Overexpression of the Transcription Factor GATA-3 Enhances the Development of Pulmonary Fibrosis
2006; Elsevier BV; Volume: 169; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.051160
ISSN1525-2191
AutoresTōru Kimura, Yukio Ishii, Keigyou Yoh, Yuko Morishima, Takashi Iizuka, Takumi Kiwamoto, Yosuke Matsuno, Shinsuke Homma, Akihiro Nomura, Tohru Sakamoto, Satoru Takahashi, Kiyohisa Sekizawa,
Tópico(s)Neonatal Respiratory Health Research
ResumoRecent studies have demonstrated that Th2 cytokines, such as interleukin-4 and interleukin-13, enhance fibrotic processes by activating fibroblast proliferation and collagen production, whereas interferon-γ, a Th1 cytokine, inhibits these processes. Th1 and Th2 cells both differentiate from common T precursor cells, with transcription factor GATA-3 a key regulator of Th2 differentiation. In the present study, therefore, we examined the effects of GATA-3 overexpression on the development of pulmonary fibrosis in a mouse model. Wild-type C57BL/6 mice and GATA-3-overexpressing (GATA-3-tg) mice of the same background were intratracheally treated with bleomycin. The survival rate after bleomycin was significantly decreased in GATA-3-tg mice compared with wild-type mice. The degree of pulmonary fibrosis was much greater in GATA-3-tg mice than in wild-type mice 28 days after bleomycin treatment. Lung interferon-γ concentration was significantly decreased in GATA-3-tg mice compared with wild-type mice by 7 days after either saline or bleomycin treatment. The concentration of transforming growth factor-β, a fibrogenic cytokine, was significantly higher in GATA-3-tg mice than in wild-type mice. Exogenous administration of interferon-γ to GATA-3-tg mice improved the degree of pulmonary fibrosis and thus increased survival. These results indicate that overexpression of GATA-3 enhances the development of pulmonary fibrosis, possibly by reducing interferon-γ levels in the lung. Recent studies have demonstrated that Th2 cytokines, such as interleukin-4 and interleukin-13, enhance fibrotic processes by activating fibroblast proliferation and collagen production, whereas interferon-γ, a Th1 cytokine, inhibits these processes. Th1 and Th2 cells both differentiate from common T precursor cells, with transcription factor GATA-3 a key regulator of Th2 differentiation. In the present study, therefore, we examined the effects of GATA-3 overexpression on the development of pulmonary fibrosis in a mouse model. Wild-type C57BL/6 mice and GATA-3-overexpressing (GATA-3-tg) mice of the same background were intratracheally treated with bleomycin. The survival rate after bleomycin was significantly decreased in GATA-3-tg mice compared with wild-type mice. The degree of pulmonary fibrosis was much greater in GATA-3-tg mice than in wild-type mice 28 days after bleomycin treatment. Lung interferon-γ concentration was significantly decreased in GATA-3-tg mice compared with wild-type mice by 7 days after either saline or bleomycin treatment. The concentration of transforming growth factor-β, a fibrogenic cytokine, was significantly higher in GATA-3-tg mice than in wild-type mice. Exogenous administration of interferon-γ to GATA-3-tg mice improved the degree of pulmonary fibrosis and thus increased survival. These results indicate that overexpression of GATA-3 enhances the development of pulmonary fibrosis, possibly by reducing interferon-γ levels in the lung. Pulmonary fibrosis is an end-stage disorder with a poor prognosis. Although the fibrotic process is not yet fully understood, initial immune and inflammatory responses to the repeated stimuli lead to tissue injury and progressive fibrosis.1Ward PA Hunninghake GW Lung inflammation and fibrosis.Am J Respir Crit Care Med. 1998; 157: S123-S129Crossref PubMed Scopus (204) Google Scholar Histological evaluation of lung tissue from patients with pulmonary fibrosis shows various degrees of inflammation in which many types of inflammatory cells, including macrophages, neutrophils, and lymphocytes, have infiltrated. Bleomycin is an anti-tumor drug often used as an inducing agent in models of pulmonary fibrosis. Intratracheal administration of bleomycin in rodents induces progressive lung inflammation followed by varying degrees of fibrosis that models human pulmonary fibrosis such as idiopathic pulmonary fibrosis.2Adamson IY Bowden DH The pathogenesis of bleomycin-induced pulmonary fibrosis in mice.Am J Pathol. 