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(Chest. 2003;123:422S-424S.)
© 2003 American College of Chest Physicians

Interleukin-13 Stimulates the Proliferation of Lung Myofibroblasts via a Signal Transducer and Activator of Transcription-6-Dependent Mechanism*

A Possible Mechanism for the Development of Airway Fibrosis in Asthma

Jennifer L. Ingram, PhD; Annette Rice, BS; Kristen Geisenhoffer, BS; David K. Madtes, MD and James C. Bonner, PhD

* From the Laboratory of Pulmonary Pathobiology (Drs. Ingram and Bonner, Ms. Rice, and Ms. Geisenhoffer), National Institute of Environmental Health Science, Research Triangle Park, NC; and Fred Hutchinson Cancer Research Center (Dr. Madtes), Seattle, WA.

Correspondence to: James C. Bonner, PhD, the National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709; e-mail: bonnerj{at}niehs.nih.gov


   Introduction
TOP
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Subepithelial fibrosis is increasingly recognized as a component of airway remodeling during the pathogenesis of asthma. It is well known that the airway fibrotic response is due to myofibroblast proliferation and the subsequent deposition of collagen by these cells.1 However, it is unclear which soluble factors initiate and perpetuate the growth of myofibroblasts surrounding small airways. Interleukin (IL)-13, a T helper type 2 cytokine, has been proposed as a major mediator of airway remodeling in asthma patients.2 IL-13 levels are elevated in the lungs of patients with asthma and pulmonary fibrosis.3 Furthermore, the overexpression of IL-13 in murine lungs results in an asthma-like phenotype that includes airway fibrosis.4 Many of the effects of IL-13 are mediated via the transcription factor STAT-6, and mice deficient in STAT-6 are protected from IL-13-induced airway remodeling.5 Although the pathology of IL-13-induced airway remodeling has been well-characterized, little is known regarding the effect of IL-13 on myofibroblast growth. In this study, we investigated IL-13 as a possible mitogen for lung myofibroblasts.


   Materials and Methods
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 Introduction
 Materials and Methods
Results and Discussion
 References
 
Primary passage rat pulmonary myofibroblasts were isolated from male Sprague-Dawley rats, as described previously.6 Primary passage pulmonary fibroblasts were collected from Balb/c (wild-type) or STAT-6-/- mice (Jackson Laboratory; Bar Harbor, ME). Human lung fibroblasts (16 Lu) were purchased from American Type Culture Collection (Manassas, VA). All cell types were seeded into 175-cm2 plastic culture dishes and grown to confluence in 20% fetal bovine serum/Dulbecco modified Eagle medium, then were trypsinized and seeded into 24-well plates. Confluent, quiescent cells were treated for 48 h with 100 ng/mL IL-13 in the presence of 5 µCi/mL [3H]thymidine. The thymidine incorporation data for each treatment were normalized to those of the untreated cells (Table 1 ).


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  		Table 1.. Mitogenic Effects of IL-13 on Lung Myofibroblasts From Various Species and Dependence Upon STAT-6*

 

   Results and Discussion
TOP
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Using [3H]thymidine incorporation assays, we found that IL-13 stimulated DNA synthesis of rat, mouse, and human lung myofibroblasts in a concentration-dependent and time-dependent manner (Table 1) . IL-13-induced growth was dependent on the activation of the STAT-6 transcription factor, since myofibroblasts isolated from the lungs of STAT-6-deficient (ie, STAT-6-/-) mice did not respond to IL-13 in mitogenesis assays. The STAT-6-/- cells underwent mitogenesis in the presence of platelet-derived growth factor (PDGF)-BB, indicating that these cells would proliferate in the presence of mitogens that acted independently of STAT-6. In contrast, IL-13 stimulated the growth of lung myofibroblasts isolated from wild-type Balb/cJ mice. The effect of IL-13 on myofibroblast growth was due to the release of a soluble mitogen, as conditioned medium from rat lung myofibroblasts treated transiently with IL-13 possessed mitogenic activity. In addition, conditioned medium from IL-13-treated Balb/cJ cells stimulated the mitogenesis of STAT-6-/- myofibroblasts. Therefore, growth stimulated by the soluble mitogen was not dependent on the STAT-6 pathway, even though the release of the soluble mitogen in response to IL-13 treatment was STAT-6-dependent. A previous report by Booth and coworkers7 identified transforming growth factor (TGF)-{alpha} as a soluble factor that mediated the IL-13-induced growth of normal human bronchial epithelial cells. However, mouse lung myofibroblasts isolated from TGF-{alpha}-/- mice underwent mitogenesis in the presence of IL-13, indicating that the soluble mitogen released by IL-13-stimulated myofibroblasts was not TGF-{alpha}.

