was 357 (± 90 nM; Figure 3A).


Figure 3
Figure 3
Figure 3
Figure 3
Figure 3
View larger version (178K):
[in this window]
[in a new window]
  		Figure 3. ASM functional response to CCR7 activation by CC chemokine ligand (CCL) 19. (A) Example of increased cytosolic free Ca2+ ([Ca2+]i) after CCL19 activation, as illustrated by increased intracellular green fluorescence by FURA-2–loaded ASM in the right panel compared to the left panel; below, corresponding graph showing transient rise in [Ca2+]i after activation by CCL19 (arrow represents point at which CCL19 [100 ng/ml] was added to cells). (B) Concentration-dependent chemotaxis of ASM towards CCL19 with platelet-derived growth factor (PDGF; 40 ng) as a positive control. Data are presented as means ± SEM for five donors and comparisons were made to insulin/transferrin/sodium selenite (ITS) (ITS+3; Sigma, St. Louis, MO) alone. (C) Migration of ASM towards CCL19 was inhibited by anti-CCL19 neutralizing antibody. Data are presented as means ± SEM for three donors. (D) Representative pictures of ASM chemotaxis seen after 6 h in 1 high-powered field (hpf) with no CCL19, upper left picture, 100 ng CCL19, upper right, 100 ng CCL19 + isotype control, lower left, and 100 ng CCL19 + anti-CCL19, lower right. The black line shows the position of the cells at 0 h. (E) Migration of fibroblasts towards CCL19 was inhibited by anti-CCL19 neutralizing antibody. Data are presented as means ± SEM for three donors. *p < 0.05; **p < 0.01.

 
Chemotaxis.
CCL19 mediated ASM (n = 5) migration and exhibited a bell-shaped concentration–response curve (Figure 3B). The number of ASM cells per high-powered field that migrated to CCL19 (100 ng) was blocked using a CCL19 neutralizing antibody (11.6 ± 0.9 vs. 8.4 ± 0.7; p < 0.01; Figures 3C and 3D). We confirmed that this directional movement was predominately due to chemotaxis rather than chemokinesis, as addition of CCL19 to the ITS media did not result in significantly more ASM movement than ITS media alone (n = 3; data not shown). CCL19 also mediated fibroblast migration in a concentration-dependent manner (data not shown). Maximal migration was to 150 ng/ml CCL19 (14.7 ± 0.8 cells/high-powered field [hpf] vs. 11.9 ± 0.7 cells/hpf in control; p < 0.05), which was inhibited by a CCL19 neutralizing antibody (11.6 ± 0.5 cells/hpf vs. 14.1 ± 0.8 cells/hpf with isotype control; p < 0.05; Figure 3E; n = 3).

Proliferation.
The number of ASM cells cultured for 24 or 96 h in the presence of CCL19 (25–100 ng/ml) was not significantly different from ASM cells cultured in media alone. This lack of effect on ASM proliferation by CCL19 was confirmed by the MTS assay (data not shown).

CCL19 Expressed by Mast Cells, ASM, and Bronchial Vessels in Asthma
We found that the number of CCL19+ cells in the bronchial submucosa was increased in mild asthma (GINA I-II; n = 7; 5 [IQR, 9] cells/mm2) and moderate to severe asthma (GINA III-IV; n = 6; 2 [IQR, 2] cells/mm2) compared with normal controls (0 [IQR, 1] cells/mm2; n = 7; p = 0.03; Figure 4A). The number of mast cells was similar in the bronchial submucosa in those subjects with mild asthma (22 [IQR, 9] cells/mm2], moderate to severe asthma (18 [IQR, 12] cells/mm2), and normal control subjects (20 [IQR, 12] cells/mm2). CCL19, but not CCL21 expression (data not shown), was colocalized to mast cells (Figures 4B–4D). In the subjects with asthma, 83 ± 5% of the CCL19+ cells colocalized to tryptase+ cells, and 17 ± 4% of tryptase+ cells were also CCL19+. CCL19+ cells were observed within the ASM bundle in mild to moderate asthma 2 (IQR, 1) cells/mm2 (n = 7), but not in the normal controls (n = 4; Figure 4E). CCL19+ mast cells could not be identified in the ASM bundle in severe asthma due to the high expression of CCL19 by the ASM cells in these subjects.