1974; 77: 185-197PubMed Google Scholar, 3Snider GL Hayes JA Korthy AL Chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin: pathology and stereology.Am Rev Respir Dis. 1978; 117: 1099-1108PubMed Google Scholar, 4Sleijfer S Bleomycin-induced pneumonitis.Chest. 2001; 120: 617-624Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar It is interesting that the accumulation of lymphocytes in the lung is observed 5 to 7 days after bleomycin administration, preceding the development of pulmonary fibrosis.5Chandler DB Hyde DM Giri SN Morphometric estimates of infiltrative cellular changes during the development of bleomycin-induced pulmonary fibrosis in hamsters.Am J Pathol. 1983; 112: 170-177PubMed Google Scholar, 6Thrall RS Barton RW A comparison of lymphocyte populations in lung tissue and in bronchoalveolar lavage fluid of rats at various times during the development of bleomycin-induced pulmonary fibrosis.Am Rev Respir Dis. 1984; 129: 279-283PubMed Google Scholar Among lymphocytes, T cells are the most likely to participate in the development of bleomycin-induced pulmonary fibrosis. Depletion of T cells by treatment with anti-CD4 and anti-CD8 antibodies inhibits the fibrotic response to bleomycin.7Piguet PF Collart MA Grau GE Kapanci Y Vassalli P Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis.J Exp Med. 1989; 170: 655-663Crossref PubMed Scopus (509) Google Scholar Bleomycin-induced pulmonary inflammation and fibrosis are not observed in athymic nude mice.8Schrier DJ Phan SH McGarry BM The effects of the nude (nu/nu) mutation on bleomycin-induced pulmonary fibrosis: a biochemical evaluation.Am Rev Respir Dis. 1983; 127: 614-617Crossref PubMed Scopus (116) Google Scholar The mechanisms by which T cells contribute to the fibrotic response have not been clarified but likely involve the production of T-cell-derived cytokines. Researchers have demonstrated that the specific cytokine phenotype may provide fundamental mechanisms for regulation of the fibrotic process. For example, interleukin (IL)-4 and IL-13 enhance the fibrotic process by augmenting fibroblast proliferation and collagen production.9Liu X Kohyama T Wang H Zhu YK Wen FQ Kim HJ Romberger DJ Rennard SI Th2 cytokine regulation of type I collagen gel contraction mediated by human lung mesenchymal cells.Am J Physiol. 2002; 282: L1049-L1056Crossref PubMed Scopus (72) Google Scholar, 10Sempowski GD Derdak S Phipps RP Interleukin-4 and interferon-gamma discordantly regulate collagen biosynthesis by functionally distinct lung fibroblast subsets.J Cell Physiol. 1996; 167: 290-296Crossref PubMed Scopus (147) Google Scholar, 11Postlethwaite AE Holness MA Katai H Raghow R Human fibroblasts synthesize elevated levels of extracellular matrix proteins in response to interleukin 4.J Clin Invest. 1992; 90: 1479-1485Crossref PubMed Scopus (438) Google Scholar Conversely, interferon-γ (IFN-γ) induces anti-fibrotic effects directly by suppressing fibroblast activity such as proliferation and collagen production11Postlethwaite AE Holness MA Katai H Raghow R Human fibroblasts synthesize elevated levels of extracellular matrix proteins in response to interleukin 4.J Clin Invest. 1992; 90: 1479-1485Crossref PubMed Scopus (438) Google Scholar, 12Gurujeyalakshmi G Giri SN Molecular mechanisms of antifibrotic effect of interferon-γ in bleomycin-mouse model of lung fibrosis: downregulation of TGF-β and procollagen I and III gene expression.Exp Lung Res. 1995; 21: 791-808Crossref PubMed Scopus (283) Google Scholar, 13Hyde DM Henderson TS Giri SN Tyler NK Stovall MY Effect of murine gamma interferon on the cellular responses to bleomycin in mice.Exp Lung Res. 1988; 14: 687-704Crossref PubMed Scopus (88) Google Scholar or indirectly by attenuating the effects of IL-4 and IL-13.14Saito A Okazaki H Sugawara I Yamamoto K Takizawa H Potential action of IL-4 and IL-13 as fibrogenic factors on lung fibroblasts in vitro.Int Arch Allergy Immunol. 