| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 20, 2004 as doi:10.1096/fj.03-1492fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA; and
* Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
1Correspondence: CIIT Centers for Health Research, P.O. Box 12137, Research Triangle Park, NC 27709, USA. E-mail: jbonner{at}ciit.org
SPECIFIC AIMS
The aim of this study was to determine the mechanism by which interleukin-13 (IL-13) stimulates the proliferation of lung fibroblasts, the central cell type that contributes to the progression of airway fibrosis in diseases such as asthma and chronic bronchitis. We hypothesized that IL-13 acts through the STAT-6 signaling pathway to trigger the release of a soluble, autocrine growth factor to stimulate fibroblast mitogenesis.
PRINCIPAL FINDINGS
1. IL-13 stimulates mitogenesis of rat, mouse, and human lung fibroblasts
Cultures of rat, mouse, or human lung fibroblasts were treated with increasing concentrations of IL-13 and assayed for incorporation of [3H]thymidine over 24 or 48 h. Although IL-13 stimulated a modest dose-dependent increase in cell proliferation at 24 h, the mitogenic effect of IL-13 was much more pronounced after 48 h. PDGF-BB, a well-characterized mitogen for lung fibroblasts, had a greater effect on mitogenesis at 24 h than at 48 h. The delayed kinetics of IL-13-stimulated mitogenesis suggested a secondary factor was mediating the proliferative event.
2. IL-13 stimulates the release of a 
30 kDa soluble mitogen
Conditioned medium was collected at various times and assayed for mitogenic activity on fresh cultures of rat lung fibroblasts in the presence of [3H]thymidine for 24 h. Conditioned medium collected 1 to 6 h after IL-13 treatment maximally stimulated mitogenesis in the 24 h [3H]thymidine assay. These data indicated that these cells released a soluble mitogen within hours after exposure to IL-13; this time lag required for the release of a secondary mediator explained the delayed kinetics of IL-13-stimulated mitogenesis. We further characterized this soluble mediator using size exclusion/gel filtration chromatography and found that the fraction containing the majority of mitogenic activity eluted with a molecular mass of 30 kDa.
3. IL-13 mediates mitogenesis of lung fibroblasts through an autocrine pathway involving release of PDGF-AA
Earlier we had shown that platelet-derived growth factor-AA (PDGF-AA) is a 30 kDa mitogen produced by rat lung fibroblasts, and a recent cDNA microarray study provided evidence that the PDGF-A chain gene was a candidate for a 30 kDa secreted factor in human lung fibroblasts treated with IL-13. Therefore, we postulated that the identity of the soluble mediator secreted in response to IL-13 treatment was PDGF-AA. To test this, we measured IL-13-induced [3H]thymidine uptake in the presence of a PDGF-AA-specific neutralizing antibody (Fig. 1
A). We found that IL-13-stimulated mitogenesis was blocked in a concentration-dependent manner by anti-PDGF-AA. We performed Western blot analysis on IL-13-stimulated human lung fibroblasts and found a dose-dependent induction of PDGF-AA protein (Fig. 1B
). Moreover, we observed that immunoreactive PDGF-AA in supernatants from IL-13-stimulated fibroblasts coeluted with the major peak of mitogenic activity at 30 kDa by size exclusion chromatography. IL-13 was incapable of stimulating mitogenesis of mouse embryonic fibroblasts carrying the Patch mutation. These mutants have a deletion of the gene encoding PDGFR
, the receptor that selectively binds PDGF-AA. However, mouse embryonic fibroblasts with the Patch mutation that had been transgenically modified to express PDGFR, proliferated in response to IL-13. Collectively, these data indicated that IL-13-mediated lung fibroblast mitogenesis was dependent on autocrine release of PDGF-AA.