Figure 4
Figure 4
View larger version (63K):
[in this window]
[in a new window]
  		Figure 4. CCL19 expressed by mast cells in vivo. (A) The number of bronchial submucosal cells that expressed CCL19 in subjects with asthma and healthy control subjects. Horizontal bars represent medians. The number of submucosal CCL19+ cells was increased in asthma (*p = 0.028, Kruskal-Wallis). Representative photomicrographs of a bronchial biopsy from a subject with asthma stained with (B) isotype control (original magnification: x100), (C) tryptase (original magnification: x400), and (D) CCL19 (original magnification: x400) are sequential sections, and illustrate that CCL19 was colocalized to mast cells. (E) CCL19+ cells within the ASM bundle in a patient with mild asthma (original magnification: x200). Arrows highlight positively stained cells.

 
The proportion of HLMC cells that expressed CCL19 on their cell surface was 28 ± 5% (n = 7), and by permeabilized HLMC cells was 58 ± 5% (n = 4). Likewise, HMC-1 cells expressed CCL19 on their cell surface 42 ± 2% (n = 3), and intracellularly 61 ± 1% (n = 3) (Figures 5A–5C and 5E). Neither HLMC nor HMC-1 cells expressed CCL21, although expression of CCL21 by PBMC was high (data not shown). CCL19 expression by HLMC and HMC-1 cells was confirmed by immunofluorescence (Figure 5D).


Figure 5
View larger version (27K):
[in this window]
[in a new window]
  		Figure 5. CCL19 expressed by mast cells in vitro. Cell surface CCL19 expression by human lung mast cells (HLMCs) and human mast cell (HMC)-1 was assessed by flow cytometry. The example dot plots illustrate: (A) HLMC dot plot for the mouse IgG1 double negative, (B) HLMCs that were CD117+, and (C) HLMCs that were CD117+ and CCL19+. CCL19 expression by HLMCs was confirmed by immunofluorescence (nuclei stained blue, CD117 red, and CCL19 green), as shown by (D) upper left panel, isotype control, upper right, tryptase, lower left, CCL19, and lower right, composite image illustrating CCL19+ HLMCs. (E) Fluorescent histograms showing CCL19 expression by HMC-1 cells (black line), with corresponding isotype control (gray line) as assessed by flow cytometry. (F) CCL19 expression by HMC-1, shown by immunofluorescence: left: isotype control, and right: CCL19+ HMC-1 cells.

 
CCL19 was not measurable in mast cell supernatants from resting cells or cells activated by IgE/anti-IgE for 4 and 24 h. However, CCL19 was measurable in HLMC lysates (454 ± 55 pg/106 cells; n = 4) and HMC-1 cells (102 ± 13 pg/106 cells; n = 4).

CCL19 was constitutively expressed by ASM in bronchial biopsies from four out of four patients with severe asthma (GINA IV), but was not expressed by ASM in subjects with mild to moderate asthma (GINA I-III) (n = 8; Figure 6A). CCL19 was also expressed by vessels in the bronchial submucosa in asthma of all severities (Figure 6B), but not in normal control subjects.


Figure 6
Figure 6
View larger version (55K):
[in this window]
[in a new window]
  		Figure 6. CCL19 is expressed by ASM in severe asthma and ex vivo ASM cells. Representative photomicrographs of a bronchial biopsy illustrating: (A) CCL19+ ASM bundle (arrow) in a patient with severe asthma, and (B) CCL19+ vessel in a patient with mild asthma (arrow; original magnification: x200). The example fluorescent histograms represent populations of (C) primary cultured ASM cells that were CCL19+ (black line) plotted with the corresponding isotype control (gray line). CCL19 expression by ASM was confirmed by immunofluorescence (nuclei stained blue, CCL19 stained green), as shown by (D) left panel: isotype control, and (E) right panel: CCL19. (F) The concentration of CCL19 in ASM lysate (open bars) and supernatant (closed bars). There was a time-dependent increase in the release of CCL19 by ASM after wounding.