2003; 132: 168-176Crossref PubMed Scopus (132) Google Scholar Interestingly, these cytokines are prototypic cytokines participating in type 1 (Th1) and type 2 (Th2) immune responses. These findings therefore suggest that Th1 differentiation (and expression of IFN-γ) attenuates the development of pulmonary fibrosis whereas Th2 differentiation (and expression of IL-4 and IL-13) aggravates it. Th1 and Th2 cells are differentiated from common T-precursor cells.15Abbas AK Murphy KM Sher A Functional diversity of helper T lymphocytes.Nature. 1996; 383: 787-793Crossref PubMed Scopus (3908) Google Scholar, 16Liblau RS Singer SM McDevitt HO Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases.Immunol Today. 1995; 16: 34-38Abstract Full Text PDF PubMed Scopus (1131) Google Scholar The differentiation requires the activity of distinct transcription factors. Among a variety of key molecules governing Th1/Th2 differentiation, GATA-3 has been implicated in Th2 commitment.17Zhang DH Cohn L Ray P Bottomly K Ray A Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene.J Biol Chem. 1997; 272: 21597-21603Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 18Zheng DH Flavell RA The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells.Cell. 1997; 89: 587-596Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar, 19Ouyang W Ranganath SH Weindel K Bhattacharya D Murphy TL Sha WC Murph KM Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism.Immunity. 1998; 9: 745-755Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 20Usui T Nishikomori R Kitani A Strober W GATA-3 suppresses Th1 development by downregulation of Stat4 and not through effects on IL-12Rbeta2 chain or T-bet.Immunity. 2003; 18: 415-428Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar Under physiological conditions, GATA-3 is selectively expressed in Th2 but not Th1 cells.17Zhang DH Cohn L Ray P Bottomly K Ray A Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene.J Biol Chem. 1997; 272: 21597-21603Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 18Zheng DH Flavell RA The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells.Cell. 1997; 89: 587-596Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar, 21Zhang DH Yang L Ray A Differential responsiveness of the IL-5 and IL-4 genes to transcription factor GATA-3.J Immunol. 1998; 161: 3817-3821Crossref PubMed Google Scholar Transgenic and retroviral expression of GATA-3 induces a Th2 cytokine profile in Th1 cells,17Zhang DH Cohn L Ray P Bottomly K Ray A Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene.J Biol Chem. 1997; 272: 21597-21603Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 18Zheng DH Flavell RA The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells.Cell. 1997; 89: 587-596Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar whereas dominant-negative GATA-3 down-regulates this profile in Th2 clones.21Zhang DH Yang L Ray A Differential responsiveness of the IL-5 and IL-4 genes to transcription factor GATA-3.J Immunol. 1998; 161: 3817-3821Crossref PubMed Google Scholar We recently established GATA-3-overexpressing transgenic mice.22Yoh K Shibuya K Morito N Nakano T Ishizaki K Shimohata H Nose M Izui S Shibuya A Koyama A Engel JD Yamamoto M Takahashi S Transgenic overexpression of GATA-3 in T lymphocytes improved autoimmune glomerulonephritis in mice with a BXSB/MpJ-Yaa genetic background.J Am Soc Nephrol. 2003; 14: 2494-2502Crossref PubMed Scopus (33) Google Scholar In the present study, using these mice, we evaluated the role of GATA-3 overexpression in the development of bleomycin-induced pulmonary fibrosis. Wild-type C57BL/6 mice were obtained from Charles River Laboratories (Kanagawa, Japan). Heterozygous GATA-3-overexpressing transgenic (GATA-3-tg) mice generated in our laboratory22Yoh K Shibuya K Morito N Nakano T Ishizaki K Shimohata H Nose M Izui S Shibuya A Koyama A Engel JD Yamamoto M Takahashi S Transgenic overexpression of GATA-3 in T lymphocytes improved autoimmune glomerulonephritis in mice with a BXSB/MpJ-Yaa genetic background.J Am Soc Nephrol. 