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(American Journal of Pathology. 2006;169:96-104.)
© 2006 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2006.051160

Overexpression of the Transcription Factor GATA-3 Enhances the Development of Pulmonary Fibrosis

Toru Kimura*, Yukio Ishii*, Keigyou Yoh{dagger}, Yuko Morishima*, Takashi Iizuka*, Takumi Kiwamoto*, Yosuke Matsuno*, Shinsuke Homma*, Akihiro Nomura*, Tohru Sakamoto*, Satoru Takahashi{ddagger} and Kiyohisa Sekizawa*

From the Departments of Respiratory Medicine* and Nephrology,{dagger} Institute of Clinical Medicine, and the Laboratory Animal Resource Center,{ddagger} University of Tsukuba, Tsukuba, Japan


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
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-{gamma}, 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-{gamma} 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-ß, a fibrogenic cytokine, was significantly higher in GATA-3-tg mice than in wild-type mice. Exogenous administration of interferon-{gamma} 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-{gamma} 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.1 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.2-4 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.5,6

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.7 Bleomycin-induced pulmonary inflammation and fibrosis are not observed in athymic nude mice.8 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.9-11 Conversely, interferon-{gamma} (IFN-{gamma}) induces anti-fibrotic effects directly by suppressing fibroblast activity such as proliferation and collagen production11-13 or indirectly by attenuating the effects of IL-4 and IL-13.14 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-{gamma}) 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.15,16 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.17-20 Under physiological conditions, GATA-3 is selectively expressed in Th2 but not Th1 cells.17,18,21 Transgenic and retroviral expression of GATA-3 induces a Th2 cytokine profile in Th1 cells,17,18 whereas dominant-negative GATA-3 down-regulates this profile in Th2 clones.21 We recently established GATA-3-overexpressing transgenic mice.22 In the present study, using these mice, we evaluated the role of GATA-3 overexpression in the development of bleomycin-induced pulmonary fibrosis.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals

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 laboratory22 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.

Histopathological Assessment

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.23 After the examination of 30 randomly chosen regions in each sample at a magnification of x100, the mean score of all of the fields was taken as the fibrosis score in each sample.

Assessment of Collagen Synthesis

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.24

Bronchoalveolar Lavage

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).

Flow Cytometry

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-{gamma} was also determined by flow cytometric intracellular cytokine analysis as previously described.25

Measurement of Cytokines

The concentrations of IFN-{gamma}, 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)-ß was also determined by enzyme-linked immunosorbent assay (R&D Systems).

Treatment with IFN-{gamma}

For continuous exposure of IFN-{gamma}, 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 x 108 U/ml mouse recombinant IFN-{gamma} (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.

Statistics

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.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Assessment of Pulmonary Fibrosis

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) .


Figure 1
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  		Figure 1. The survival rate is decreased in GATA-3-overexpressing mice after bleomycin treatment. The survival rates of wild-type mice (WT, circles) or GATA-3-overexpressing mice (GATA-3, squares) after intratracheal administration of 5 mg/kg bleomycin (bleo, filled) or saline (saline, open). n = 20 in each group. *Significant difference (P < 0.05) between the WT-bleo and GATA-3-bleo groups.

 
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).


Figure 2
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  		Figure 2. Bleomycin-induced pulmonary fibrosis is aggravated in GATA-3-overexpressing mice. A: Photomicrograph of the lung tissues from wild-type mice (WT) and GATA-3-overexpressing mice (GATA-3) 28 days after the intratracheal administration of 5 mg/kg bleomycin (bleo) or saline. Masson’s trichrome staining. The score of fibrosis (B) and lung hydroxyproline contents (C) of wild-type mice (WT) and GATA-3-overexpressing mice (GATA-3) 28 days after intratracheal administration of 5 mg/kg bleomycin (bleo) or saline. n = 6 in each group. *Significant difference (P < 0.05) between the WT-bleo and GATA-3-bleo groups. Scale bar = 100 µm.

 
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.

Assessment of Pulmonary Inflammation after Bleomycin Treatment

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.