 
ASM cells expressed CCL19, but not CCL21, on their cell surface (59 ± 7%; n = 8). CCL19 expression by ASM cells was confirmed by immunofluorescence (Figures 6C–6E).

CCL19 was measurable in ASM supernatants from unstimulated ASM at low concentrations (7 ± 3 pg/106 cells), but was not affected by stimulation with IL-1beta, IFN-{gamma}, and TNF-{alpha}, alone or in combination. The CCL19 concentration in ASM cell lysates was 42 ± 4 pg/106 cells. Interestingly, after wounding of ASM cells, there was a significant time-dependent release of CCL19. The CCL19 concentration in supernatants 1 h after wounding was 25 ± 8 pg/106 cells (p = 0.026; Figure 6F).

Mast Cell–derived CCL19 Promotes ASM Migration
ASM cells (n = 4) migrated toward HMC-1 lysates derived in a proportion of 1:5 or 10 HMC-1:ASM cells (Figure 7A). This migration was predominately CCL19 mediated, as evidenced by a marked reduction in migration in the presence of a CCL19 neutralizing antibody (12 ± 0.8 cells/hpf vs. 9.4 ± 0.7; p < 0.05; Figure 7B).


Figure 7
Figure 7
View larger version (27K):
[in this window]
[in a new window]
  		Figure 7. Mast cell–derived CCL19 promotes ASM migration. (A) HMC-1 lysates were impregnated onto blotting paper at ratios within the range: lysate from 1 HMC-1 cell per 2–20 ASM cells, and migration of ASM was assessed after 6 h. Data are presented as means ± SEM for four donors, except 1:5 (n = 3 donors). Comparisons were made with ITS alone. (B) Effect of anti-CCL19 on the chemotactic response to HMC-1 lysates was assessed by adding HMC-1 lysate (1:5–1:10) to the blotting paper, as described previously here, in the presence of 5 µg/ml anti-CCL19 or isotype control added to the media at 0 h. Data are presented as means ± SEM for four donors. Statistical differences were assessed using unpaired, two-tailed t tests; *p < 0.05; **p < 0.01.

 
When the ASM cells were wounded in the presence of HMC-1 cells, no significant increase in the wounding response was seen above ITS media alone (data not shown).

ASM-derived CCL19 Mediates Wound Repair
Wound healing was promoted by 10 ng/ml PDGF (29.7 ± 1.2 cells/hpf vs. 24.5 ± 0.8 in control; p < 0.01; n = 4). The wound healing response of ASM cells (n = 3) was unaffected by addition of recombinant CCL19. Interestingly, the wound-healing response seen in the presence of 100 ng/ml CCL19 with the CCL19 neutralizing antibody was significantly reduced, such that the number of ASM cells that migrated into the wound was less than that for ITS media alone (Figures 8A and 8B). This suggested that ASM-derived CCL19 promoted wound healing. Indeed, in the presence of ITS media alone, the anti-CCL19 antibody significantly reduced the wound-healing response (22 ± 1.0 cells/hpf vs. 18.0 ± 1.0; p < 0.01; Figure 8C).


Figure 8
Figure 8
Figure 8
View larger version (142K):
[in this window]
[in a new window]
  		Figure 8. ASM-derived CCL19 mediates wound repair. (A) ASM cells were wounded in the presence of CCL19 (50–200 ng/ml). No statistical differences were seen compared to ITS alone. The wound-healing response in the presence of 100 ng/ml CCL19 + 5 µg/ml anti-CCL19 was attenuated compared with 100 ng/ml CCL19 + 5 µg/ml isotype control. (B) Representative pictures of the wound-healing response seen after 6 h with ITS alone (upper left), CCL19 (upper right), CCL19 + isotype control (lower left), and CCL19 + anti-CCL19 (lower right). The black lines show the position of the cells at 0 h. (C) ASM cells wounded in the presence of 5 µg/ml CCL19 neutralizing antibody or isotype control showed that ASM-derived CCL19 mediated wound repair. Data are presented as means ± SEM for three donors. Statistical differences were assessed using unpaired, two-tailed t tests; *p < 0.05; **p < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We demonstrate, for the first time, that ASM cells, myofibroblasts, and fibroblasts express CCR7, and that vessels in the bronchial submucosa, mast cells, and ASM itself are important sources of the CCR7 ligand, CCL19. Importantly, we found that CCL19 expression by mast cells was increased in asthma, independent of disease severity, and was constitutively expressed by ASM in severe disease. CCL19 derived from mast cells and ASM mediated ASM migration and repair. These observations, together with our previous finding that mast cell numbers were increased in the ASM bundles in asthma (4), provide evidence that activation of CCR7+ ASM or myofibroblasts by CCL19 derived from mast cells and/or ASM may contribute to the development of increased ASM mass in asthma.