2003; 14: 2494-2502Crossref PubMed Scopus (33) Google Scholar were backcrossed with C57BL/6 mice for eight generations. All mice used in this study were 6 to 8 weeks of age and were maintained in our animal facilities under specific pathogen-free conditions. All animal studies were approved by the institutional review board at our facility. Mice were administered bleomycin (5 mg/kg; Calbiochem, San Diego, CA) or saline intratracheally. The lungs were removed 28 days after bleomycin or saline administration. After fixation, the lungs were embedded in paraffin. The sections were then stained with Masson's trichrome stain. The grade of pulmonary fibrosis was scored on a scale of 0 to 8 using a previously described scoring method.23Ashcroft T Simpson JM Timbrell V Simple method of estimating severity of pulmonary fibrosis on a numerical scale.J Clin Pathol. 1988; 41: 467-470Crossref PubMed Scopus (1157) Google Scholar After the examination of 30 randomly chosen regions in each sample at a magnification of ×100, the mean score of all of the fields was taken as the fibrosis score in each sample. Collagen synthesis was assessed using a hydroxyproline assay. The mice were anesthetized, and the lungs were removed 28 days after bleomycin or saline administration. Hydroxyproline content was measured as reported previously.24Woessner LF The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid.Arch Biochem Biophys. 1961; 93: 440-447Crossref PubMed Scopus (3409) Google Scholar One and seven days after bleomycin or saline administration, the lungs were lavaged with six sequential aliquots of 1 ml of phosphate-buffered saline (PBS). The remaining pooled bronchoalveolar lavage was centrifuged and resuspended in PBS. Cells were counted using a hemocytometer, and a differential cell count was performed by standard light microscopic techniques based on staining with Diff-Quik (American Scientific Products, McGraw Park, IL). Seven days after bleomycin or saline administration, the lungs were removed, minced, and incubated with RPMI 1640 containing 10% fetal bovine serum and 75 U/ml collagenase (type 1; Sigma Chemical Co., St. Louis, MO) at 37°C for 90 minutes. The cells were then filtered through 20-μm nylon mesh. The cell suspensions were stained with anti-CD4 (BD PharMingen, San Diego, CA), anti-CXCR3 (R&D Systems, Minneapolis, MN), anti-CCR3 (R&D Systems), and anti-CD25 antibodies (BD PharMingen), respectively. After staining, the cells were analyzed by flow cytometry using the FACSCaliber with CellQuest software (BD Biosciences, San Jose, CA). Intracellular production of IFN-γ was also determined by flow cytometric intracellular cytokine analysis as previously described.25Murphy E Shibuya K Hosken N Openshaw P Maino V Davis K Murphy K O'Garra A Reversibility of T helper 1 and 2 populations is lost after long-term stimulation.J Exp Med. 1996; 183: 901-913Crossref PubMed Scopus (387) Google Scholar The concentrations of IFN-γ, IL-4, IL-5, and IL-13 in the lung homogenates were determined by enzyme-linked immunosorbent assay according to the manufacturer's instructions (Endogen, Cambridge, MA). A concentration of the active form of transforming growth factor (TGF)-β was also determined by enzyme-linked immunosorbent assay (R&D Systems). For continuous exposure of IFN-γ, Alzet osmotic pumps (model no. 1002; Durect, Cupertino, CA) with a 14-day pumping capacity and an infusion rate of 0.25 μl/hour were used. Pumps were filled with 1.7 × 108 U/ml mouse recombinant IFN-γ (BD PharMingen) diluted in PBS. One day before bleomycin administration, pumps were implanted into the intrascapular subcutaneous space. In control mice, PBS was continuously administered using the same pumps. Data are expressed as the mean ± SEM. Comparisons of data among the experimental groups were performed using analysis of variance and Scheffé's test. The survival curves were analyzed using the log-rank test. Values of P < 0.05 were considered to be statistically significant. We first evaluated the survival of mice after bleomycin administration. Thirty-five percent of the wild-type C57BL/6 mice and sixty-five percent of the GATA-3-tg mice died by 28 days after bleomycin administration (Figure 1). The survival rate after bleomycin treatment was significantly decreased in GATA-3-tg mice compared with wild-type mice. No