Experiments then were conducted to identify the soluble mitogen that was released by myofibroblasts following IL-13 treatment. Size exclusion gel filtration chromatography of conditioned medium from rat lung myofibroblasts treated with IL-13 revealed a peak of mitogenesis that corresponded to a protein of approximately 30 kd. PDGF-AA is a 30-kd protein and is a mitogen for lung myofibroblasts.8 Furthermore, a recent report by Lee et al9 showed that the PDGF-A gene was induced approximately 2-fold in normal human lung fibroblasts on exposure to IL-13. Therefore, we hypothesized that PDGF-AA was the soluble mitogen induced by IL-13 in lung fibroblasts. Western blot analysis demonstrated that PDGF-AA protein was up-regulated in the conditioned media from IL-13-treated myofibroblasts in a dose-dependent manner. Also, a PDGF-AA neutralizing antibody attenuated IL-13-stimulated mitogenesis. IL-13 has no effect on the expression of the PDGF-{alpha} receptor in rat lung fibroblasts, however, IL-1ß has been shown to up-regulate PDGF-{alpha} receptor and to initiate the mitogenesis of these cells through a PDGF-AA autocrine loop.10 Indeed, we showed that IL-1ß synergistically enhanced IL-13-induced mitogenesis and that this was due to IL-1ß-induced up-regulation of the PDGF-{alpha} receptor, which selectively binds PDGF-AA.

Our results showed that IL-13 stimulates the proliferation of myofibroblasts and that this activity is dependent on STAT-6. Furthermore, IL-13-stimulated mitogenesis appears to be mediated by the secretion of PDGF-AA, thus activating myofibroblast growth through an autocrine mechanism. We also demonstrated for the first time that IL-13 acts in synergy with IL-1ß to stimulate myofibroblast growth by coordinately up-regulating PDGF-AA and the PDGF-{alpha} receptor, respectively. This work will provide a greater understanding of the pathogenesis of airway fibrosis and identifies the PDGF-AA/PDGF-{alpha} receptor growth regulatory axis as a potential therapeutic target for asthma.


   Footnotes
 
Abbreviations: IL = interleukin; PDGF = platelet-derived growth factor; STAT = signal transducer and activator of transcription; TGF = transforming growth factor


   References
TOP
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Brewster, CEP, Howarth, PH, Djukanovic, DR, et al (1990) Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 3,507-511
  2. Wills-Karp, M, Luyimbazi, J, Xu, X, et al Interleukin-13: central mediator of allergic asthma. Science 1998;282,2258-2261[Abstract/Free Full Text]
  3. Hancock, A, Armstrong, L, Gama, R, et al Production of interleukin 13 by alveolar macrophages from normal and fibrotic lung. Am J Respir Cell Mol Biol 1998;18,60-65[Abstract/Free Full Text]
  4. Zhu, Z, Homer, RJ, Wang, Z, et al Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J Clin Invest 1999;103,779-788[ISI][Medline]
  5. Kuperman, D, Schofield, B, Wills-Karp, M, et al Signal transducer and activator of transcription factor 6 (Stat6)-deficient mice are protected from antigen-induced airway hyperresponsiveness and mucus production. J Exp Med 1998;187,939-948[Abstract/Free Full Text]
  6. Coin, PG, Lindroos, PM, Bird, GS, et al Lipopolysaccharide up-regulates platelet-derived growth factor (PDGF) {alpha}-receptor expression in rat lung myofibroblasts and enhances response to all PDGF isoforms. J Immunol 1996;156,4797-4806[Abstract]
  7. Booth, BW, Adler, KB, Bonner, JC, et al Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factor-{alpha}. Am J Respir Cell Mol Biol 2001;25,739-743[Abstract/Free Full Text]
  8. Raines, EW, Dower, SK, Ross, R Interleukin-1 mitogenic activity for fibroblasts and smooth muscle cells is due to PDGF-AA. Science 1989;243,393-396[Abstract/Free Full Text]
  9. Lee, JH, Kaminski, N, Dolganov, G, et al Interleukin-13 induces dramatically different transcriptional programs in three human airway cell types. Am J Respir Cell Mol Biol 2001;25,474-485[Abstract/Free Full Text]
  10. Lindroos, PM, Coin, PG, Osornio-Vargas, AR, et al Interleukin 1ß (IL-1ß) and the IL-1ß-{alpha}2-macroglobulin complex upregulate the platelet-derived growth factor {alpha}-receptor on rat pulmonary fibroblasts. Am J Respir Cell Mol Biol 1995;13,455-465[Abstract]



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