CCR7 is involved in the organization of normal lymphoid tissue during development (21). It is expressed by naive and central memory T cells (22) and by mature dendritic cells (21). CCR7 is also highly expressed by malignant breast cells, and has been implicated in metastatic breast cancer (23). In addition to its role in inflammatory cell trafficking, CCR7 has been implicated in tissue repair mechanisms. Peripheral blood fibrocytes (9) and hepatic stellate cells (24) also express CCR7. Fibrocytes are peripheral blood fibroblast precursors that migrate to areas of tissue injury and participate in normal wound repair mechanisms (9, 25). They were found to be the major source of matrix proteins in an animal model of pulmonary fibrosis (11), although their exact role in interstitial lung disease in humans is unclear (26, 27). We report here, for the first time, that ASM and myofibroblasts in bronchial biopsies express CCR7. CCR7+/{alpha}-SMA+ myofibroblasts were only observed in asthma, and were typically located between vessels and the ASM bundle. These myofibroblasts may be derived from peripheral blood fibrocytes or other progenitors within the airway wall. A fibrocytic origin is supported by the finding that, after allergen challenge in patients with asthma, there was an increase in fibrocyte-like {alpha}-SMA+ cells in the subepithelium (10). We demonstrated that primary ASM and fibroblasts expressed CCR7, and that there was no difference in the CCR7 expression by ASM in patients with asthma compared with healthy control subjects. CCR7 expressed by ASM and fibroblasts was functional, as evidenced by increased intracellular calcium and concentration-dependent chemotaxis after activation by CCL19 that was inhibited by a specific CCL19 neutralizing antibody.

If CCR7 expression by ASM is important in asthma, then the CCR7 ligands, CCL19 and CCL21, should be differentially expressed in health and disease. In bronchial biopsies, we found that CCL19 immunoreactivity was markedly increased in asthma. This immunoreactive CCL19 was localized predominately to submucosal vessels and mast cells located in both the submucosa and ASM in mild disease; ASM also demonstrated strong CCL19 expression in severe disease. The distribution of CCL19 expression in the bronchial mucosa provides a biologically plausible mechanism for the recruitment of CCR7+ ASM cells or their progenitors to the ASM bundle. In asthma, CCL19+ vessels may be important in trafficking of CCR7+ fibrocytes to the airway, and CCL19 expressed by mast cells and ASM may direct ASM migration toward the ASM bundle. Kulka and colleagues reported recently that human mast cells expressed CCL19 mRNA and protein (28), and we found that CCL19 was measurable in HLMC and HMC-1 lysates. Importantly, CCL19 was also expressed by primary ASM, and secretion was increased in response to wounding. CCL19 was highly expressed, both on the cell surface and in the cytoplasm of mast cells and ASM. It is well established that mast cells are capable of expressing chemokines and cytokines bound to glycosaminoglycans on the cell surface (29), and the CCR7 ligands in particular can bind in this fashion. Mast cells adhere to ASM (30), so localization of CCL19 on the surface of ASM and mast cells is predicted to lead to a high concentration of this chemokine at the cell–cell interface and, therefore, is likely to play a key role in mast cell–ASM and ASM–ASM cross-talk. In contrast to CCL19, CCL21 was not expressed by mast cells or ASM. This is consistent with an earlier observation that in atherosclerosis CCL19 mRNA, but not CCL21, was highly expressed by vascular smooth muscle (31).