mice died in the saline-administered control group of either genotype (Figure 1). We next assessed the development of bleomycin-induced pulmonary fibrosis in both wild-type mice and GATA-3-tg mice. Figure 2 shows the lung histology 28 days after bleomycin or saline administration. Masson's trichrome stain revealed mild thickening of alveolar septa and collagen deposition 28 days after bleomycin administration in the lungs of wild-type mice (Figure 2A, WT-bleo). The degree of pulmonary fibrosis was much greater in GATA-3-tg mice than in wild-type mice. Dense fibrosis with prominent collagen deposition was observed 28 days after bleomycin administration in the lung of GATA-3-tg mice (Figure 2A, GATA-3-bleo). No abnormal alveolar architecture was observed in the lungs of the saline-administered control group in either wild-type mice or GATA-3-tg mice (Figure 2A, WT-saline and GATA-3-saline). We then assessed the degree of pulmonary fibrosis using a scoring method. In mice of both genotypes, the scores of fibrotic lesions were significantly increased 28 days after bleomycin administration, compared with saline-administered controls (Figure 2B). However, the score was significantly higher in GATA-3-tg mice than in wild-type mice at that time (Figure 2B). We further assessed the degree of pulmonary fibrosis by measuring the lung hydroxyproline content, which we found to be significantly increased 28 days after bleomycin administration in both genotypes (Figure 2C). However, the concentration was significantly higher in GATA-3-tg mice than in wild-type mice at that time (Figure 2C). These results clearly indicate that the degree of bleomycin-induced pulmonary fibrosis was much greater in mice overexpressing the GATA-3 gene. To evaluate the inflammatory phenotype in the early phase of bleomycin treatment, the lungs were lavaged 1 day and 7 days after bleomycin administration in both wild-type mice and GATA-3-tg mice. The number of lavageable macrophages increased significantly 7 days after bleomycin administration in mice of both genotypes, compared with mice before bleomycin administration (Figure 3A). Similarly, the number of lymphocytes was increased significantly 7 days after bleomycin administration in the lungs of mice of both genotypes (Figure 3B). The number of neutrophils was significantly elevated 1 day after bleomycin administration in the lungs of mice of both genotypes (Figure 3C). However, these numbers were not different between wild-type mice and GATA-3-tg mice at any time points (Figure 3, A–C). These results indicate that the degree of initial inflammation induced by bleomycin was not different between wild-type mice and GATA-3-tg mice. GATA-3 is a transcription factor that regulates Th2 differentiation. We therefore assessed Th1/Th2 cytokine balance in the lungs of both wild-type mice and GATA-3-tg mice. As indicated above, lymphocytes are predominantly infiltrated into the lung 7 days after bleomycin administration in this model. Accordingly, we determined the concentration of IFN-γ, IL-4, IL-5, and IL-13 in lung homogenates at that time point. The concentration of IFN-γ in the lung homogenates was significantly lower in GATA-3-tg mice than in wild-type mice under both saline- and bleomycin-treated conditions (Figure 4A). However, the concentrations of IL-4, IL-5, and IL-13 in the lung homogenates were not significantly different between mouse genotypes or by treatment with bleomycin (Figure 4, B–D). Because GATA-3 is a transcription factor mainly expressed in T cells, we next assessed the production of IFN-γ in CD4+ T cells obtained from the lungs of wild-type mice and GATA-3-tg mice 7 days after administration of bleomycin or saline. In the saline-administered control group, less than 10% of the CD4+ T cells were positive for IFN-γ, and there was no difference in IFN-γ production between wild-type mice and GATA-3-tg mice (Figure 5A). The proportion of IFN-γ-producing CD4+ T cells increased significantly in wild-type mice after bleomycin administration (Figure 5A). However, the proportion of IFN-γ-producing CD4+ T cells was still low in GATA-3-tg mice after bleomycin administration (Figure 5A). These results indicate