Figure 3
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  		Figure 3. The degree of initial pulmonary inflammation induced by bleomycin is not different between wild-type mice and GATA-3-overexpressing mice. The number of macrophages (A), lymphocytes (B), and neutrophils (C) in bronchoalveolar lavage fluid of wild-type mice (WT) and GATA-3-overexpressing mice (GATA-3) before and 1 day and 7 days after administration of 5 mg/kg bleomycin. n = 6 in each group. *Significant difference (P < 0.05) compared with mice before bleomycin administration.

 
Assessment of Lung Cytokine Production during the Development of Pulmonary Fibrosis

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-{gamma}, IL-4, IL-5, and IL-13 in lung homogenates at that time point. The concentration of IFN-{gamma} 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) .


Figure 4
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  		Figure 4. The concentration of IFN-{gamma} is reduced in the lungs of GATA-3-overexpressing mice. The concentrations of IFN-{gamma} (A), IL-4 (B), IL-5 (C), and IL-13 (D) in lung homogenates of wild-type mice (WT) and GATA-3-overexpressing mice (GATA-3) 7 days after intratracheal administration of 5 mg/kg bleomycin (bleo) or saline. n = 6 in each group. *Significant difference (P < 0.05) between WT and GATA-3 mice.

 
Because GATA-3 is a transcription factor mainly expressed in T cells, we next assessed the production of IFN-{gamma} 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-{gamma}, and there was no difference in IFN-{gamma} production between wild-type mice and GATA-3-tg mice (Figure 5A) . The proportion of IFN-{gamma}-producing CD4+ T cells increased significantly in wild-type mice after bleomycin administration (Figure 5A) . However, the proportion of IFN-{gamma}-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-{gamma} by stimulation with bleomycin was suppressed in CD4+ T cells of GATA-3-tg mice.


Figure 5
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  		Figure 5. IFN-{gamma}-producing CD4-positive T cells are decreased in the lungs of GATA-3-overexpressing mice. A: The proportion of IFN-{gamma}-producing cells in lung CD4-positive T cells of wild-type mice (WT) and GATA-3-overexpressing mice (GATA-3) 7 days after intratracheal administration of 5 mg/kg bleomycin (bleo) or saline. n = 3 in each group. *Significant difference (P < 0.05) between the WT-bleo and GATA-3-bleo groups. B: The proportion of CXCR-3-positive cells (top), CCR3-positive cells (middle), and CD25-positive cells (bottom) in CD4-positive T cells obtained from the lungs of wild-type mice (WT) and GATA-3-overexpressing mice (GATA-3) 7 days after intratracheal administration of 5 mg/kg bleomycin (bleo). n = 3 in each group. *Significant difference (P < 0.05) between the WT-bleo and GATA-3-bleo groups. C: The proportion of IFN-{gamma}-producing cells in CXCR-3-positive cells (top), in CCR3-positive cells (middle), and in CD25-positive cells (bottom) obtained from the lungs of wild-type mice (WT) and GATA-3-overexpressing mice (GATA-3) 7 days after intratracheal administration of 5 mg/kg bleomycin (bleo). n = 3 in each group. *Significant difference (P < 0.05) between the WT-bleo and GATA-3-bleo groups.

 
We further assessed the production of IFN-{gamma} 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-{gamma}-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-{gamma}-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-{gamma}-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-{gamma} was suppressed in lung CD4+ T cells, especially Th1 cells and regulatory T cells, of mice overexpressing the GATA-3 gene.

Regulation of TGF-ß Expression by GATA-3

It has been demonstrated that the expression of TGF-ß, a fibrogenic cytokine, is in part regulated by IFN-{gamma}. We therefore evaluated the concentration of the active form of TGF-ß 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-ß 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) .


Figure 6
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  		Figure 6. The level of the active form of TGF-ß is increased in the lungs of GATA-3-overexpressing mice. The concentrations of TGF-ß1 in lung homogenates of wild-type mice (WT) and GATA-3-overexpressing mice (GATA-3) 7 days after intratracheal administration of 5 mg/kg bleomycin (bleo) or saline. n = 4 in each group. *Significant difference (P < 0.05) between the WT-bleo and GATA-3-bleo groups.