One criticism of our study is that the absolute concentration of CCL19 in mast cell lysates and ASM supernatants was low, and our data would predict that these concentrations are insufficient to mediate ASM chemotaxis. However, the CCL19 concentration may have been underestimated due to masking of CCL19 by binding to glycosaminoglycans (29). Indeed, our view that mast cell and ASM-derived CCL19 is functionally important is supported by a number of observations. Mast cell lysates were chemotactic for ASM in a dose-dependent manner, with migration maximal to a ratio of mast cells to ASM of 1:10, which was similar to the proportion of mast cells in the ASM bundle (4). This effect was completely abrogated by CCL19 neutralization, suggesting that the CCR7/CCL19 axis is the dominant pathway for mast cell–mediated ASM migration. ASM wound repair was partially attenuated by neutralizing CCL19, supporting a role for CCL19 in ASM repair mechanisms. Interestingly, CCL19 did not mediate proliferation, suggesting that the effect of CCL19 on ASM is likely to be reserved to chemotaxis.

The view that ASM hyperplasia may be a consequence of fibrocytes trafficking to the airway is consistent with mechanisms proposed in skin wound healing (25), and is analogous to the current concepts of cardiac myocyte progenitors contributing to cardiac repair after myocardial infarction (32). However, in addition to CCL19 activation of CCR7 mediating ASM migration, other chemokines may play a role. Indeed, asthmatic ASM expresses functional CCR3 (33). CCR3 ligands, such as CCL11, are produced by ASM (12) and are increased in severe disease (34). Therefore, this chemokine may also be important in mediating migration of ASM in the bronchial mucosa, but the role of CCR3 in the recruitment of ASM progenitors to the airway is uncertain. Fibrocytes do express CCR3 (9), but only CCR7 (9) and CXCR4 (11) have been reported to be functional. Interestingly, mast cell tryptase degrades ASM-derived CCL11 (35), and CXCL10, which is elevated in asthma (12), is a natural antagonist for CCR3 (36). Therefore, mast cells within the ASM bundle in asthma may inactivate the CCL11 released by ASM, calling in to question the importance of the CCR3/CCL11 axis in ASM migration. In addition, the CXCR4 ligand, CXCL12, is preferentially expressed by the bronchial epithelium (12), and this may provide a mechanism for the enhanced subepithelial (37) and luminal myofibroblasts (38) observed in asthma.

In conclusion, our findings strongly support a role for recruitment of ASM progenitors to the ASM bundle in asthma mediated by the activation of CCR7 by CCL19 expressed by bronchial submucosal vessels, mast cells, and possibly ASM itself. Targeting the migration of ASM and its progenitors to the ASM bundle in asthma may provide novel therapies to modulate the increased ASM mass observed in asthma.


   Acknowledgments
 
The authors thank Millennium for kindly providing the CCR7 mAb and Miss N Neale for technical support.


   FOOTNOTES
 
Supported by Asthma UK and the Department of Health Clinician Scientist Award.

* These investigators contributed equally to this article. Back

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

Originally Published in Press as DOI: 10.1164/rccm.200603-394OC on September 7, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 17, 2006; accepted in final form August 8, 2006