that the production of IFN-γ by stimulation with bleomycin was suppressed in CD4+ T cells of GATA-3-tg mice. We further assessed the production of IFN-γ in the subpopulation of CD4+ T cells in the lungs of mice of both genotypes. The proportion of CD4+CXCR3+ cells, which represents Th1 cells, in CD4+ T cells was significantly higher in the lungs of wild-type mice than in those of GATA-3-tg mice at 7 days after bleomycin administration (Figure 5B, top panels). Moreover, the proportion of IFN-γ-positive cells in CD4+CXCR3+ cells was higher in wild-type mice than in GATA-3-tg mice (Figure 5C, top panels). The proportion of CD4+CCR3+ cells, which represent Th2 cells, in CD4+ T cells was less than 5% in the lungs of both genotypes (Figure 5B, middle panels). The proportion of IFN-γ-positive cells in CD4+CCR3+ cells was not different between wild-type mice and GATA-3-tg mice (Figure 5C, middle panels). Although the proportion of CD4+CD25+ regulatory T cells in CD4+ T cells was not different between wild-type mice and GATA-3-tg mice (Figure 5B, bottom panels), the proportion of IFN-γ-positive cells in CD4+CD25+ regulatory T cells was significantly higher in wild-type mice than in GATA-3-tg mice (Figure 5C, bottom panels). These results indicate that the production of IFN-γ was suppressed in lung CD4+ T cells, especially Th1 cells and regulatory T cells, of mice overexpressing the GATA-3 gene. It has been demonstrated that the expression of TGF-β, a fibrogenic cytokine, is in part regulated by IFN-γ. We therefore evaluated the concentration of the active form of TGF-β in lung homogenates of wild-type mice and GATA-3-tg mice 7 days after the administration of bleomycin or saline. The protein level of the TGF-β active form was increased significantly in mice of both genotypes after bleomycin administration, compared with the corresponding control groups (Figure 6). However, the concentration of this form was significantly higher in GATA-3-tg mice than in wild-type mice after bleomycin administration (Figure 6). Because the concentration of IFN-γ was significantly lower in the lungs of GATA-3-tg mice than in those of wild-type mice, we next assessed whether supplementation with IFN-γ would reduce the development of bleomycin-induced pulmonary fibrosis. Mouse recombinant IFN-γ was administered continuously for 2 weeks via a miniosmotic pump beginning 1 day before the treatment with bleomycin or saline. We first evaluated the concentration of IFN-γ in lung homogenates 8 days after the initiation of IFN-γ administration to clarify whether the amount of exogenously administered IFN-γ was sufficient to increase the lung IFN-γ level. We confirmed that the lung IFN-γ level in GATA-3-tg mice was increased significantly to the level of wild-type mice by exogenous administration of IFN-γ (Figure 7A). We next assessed the effects of exogenously administered IFN-γ on the development of pulmonary fibrosis using this model. Exogenous administration of IFN-γ significantly improved the survival rate of GATA-3-tg mice after bleomycin administration: 80% of IFN-γ-treated GATA-3-tg mice had survived at 28 days after bleomycin treatment (Figure 7B). No mice died in the saline-administered control groups, irrespective of whether or not they were treated with IFN-γ (Figure 7B). Lung histology showed that the degree of pulmonary fibrosis was much lower in IFN-γ-treated GATA-3-tg mice than in IFN-γ-untreated controls 28 days after bleomycin administration (Figure 7C). No abnormal alveolar architecture was observed in the lungs of the saline-administered control groups, irrespective of whether or not they were simultaneously treated with IFN-γ (Figure 7C). Both the lung fibrotic score and the lung hydroxyproline content were also significantly reduced in IFN-γ-treated GATA-3-tg mice to the level of wild-type mice 28 days after bleomycin administration (Figure 7, D and E, respectively). These results indicate that the supplementation of IFN-γ reduced the development of pulmonary fibrosis in mice overexpressing the GATA-3 gene. In the present study, we demonstrated for the first time that the development of bleomycin-induced pulmonary fibrosis is much more severe in