|
4. IL-13-stimulated lung fibroblast proliferation and PDGF-AA secretion is dependent on STAT-6 signaling
Both IL-13 and IL-4 have been shown to exert many of their effects on airway remodeling by activating the signal transducer and activator of trancription-6 (STAT-6) pathway. To determine whether IL-13-induced lung fibroblast proliferation was dependent on STAT-6, we used mouse lung fibroblast cultures derived from STAT-6 null mice. IL-13 did not stimulate mitogenesis in STAT-6-deficient mouse lung fibroblasts, but did induce cell growth of wild-type mouse lung fibroblasts. Therefore, STAT-6 was required for IL-13-stimulated mitogenesis. In contrast, STAT-6 was not required for PDGF-AA-induced mitogenesis. Conditioned media from wild-type fibroblasts treated with IL-13 effectively stimulated mitogenesis of STAT-6-deficient lung fibroblasts, demonstrating that while STAT-6 was required for IL-13-induced release of the soluble mitogen we identified as PDGF-AA, cell growth stimulated by PDGF-AA is independent of STAT-6.
5. IL-1ß acts synergistically with IL-13 to enhance mitogenesis by up-regulation of PDGFR
We previously showed that IL-1ß is a potent inducer of PDGFR, the receptor for PDGF-AA. We hypothesized that IL-1ß could enhance IL-13-induced lung fibroblast proliferation by increasing PDGFR. Therefore, we investigated the ability of IL-13 to up-regulate expression of PDGFR by rat lung fibroblasts. Western analysis showed that IL-13 does not cause increased levels of PDGFR protein (Fig. 2
A). Similarly, numbers of PDGFR expressed on the cell surface were not up-regulated after IL-13 treatment as determined by [125I]PDGF-AA binding assay (Fig. 2B
). IL-1ß caused up-regulation of both PDGFR protein expression levels and strongly elevated [125I]PDGF-AA receptor binding. Pretreatment of rat lung fibroblasts with IL-1ß in order to up-regulate PDGFR followed by IL-13 treatment resulted in a dose-dependent synergistic enhancement of fibroblast proliferation (Fig. 2C
). This enhancement was due to the coordinated up-regulation of PDGFR and its ligand, PDGF-AA, by IL-1ß and IL-13, respectively.
|
CONCLUSIONS
In this study, PDGF-AA was identified as an autocrine growth factor that mediated IL-13-stimulated lung fibroblast proliferation. PDGF-AA release after IL-13 exposure was dependent on STAT-6 signaling. IL-13-stimulated mitogenesis was not mediated via increased expression of PDGFR, but up-regulation of PDGFR by IL-1ß amplified IL-13-stimulated mitogenesis.
Although IL-13 has been implicated in chronic airway remodeling associated with asthma, this study is the first to demonstrate a signaling mechanism for IL-13-stimulated fibroblast mitogenesis and to identify PDGF-AA as a mediator. In our postulated pathway for IL-13-induced fibroblast growth (Fig. 3
), Th2 cells migrate to an area of airway inflammation and secrete IL-13, which binds to specific receptors located on the surface of fibroblasts. This results in the activation of STAT-6, which then triggers the release of PDGF-AA by the cell to stimulate cell growth in an autocrine manner. IL-1ß, which is also increased during airway inflammation, causes up-regulation of PDGFR levels at the cell surface, and thereby amplifies IL-13-induced cell proliferation through increased PDGF-AA binding and signaling.
|
Elucidation of the mechanism by which IL-13 elicits fibroblast proliferation in vitro has clear clinical relevance for our understanding of asthma pathogenesis. Chronic remodeling in asthma features airway fibrosis, which is defined by fibroblast proliferation and collagen deposition. Our study provides a novel mechanism of fibroblast growth driven by IL-13 that has implications for chronic airway remodeling in asthma, and we identify possible therapeutic targets (i.e., PDGF-AA, PDGFR) for treating airway fibrosis associated with asthma and chronic bronchitis.