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hartert TV, Peebles RS Jr. Epidemiology of asthma: the year in review. Curr Opin Pulm Med 2000;6:4–9.[CrossRef][Medline]
  2. Kay AB. Pathology of mild, severe, and fatal asthma. Am J Respir Crit Care Med 1996;154:S66–S69.[Medline]
  3. Wardlaw AJ, Brightling C, Green R, Woltmann G, Pavord I. Eosinophils in asthma and other allergic diseases. Br Med Bull 2000;56:985–1003.[Abstract/Free Full Text]
  4. Brightling CE, Bradding P, Symon FA, Holgate ST, Wardlaw AJ, Pavord ID. Mast-cell infiltration of airway smooth muscle in asthma. N Engl J Med 2002;346:1699–1705.[Abstract/Free Full Text]
  5. El-Shazly A, Berger P, Girodet P, Ousova O, Fayon M, Vernejoux J, Marthan R, Tunon-de-Lara M. Fraktalkine produced by airway smooth muscle cells contributes to mast cell recruitment in asthma. J Immunol 2006;176:1860–1868.[Abstract/Free Full Text]
  6. Woodruff PG, Dolganov GM, Ferrando RE, Donnelly S, Hays SR, Solberg OD, Carter R, Wong HH, Cadbury PS, Fahy JV. Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression. Am J Respir Crit Care Med 2004;169:1001–1006.[Abstract/Free Full Text]
  7. Benayoun L, Druilhe A, Dombret MC, Aubier M, Pretolani M. Airway structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med 2003;167:1360–1368.[Abstract/Free Full Text]
  8. Johnson PR, Roth M, Tamm M, Hughes M, Ge Q, King G, Burgess JK, Black JL. Airway smooth muscle cell proliferation is increased in asthma. Am J Respir Crit Care Med 2001;164:474–477.[Abstract/Free Full Text]
  9. Abe R, Donnelly SC, Peng T, Bucala R, Metz CN. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol 2001;166:7556–7562.[Abstract/Free Full Text]
  10. Schmidt M, Sun G, Stacey MA, Mori L, Mattoli S. Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol 2003;171:380–389.[Abstract/Free Full Text]
  11. Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY, Belperio JA, Keane MP, Strieter RM. Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest 2004;114:438–446.[CrossRef][Medline]
  12. Brightling CE, Ammit AJ, Kaur D, Black JL, Wardlaw AJ, Hughes JM, Bradding P. The CXCL10/CXCR3 axis mediates human lung mast cell migration to asthmatic airway smooth muscle. Am J Respir Crit Care Med 2005;171:1103–1108.[Abstract/Free Full Text]
  13. Kaur D, Woodman L, Okayama Y, Bradding P, Brightling CE. The CCR7 ligand CCL19, but not CCL21, is expressed by mast cells: implications for mast cell–airway smooth muscle cell interactions in asthma [abstract]. Am J Respir Crit Care Med 2005;2:A337.
  14. Saunders R, Kaur D, Siddiqui S, Woodman L, Bradding P, Brightling CE. CCL19 derived from airway smooth muscle and mast cells mediates airway smooth muscle migration and repair [abstract]. Proc Am Thorac Soc 2006;3:A281.
  15. Global Initiative for Asthma (GINA) guideline. 2005 Available from: http://www.ginasthma.com (accessed July 31, 2006).
  16. British Thoracic Society. British Thoracic Society guidelines on diagnostic flexible bronchoscopy. Thorax 2001;56:i1–21.[Free Full Text]
  17. Britten KM, Howarth PH, Roche WR. Immunohistochemistry on resin sections: a comparison of resin embedding techniques for small mucosal biopsies. Biotech Histochem 1993;68:271–280.[Medline]
  18. Sanmugalingam D, Wardlaw AJ, Bradding P. Adhesion of human lung mast cells to bronchial epithelium: evidence for a novel carbohydrate-mediated mechanism. J Leukoc Biol 2000;68:38–46.[Abstract/Free Full Text]
  19. Duffy SM, Leyland ML, Conley EC, Bradding P. Voltage-dependent and calcium-activated ion channels in the human mast cell line HMC-1. J Leukoc Biol 2001;70:233–240.[Abstract/Free Full Text]
  20. Brightling CE, Kaur D, Berger P, Morgan A, Wardlaw AJ, Bradding P. Differential expression of CCR3 and CXCR3 by human lung and bone marrow–derived mast cells: implications for tissue mast cell migration. J Leukoc Biol 2005;77:759–766.[Abstract/Free Full Text]
  21. Luther SA, Bidgol A, Hargreaves DC, Schmidt A, Xu Y, Paniyadi J, Matloubian M, Cyster JG. Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis. J Immunol 2002;169:424–433.[Abstract/Free Full Text]
  22. Campbell JJ, Murphy KE, Kunkel EJ, Brightling CE, Soler D, Shen Z, Boisvert J, Greenberg HB, Vierra MA, Goodman SB, et al. CCR7 expression and memory T cell diversity in humans. J Immunol 2001;166:877–884.[Abstract/Free Full Text]
  23. Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001;410:50–56.[CrossRef][Medline]