 
Effects of IFN-{gamma} Supplementation

Because the concentration of IFN-{gamma} 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-{gamma} would reduce the development of bleomycin-induced pulmonary fibrosis. Mouse recombinant IFN-{gamma} 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-{gamma} in lung homogenates 8 days after the initiation of IFN-{gamma} administration to clarify whether the amount of exogenously administered IFN-{gamma} was sufficient to increase the lung IFN-{gamma} level. We confirmed that the lung IFN-{gamma} level in GATA-3-tg mice was increased significantly to the level of wild-type mice by exogenous administration of IFN-{gamma} (Figure 7A) .


Figure 7
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  		Figure 7. Exogenous administration of IFN-{gamma} attenuates the development of pulmonary fibrosis in GATA-3-overexpressing mice. A: The concentration of IFN-{gamma} in the lungs of wild-type mice (WT), GATA-3-overexpressing mice (GATA-3), and GATA-3-overexpressing mice treated with mouse recombinant IFN-{gamma} (GATA-3-IFN-{gamma}) 7 days after intratracheal administration of 5 mg/kg bleomycin. n = 4 in each group. *Significant difference (P < 0.05) between the GATA-3 and GATA-3-IFN-{gamma} groups. B: Survival of GATA-3-overexpressing mice treated with (squares) or without (circles) IFN-{gamma} after intratracheal administration with 5 mg/kg bleomycin (bleo; closed) or saline (saline; open). n = 20 in each group. *Significant difference (P < 0.05) between the GATA-3-bleo and GATA-3-bleo-IFN-{gamma} groups. C: Photomicrographs of lung tissues from GATA-3-overexpressing mice treated with or without IFN-{gamma} 28 days after the intratracheal administration of 5 mg/kg bleomycin (bleo) or saline. Masson’s trichrome stain D: The fibrotic score (left) and lung hydroxyproline contents (right) of wild-type mice (WT), GATA-3-overexpressing mice (GATA-3), and GATA3-overexpressing mice treated with IFN-{gamma} (GATA-3+IFN-{gamma}) 28 days after intratracheal administration of 5 mg/kg bleomycin. n = 4 in each group. *Significant difference (P < 0.05) between WT and GATA-3 mice. **Significant difference (P < 0.05) between the GATA-3 and GATA-3-IFN-{gamma} groups. Scale bar = 100 µm.

 
We next assessed the effects of exogenously administered IFN-{gamma} on the development of pulmonary fibrosis using this model. Exogenous administration of IFN-{gamma} significantly improved the survival rate of GATA-3-tg mice after bleomycin administration: 80% of IFN-{gamma}-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-{gamma} (Figure 7B) . Lung histology showed that the degree of pulmonary fibrosis was much lower in IFN-{gamma}-treated GATA-3-tg mice than in IFN-{gamma}-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-{gamma} (Figure 7C) . Both the lung fibrotic score and the lung hydroxyproline content were also significantly reduced in IFN-{gamma}-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-{gamma} reduced the development of pulmonary fibrosis in mice overexpressing the GATA-3 gene.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
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.26,27 It has been demonstrated that anti-sense GATA-3 inhibited the expression of all Th2 cytokine genes in the Th2 clone D10.18 In transgenic mice, elevated GATA-3 in CD4+ T cells caused Th2 cytokine gene expression in developing Th1 cells.18 GATA-3 has been reported to transactivate the IL-5 promoter with only limited effects on IL-4 gene transcription.18,21,28 It has also been reported that GATA-3 regulates the locus accessibility of the IL-4 and IL-13 genes with chromatin remodeling.29,30 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-{gamma} 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-{gamma} production during in vitro Th1 differentiation of naïve CD4+ T cells through down-regulation of IL-12 receptor ß2 and IFN-{gamma} production.19,31 In contrast, IFN-{gamma} production in CD4+ T cells of GATA-3-deficient mice was increased even under Th2 condition.32 Furthermore, Kaminuma and colleagues33 demonstrated that GATA-3 suppresses IFN-{gamma} gene transcription via the down-regulation of Stat4. Although it remains unclear why GATA-3 overexpression did not affect Th2 cytokine production in the present model, the genetic background of the parental strain may be an important factor for Th1/Th2 responses. C57BL/6 mice are known as a Th1 responder strain. C57BL/6 mice produce a large amount of IFN-{gamma} by several stimuli including bleomycin.34 It has also been reported that ectopic expression of GATA-3 leads to the inhibition of IFN-{gamma} production under Th1-polarizing conditions.17,19,20 Thus, GATA-3 overexpression suppresses Th1 cytokine production rather than up-regulating Th2 cytokine production under a Th1-dominant condition, such as in bleomycin-treated C57BL/6 mice.