Finally, our findings may have important implications in current treatment strategies for asthma. Dexamethasone, an anti-inflammatory corticosteroid widely used to treat asthma, is not effective in preventing fibrotic reactions in the lung. In fact, dexamethasone has been reported to stimulate the proliferation of airway fibroblasts in patients with asthma and has been demonstrated to up-regulate PDGFR in rat lung fibroblasts. These studies suggest that corticosteroid treatment for asthma could exacerbate airway fibrosis through interaction with IL-13 and the PDGF system. Moreover, our findings indicate that PDGF receptor tyrosine kinase inhibitors could hold promise for the treatment of airway fibrosis in chronic asthma. Experiments are ongoing to evaluate a possible role for signaling through PDGF-AA/PDGFR in fibroblasts from patients with mild or severe asthma.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1492fje; doi: 10.1096/fj.03-1492fje
This article has been cited by other articles:
|
|
 |
|
  P. Fernandez, S. Trzaska, T. Wilder, L. Chiriboga, M. R. Blackburn, B. N. Cronstein, and E. S. L. Chan Pharmacological Blockade of A2A Receptors Prevents Dermal Fibrosis in a Model of Elevated Tissue Adenosine Am. J. Pathol., June 1, 2008; 172(6): 1675 - 1682. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 L. Y. Leung, D. Tian, C. P. Brangwynne, D. A. Weitz, and D. J. Tschumperlin A new microrheometric approach reveals individual and cooperative roles for TGF-{beta}1 and IL-1{beta} in fibroblast-mediated stiffening of collagen gels FASEB J, July 1, 2007; 21(9): 2064 - 2073. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Bonner Lung Fibrotic Responses to Particle Exposure Toxicol Pathol, January 1, 2007; 35(1): 148 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Churg, H. Tai, T. Coulthard, R. Wang, and J. L. Wright Cigarette Smoke Drives Small Airway Remodeling by Induction of Growth Factors in the Airway Wall Am. J. Respir. Crit. Care Med., December 15, 2006; 174(12): 1327 - 1334. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Pegorier, L. A. Wagner, G. J. Gleich, and M. Pretolani Eosinophil-Derived Cationic Proteins Activate the Synthesis of Remodeling Factors by Airway Epithelial Cells J. Immunol., October 1, 2006; 177(7): 4861 - 4869. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Ingram, A. Antao-Menezes, J. B. Mangum, O. Lyght, P. J. Lee, J. A. Elias, and J. C. Bonner Opposing Actions of Stat1 and Stat6 on IL-13-Induced Up-Regulation of Early Growth Response-1 and Platelet-Derived Growth Factor Ligands in Pulmonary Fibroblasts J. Immunol., September 15, 2006; 177(6): 4141 - 4148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Hogg State of the Art. Bronchiolitis in Chronic Obstructive Pulmonary Disease Proceedings of the ATS, August 1, 2006; 3(6): 489 - 493. [Full Text] [PDF]
|
|
|
|
|
|
V. N. Lama, H. Harada, L. N. Badri, A. Flint, C. M. Hogaboam, A. McKenzie, F. J. Martinez, G. B. Toews, B. B. Moore, and D. J. Pinsky Obligatory Role for Interleukin-13 in Obstructive Lesion Development in Airway Allografts Am. J. Pathol., July 1, 2006; 169(1): 47 - 60. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Walters, A. Antao-Menezes, J. L. Ingram, A. B. Rice, A. Nyska, Y. Tani, S. R. Kleeberger, and J. C. Bonner Susceptibility of Signal Transducer and Activator of Transcription-1-Deficient Mice to Pulmonary Fibrogenesis Am. J. Pathol., November 1, 2005; 167(5): 1221 - 1229. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. P. Ennis, J. P. Cassidy, and B. P. Mahon Acellular Pertussis Vaccine Protects against Exacerbation of Allergic Asthma Due to Bordetella pertussis in a Murine Model Clin. Vaccine Immunol., March 1, 2005; 12(3): 409 - 417. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) -stimulated mitogenesis, but not PDGF-BB (
) -induced mitogenesis, by an anti-PDGF-AA neutralizing antibody. Confluent, quiescent cultures of human lung fibroblasts were incubated for 48 h in SFDM containing 100 ng/mL IL-13 and increasing concentrations of anti-PDGF-AA in the presence of [3H]thymidine. Control (
) indicates the effect of increasing concentration of anti-PDGF-AA in the absence of growth factor. Data are expressed as the mean fold increase above control (SFDM) of quadruplicate wells from a single experiment typical of three. Bars: SEM. B) PDGF-AA Western blot analysis of conditioned media from human lung fibroblasts cultures treated with increasing concentrations of IL-13. The same anti-PDGF-AA antibody used in the neutralization experiment (A) detected PDGF-AA in CM from IL-13-treated cells.