  24. Bonacchi A, Petrai I, Defranco RM, Lazzeri E, Annunziato F, Efsen E, Cosmi L, Romagnani P, Milani S, Failli P, et al. The chemokine CCL21 modulates lymphocyte recruitment and fibrosis in chronic hepatitis C. Gastroenterology 2003;125:1060–1076.[CrossRef]
  25. Mori L, Bellini A, Stacey MA, Schmidt M, Mattoli S. Fibrocytes contribute to the myofibroblast population in wounded skin and originate from the bone marrow. Exp Cell Res 2005;304:81–90.[CrossRef][Medline]
  26. Choi ES, Pierce EM, Jakubzick C, Carpenter KJ, Kunkel SL, Evanoff H, Martinez FJ, Flaherty KR, Moore BB, Toews GB, et al. Focal interstitial CC chemokine receptor 7 (CCR7) expression in idiopathic interstitial pneumonia. J Clin Pathol 2006;59:28–39.[Abstract/Free Full Text]
  27. Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH. Bone marrow–derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004;113:243–252.[CrossRef][Medline]
  28. Kulka M, Fukuishi N, Rottem M, Mekori YA, Metcalfe DD. Mast cells, which interact with Escherichia coli, up-regulate genes associated with innate immunity and become less responsive to Fc{epsilon}RI-mediated activation. J Leukoc Biol 2006;79:339–350.[Abstract/Free Full Text]
  29. Patel DD, Koopmann W, Imai T, Whichard LP, Yoshie O, Krangel MS. Chemokines have diverse abilities to form solid phase gradients. Clin Immunol 2001;99:43–52.[CrossRef][Medline]
  30. Yang W, Kaur D, Okayama Y, Ito A, Wardlaw AJ, Brightling CE, Bradding P. Human lung mast cells adhere to human airway smooth muscle, in part, via tumor suppressor in lung cancer-1. J Immunol 2006;176:1238–1243.[Abstract/Free Full Text]
  31. Reape TJ, Rayner K, Manning CD, Gee AN, Barnette MS, Burnand KG, Groot PHE. Expression and cellular localization of the CC chemokines PARC and ELC in human atherosclerotic plaques. Am J Pathol 1999;154:365–374.[Abstract/Free Full Text]
  32. Yoon YS, Lee N, Scadova H. Myocardial regeneration with bone-marrow–derived stem cells. Biol Cell 2005;97:253–263.[CrossRef][Medline]
  33. Joubert P, Lajoie-Kadoch S, Labonte I, Gounni AS, Maghni K, Wellemans V, Chakir J, Laviolette M, Hamid Q, Lamkhioued B. CCR3 expression and function in asthmatic airway smooth muscle cells. J Immunol 2005;175:2702–2708.[Abstract/Free Full Text]
  34. Pepe C, Foley S, Shannon J, Lemiere C, Olivenstein R, Ernst P, Ludwig MS, Martin JG, Hamid Q. Differences in airway remodeling between subjects with severe and moderate asthma. J Allergy Clin Immunol 2005;116:544–549.[CrossRef][Medline]
  35. Pang L, Nie M, Corbett L, Sutcliffe A, Knox AJ. Mast cell beta-tryptase selectively cleaves eotaxin and RANTES and abrogates their eosinophil chemotactic activities. J Immunol 2006;176:3788–3795.[Abstract/Free Full Text]
  36. Loetscher P, Pellegrino A, Gong JH, Mattioli I, Loetscher M, Bardi G, Baggiolini M, Clark-Lewis I. The ligands of CXC chemokine receptor 3, I-TAC, Mig, and IP10, are natural antagonists for CCR3. J Biol Chem 2001;276:2986–2991.[Abstract/Free Full Text]
  37. Gizycki M, Adelroth E, Rogers A, O'Byrne P, Jeffery PK. Myofibroblast involvement in the allergen-induced late response in mild atopic asthma. Am J Respir Cell Mol Biol 1997;16:664–673.[Abstract]
  38. Larsen K, Tufvesson E, Malmstrom J, Morgelin M, Wildt M, Andersson A, Lindstrom A, Malmstrom A, Lofdahl CG, Marko-Varga G, et al. Presence of activated mobile fibroblasts in bronchoalveolar lavage from patients with mild asthma. Am J Respir Crit Care Med 2004;170:1049–1056.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
A. M. Misior, D. A. Deshpande, M. J. Loza, R. M. Pascual, J. D. Hipp, and R. B. Penn
Glucocorticoid- and Protein Kinase A-Dependent Transcriptome Regulation in Airway Smooth Muscle
Am. J. Respir. Cell Mol. Biol., July 1, 2009; 41(1): 24 - 39.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
K. Mahn, S. J. Hirst, S. Ying, M. R. Holt, P. Lavender, O. O. Ojo, L. Siew, D. E. Simcock, C. G. McVicker, V. Kanabar, et al.
Diminished sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) expression contributes to airway remodelling in bronchial asthma
PNAS, June 30, 2009; 106(26): 10775 - 10780.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
S. Zuyderduyn, M. B. Sukkar, A. Fust, S. Dhaliwal, and J. K. Burgess
Treating asthma means treating airway smooth muscle cells
Eur. Respir. J., August 1, 2008; 32(2): 265 - 274.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
S. Siddiqui, F. Hollins, and C. E. Brightling
What can we learn about airway smooth muscle from the company it keeps?
Eur. Respir. J., July 1, 2008; 32(1): 9 - 11.
[Full Text] [PDF]