In the present study, exogenous administration of IFN-{gamma} to GATA-3-tg mice improved the degree of pulmonary fibrosis to the level of wild-type mice. Our recent study demonstrated that treatment of C57BL/6 mice with {alpha}-galactosylceramide, a selective ligand for natural killer T cells, attenuated the development of bleomycin-induced pulmonary fibrosis.35 The protective effect of {alpha}-galactosylceramide was associated with an increase in the pulmonary IFN-{gamma} level. In addition, neutralization of endogenously synthesized IFN-{gamma} with a specific antibody abrogated the protective effects of {alpha}-galactosylceramide. Similarly, Kim and colleagues36 have demonstrated that natural killer T cells attenuated pulmonary fibrosis by directly producing IFN-{gamma}. Previous studies have also demonstrated that IFN-{gamma} exerts anti-fibrotic effects in experimental animals, including a bleomycin-induced pulmonary fibrosis model.12,13 It is therefore possible to say that IFN-{gamma} plays a key role in the susceptibility of GATA-3-tg mice to bleomycin-induced pulmonary fibrosis.

To generate GATA3-tg mice, we inserted a full-length murine GATA-3 cDNA into a VA CD2 transgene cassette.22 The VA vector has been reported to directly express the inserted cDNA in all single-positive mature T cells of transgenic mice.37 Because GATA-3 is a T-cell-specific transcription factor, transgenic mice are thought to be adequate for use in evaluating T-cell-mediated regulation of pulmonary fibrosis. In the present study, intracellular cytokine analysis revealed that the suppression of IFN-{gamma} production in GATA-3-tg mice occurred in CD4+ T cells, especially Th1 cells and regulatory T cells. These findings suggest that the overexpressed GATA-3 gene actually acts on CD4+ T cells in the present model.

Whether or not T cells contribute to the pathogenesis of pulmonary fibrosis is still controversial both in humans and in experimental animals. The depletion of T cells by antibodies has been shown to inhibit the development of bleomycin-induced pulmonary fibrosis.7 Bleomycin-induced pulmonary inflammation and fibrosis were not observed in athymic nude mice.8 These findings suggest that T cells participate in the pathophysiology of fibrosis in the bleomycin model. Another study, however, has demonstrated that bleomycin-induced pulmonary fibrosis occurred in nude mice and SCID mice that lack T cells.38,39 In patients with idiopathic pulmonary fibrosis, lymphocytosis is not a characteristic feature. However, it has been shown that a significant number of both CD4+ and CD8+ T cells are infiltrated into the lung parenchyma of patients with idiopathic pulmonary fibrosis.40 Analysis of the CD4+ T-cell subpopulation has shown that patients with idiopathic pulmonary fibrosis have a significantly lower CXCR3 and a higher CCR4 expression on bronchoalveolar lavage CD4+ T cells.41 Moreover, treatment of these patients with corticosteroids results in higher CXCR3 and lower CCR4 expression compared with those in untreated patients. These findings demonstrate that an imbalance in CXCR3/CCR4, that is, an imbalance of Th1/Th2, actually occurs in the lungs of patients with idiopathic pulmonary fibrosis.