GATA-3-tg mice than in wild-type mice of the same background. GATA-3 is a member of the GATA family of zinc-finger transcription factors, which bind the GATA consensus motif.26Yamamoto M Ko LJ Leonard MW Beug H Orkin SH Engle JD Activity and tissue-specific expression of the transcription factor NF-E1 multigene family.Genes Dev. 1990; 4: 1650-1662Crossref PubMed Scopus (452) Google Scholar, 27Ko LJ Yamamoto M Leonard MW George KM Ting P Engel JD Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor σ gene enhancer.Mol Cell Biol. 1991; 11: 2778-2784Crossref PubMed Scopus (228) Google Scholar It has been demonstrated that anti-sense GATA-3 inhibited the expression of all Th2 cytokine genes in the Th2 clone D10.18Zheng DH Flavell RA The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells.Cell. 1997; 89: 587-596Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar In transgenic mice, elevated GATA-3 in CD4+ T cells caused Th2 cytokine gene expression in developing Th1 cells.18Zheng DH Flavell RA The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells.Cell. 1997; 89: 587-596Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar GATA-3 has been reported to transactivate the IL-5 promoter with only limited effects on IL-4 gene transcription.18Zheng DH Flavell RA The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells.Cell. 1997; 89: 587-596Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar, 21Zhang DH Yang L Ray A Differential responsiveness of the IL-5 and IL-4 genes to transcription factor GATA-3.J Immunol. 1998; 161: 3817-3821Crossref PubMed Google Scholar, 28Ranganath S Ouyang W Bhattarcharya D Sha WC Grupe A Peltz G Murphy KM GATA-3-dependent enhancer activity in IL-4 gene regulation.J Immunol. 1998; 161: 3822-3826Crossref PubMed Google Scholar It has also been reported that GATA-3 regulates the locus accessibility of the IL-4 and IL-13 genes with chromatin remodeling.29Takemoto N Kamogawa Y Lee HJ Kuruta H Arai K O'Garra A Arai N Miyataka S Chromatin remodeling at the IL4/IL13 intergenic regulatory region for Th2-specific cytokine gene cluster.J Immunol. 2000; 165: 6687-6691Crossref PubMed Scopus (132) Google Scholar, 30Lee GR Field PE Flavell RA Regulation of IL-4 gene expression by distal regulatory elements and GATA-3 at the chromatin level.Immunity. 2001; 14: 447-459Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar These findings suggest that GATA-3 allows the expression of Th2 cytokines by functioning as a transcription factor as well as by modifying the chromatin structure of these cytokines. In the present study, however, the lung IFN-γ level was characteristically suppressed after bleomycin administration in GATA-3-tg mice, compared with wild-type mice of the same background, while the pulmonary levels of Th2 cytokines such as IL-4, IL-5, and IL-13 were not different between them. We believe these findings are reasonable because several studies have demonstrated that GATA-3 not only transactivates Th2 cytokines but also suppresses Th1 cytokine expression. It was reported that GATA-3 significantly down-regulated IFN-γ production during in vitro Th1 differentiation of naïve CD4+ T cells through down-regulation of IL-12 receptor β2 and IFN-γ production.19Ouyang W Ranganath SH Weindel K Bhattacharya D Murphy TL Sha WC Murph KM Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism.Immunity. 1998; 9: 745-755Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 31Ferber IA Lee HJ Zonin F Heath V Mui A Arai N O'Garra A GATA-3 significantly downregulates IFN-γ production from developing Th1 cells in addition to inducing IL-4 and IL-5 levels.Clin Immunol. 1999; 91: 134-144Crossref PubMed Scopus (147) Google Scholar In contrast, IFN-γ production in CD4+ T cells of GATA-3-deficient mice was increased even under Th2 condition.32Pai SY Truitt ML Ho IC GATA-3 deficiency abrogates the development and maintenance of T helper type 2 cells.Proc Natl Acad Sci USA. 2004; 101: 1993-1998Crossref PubMed Scopus (285) Google Scholar Furthermore, Kaminuma and colleagues33Kaminuma O Kitamura F Kitamura N Miyagishi M Taira K Yamamoto K Miura O Miyatake S GATA-3 suppr
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