Home page
 J. Immunol.Home page
P. Joubert, S. Lajoie-Kadoch, M. Welman, S. Dragon, S. Letuvee, B. Tolloczko, A. J. Halayko, A. S. Gounni, K. Maghni, and Q. Hamid
Expression and Regulation of CCR1 by Airway Smooth Muscle Cells in Asthma
J. Immunol., January 15, 2008; 180(2): 1268 - 1275.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
R. B. Penn and J. L. Benovic
Regulation of Heterotrimeric G Protein Signaling in Airway Smooth Muscle
Proceedings of the ATS, January 1, 2008; 5(1): 47 - 57.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
J. K. Bentley and M. B. Hershenson
Airway Smooth Muscle Growth in Asthma: Proliferation, Hypertrophy, and Migration
Proceedings of the ATS, January 1, 2008; 5(1): 89 - 96.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
W. T. Gerthoffer
Migration of Airway Smooth Muscle Cells
Proceedings of the ATS, January 1, 2008; 5(1): 97 - 105.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
S. R. Singh and I. P. Hall
Airway Myofibroblasts and Their Relationship with Airway Myocytes and Fibroblasts
Proceedings of the ATS, January 1, 2008; 5(1): 127 - 132.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
S. Siddiqui, F. Hollins, S. Saha, and C. E. Brightling
Inflammatory cell microlocalisation and airway dysfunction: cause and effect?
Eur. Respir. J., December 1, 2007; 30(6): 1043 - 1056.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
M. C. Shepherd, S. M. Duffy, T. Harris, G. Cruse, M. Schuliga, C. E. Brightling, C. B. Neylon, P. Bradding, and A. G. Stewart
KCa3.1 Ca2+Activated K+ Channels Regulate Human Airway Smooth Muscle Proliferation
Am. J. Respir. Cell Mol. Biol., November 1, 2007; 37(5): 525 - 531.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
K. Parameswaran, K. Radford, A. Fanat, J. Stephen, C. Bonnans, B. D. Levy, L. J. Janssen, and P. G. Cox
Modulation of Human Airway Smooth Muscle Migration by Lipid Mediators and Th-2 Cytokines
Am. J. Respir. Cell Mol. Biol., August 1, 2007; 37(2): 240 - 247.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
W. C. Moore and S. P. Peters
Update in Asthma 2006
Am. J. Respir. Crit. Care Med., April 1, 2007; 175(7): 649 - 654.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200603-394OCv1
174/11/1179    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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kaur, D.
Right arrow Articles by Brightling, C. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kaur, D.
Right arrow Articles by Brightling, C. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2006 American Thoracic Society