Some researchers hypothesize that idiopathic pulmonary fibrosis is a disease in which parenchymal fibrosis is directly caused by chronic inflammation.42,43 An early hypothesis suggested that an unidentified insult initiated a cycle of chronic inflammatory injury leading to fibrosis. Although corticosteroid and cytotoxic agents have been used in patients with idiopathic pulmonary fibrosis to interrupt the inflammatory cascade before irreversible tissue injury occurred, it is now clear that these anti-inflammatory therapies provide no benefit. A newer hypothesis suggests that the pulmonary fibrosis results from sequential acute lung injury.44 The resultant wound-healing response to this lung injury culminates in pulmonary fibrosis. The fibrotic response is modified by several interacting factors, including the genetic background, environmental inflammatory triggers, and the predominant inflammatory phenotype (Th1 or Th2). Although the Th2 and Th1 phenotypes are not as well defined in idiopathic pulmonary fibrosis as they are in asthma and animal models, their potential importance is one rationale for trials of immunomodulators such as IFN-{gamma} in attempts to switch the inflammatory responses to a more Th1-like phenotype.

In the present study, surprisingly, the production of IFN-{gamma} in CD4+CD25+ regulatory T cells was decreased in the lungs of GATA-3-tg mice. Regulatory T cells are a small subset of CD4+ T cells that exhibit potent immunosuppressive properties. Because recent studies suggest that regulatory T cells protect against the development of Th2 diseases such as allergic and asthmatic diseases,45 it is likely that regulatory T cells negatively regulate the development of lung fibrosis. The development and function of CD4+CD25+ regulatory T cells are under the control of the transcription factor Foxp3, which is highly expressed in these cells.46,47 The contribution of GATA-3 to the differentiation of regulatory T cells and to the production of IFN-{gamma} in these cells should be examined in future studies.

The potential of IFN-{gamma} as a treatment for idiopathic pulmonary fibrosis has not been established, and some alternative strategies are needed, because of the recent failure in the IFN-{gamma} treatment clinical trials.48 Although there is currently no direct evidence that GATA-3 plays a role in the pathogenesis of human pulmonary fibrosis, Th1/Th2 cell regulation by transcription factors might be a useful alternative approach for the treatment of pulmonary fibrosis. Our observation that the overexpression of GATA-3 exacerbated the development of pulmonary fibrosis by inducing a relative Th2 bias suggests that inhibiting the activation of GATA-3 might be useful as a therapeutic strategy for pulmonary fibrosis. On the other hand, the transcription factor T-bet is thought to be a key regulator of Th1 differentiation. T-bet expression is strongly correlated with IFN-{gamma} expression and is specifically up-regulated in primary Th cells that differentiate along the Th1 but not the Th2 pathways.49 T-bet-deficient mice have been shown to develop spontaneous airway changes consistent with human asthma, which is a Th2 model disease.50 Finotto and colleagues51 have recently demonstrated that T-bet negatively regulates lung remodeling, such as subepithelial collagen deposition and myofibroblast transformation, by controlling the interaction with IL-13 and TGF-ß in the asthmatic airway. It is therefore likely that the regulation of T-bet gene expression influences the development of pulmonary fibrosis. Therefore, we are currently investigating the protective effects of T-bet overexpression on the development of bleomycin-induced pulmonary fibrosis. We suspect that Th1/Th2 regulation by these transcription factors has an advantage over the systemic administration of IFN-{gamma} for the treatment of pulmonary fibrosis for the following reasons: the transactivation of the target proteins occurs specifically in T cells, and thus the systemic side effects can be reduced; and it is expected that Th1/Th2 regulation will not only up-regulate IFN-{gamma} production but also suppress the production of Th2 cytokines that enhance fibrotic processes, such as IL-4 and IL-13.

Idiopathic pulmonary fibrosis is a lethal disorder for which efficacious therapeutic approaches are not readily available. Although transcription factor regulation therapy cannot be used currently for the treatment of pulmonary fibrosis, we believe that this study may provide the possibility of new therapies for the treatment of pulmonary fibrosis.


   Footnotes
 
Address reprint requests to Yukio Ishii, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305, Japan. E-mail: ishii-y{at}md.tsukuba.ac.jp

Supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan.

Accepted for publication April 11, 2006.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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