Abstract
Rationale: Hypersensitivity pneumonitis (HP) is mediated by a Th1 immune response. Transcription factor GATA binding protein-3 (GATA-3) is believed to be a key regulator of Th2 differentiation and thus might play regulatory roles in the pathogenesis of hypersensitivity pneumonitis (HP).
Objectives: We examined the effect of GATA-3 overexpression on the development of HP in mice.
Methods: Wild-type C57BL/6 mice and GATA-3–overexpressing mice of the same background were used in this study. HP was induced by repeated exposure to Saccharopolyspora rectivirgula, the causative antigen of farmer's lung.
Measurements and Main Results: Antigen exposure resulted in a marked inflammatory response with enhanced pulmonary expression of T-bet and the Th1 cytokine interferon (IFN)-γ in wild-type mice. The degree of pulmonary inflammation was much less severe in GATA-3–overexpressing mice. The induction of T-bet and IFN-γ genes was suppressed, but a significant induction of Th2 cytokines, including IL-5 and IL-13, was observed in the lungs of GATA-3–overexpressing mice after antigen exposure. Supplementation with recombinant IFN-γ enhanced lung inflammatory responses in GATA-3–overexpressing mice to the level of wild-type mice. Because antigen-induced IFN-γ production predominantly occurred in CD4+ T cells, nude mice were transferred with CD4+ T cells from either wild-type or GATA-3–overexpressing mice and subsequently exposed to antigen. Lung inflammatory responses were significantly lower in nude mice transferred with CD4+ T cells from GATA-3–overexpressing mice than in those with wild-type CD4+ T cells, with a reduction of lung IFN-γ level.
Conclusions: These results indicate that overexpression of GATA-3 attenuates the development of HP by correcting the Th1-polarizing condition.
Hypersensitivity pneumonitis is mediated by a Th1 immune response. The GATA binding protein-3 (GATA-3) transcriptional factor participates in Th2 differentiation and may be involved in regulating the pathogenesis of hypersensitivity pneumonitis (HP).
Overexpression of GATA-3 attenuates the development of HP by correcting the Th1-polarizing condition.
Th1 and Th2 cells are differentiated from common T precursor cells (13, 14). The differentiation requires the activity of distinct transcription factors. GATA binding protein-3 (GATA-3), a member of the GATA family of zinc-finger transcription factors, has been identified as a key regulator of Th2 development (15–18). In physiologic conditions, GATA-3 is selectively expressed in Th2 but not Th1 cells (15, 16, 19). Transgenic and retroviral expression of GATA-3 induces a Th2 cytokine profile in Th1 cells (15, 16), whereas dominant-negative GATA-3 down-regulates in Th2 clones (19). Several studies have demonstrated that GATA-3 not only transactivates Th2 cytokines but also suppresses Th1 cytokine expression including IFN-γ. GATA-3 significantly down-regulated IFN-γ production during in vitro Th1 differentiation of naive CD4+ T cells through down-regulation of IL-12 receptor β2 and IFN-γ production (17, 20). In contrast, IFN-γ production in CD4+ T cells of GATA-3–deficient mice was increased even under Th2 conditions (21). Because GATA-3 regulates plenty of T-cell cytokines, activation of GATA-3 may have an advantage over the targeting of single T-cell cytokines in therapeutic strategies against Th1-mediated diseases, including HP.
We recently established GATA-3–overexpressing transgenic (GATA-3-tg) mice. We used such mice in the present study to investigate the role of GATA-3 in the development of murine HP induced by SR. We found that overexpression of GATA-3 attenuates the development of HP by suppressing Th1 polarization.
Some of the results of these studies have been previously reported in the form of an abstract (22).
Details on methods are available in the online supplement.
GATA-3-tg mice were generated as previously described (23). GATA-3-tg mice were backcrossed with C57BL/6 mice for eight generations and we confirmed the sufficiency of backcrossing by analyzing the polymorphism of microsatellite DNAs. C57BL/6 nu/nu mice (B6.Cg-Foxn1nu) were purchased from Jackson Laboratory (Bar Harbor, ME). All mice used in this study were 8 to 12 weeks of age and were maintained in our animal facilities under specific pathogen–free conditions. All animal studies were approved by the University of Tsukuba institutional review board.
SR was obtained from the American Type Culture Collection (Rockville, MD). SR antigen was prepared as previously described (4, 8). HP was induced by inoculating 150 μg of SR antigen intranasally for 3 consecutive days per week for 3 weeks. Mice inoculated with saline were examined as control animals.
Lung indexes were calculated as described previously (24): lung index = [(lung weight/body weight) test animal]/[(lung weight/body weight) control animal].
Several lung paraffin sections from each mouse were stained with hematoxylin and eosin. For each mouse, 200 different areas from the sections were chosen at random and examined at ×100 magnification. Each area was judged to be normal or abnormal based on the absence or presence of inflammatory cells, respectively. The percentage of examined areas that were abnormal is shown as “percent abnormal.”
The lung was lavaged six times with 1 ml saline. Total cell counts were enumerated using a hemocytometer. Differential cell count was performed by staining with Diff-Quik (American Scientific Products, Obetz, OH).
The expression levels of GATA-3, T-bet, IFN-γ, tumor necrosis factor (TNF)-α, IL-4, IL-5, and IL-13 genes were determined by quantitative real-time reverse transcription–polymerase chain reaction using ready-made fluorogenic probes and primers (Applied Biosystems, Foster City, CA). Expression levels for amplicons were quantified using the ΔΔCT method according to the manufacturer's protocols. The expression levels were normalized against 18S ribosomal RNA.
The lungs were removed, minced, and incubated with Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum and 75 U/ml collagenase (type 1; Sigma-Aldrich, St. Louis, MO) at 37°C for 90 minutes. The cells were then filtered through 20-μm nylon mesh. Cells were stained with anti–T-cell receptor β (TCRβ), anti-natural killer 1.1 (NK1.1), anti-CD4, anti-CD8 (BD PharMingen, San Diego, CA), and anti-Ly6G (BioLegend, San Diego, CA) antibodies, and analyzed by flow cytometry. IFN-γ production in lung lymphocytes was determined by flow cytometric intracellular cytokine analysis as previously described (25).
GATA-3-tg mice were given 1.25 μg of murine recombinant IFN-γ (R&D Systems, Minneapolis, MN) or diluent intratracheally after every SR inoculation.
Splenic cells were collected from wild-type mice and GATA-3-tg mice. CD4+ T cells were enriched using magnetic beads (MACS; Miltenyi Biotech, Auburn, CA). The purity of isolated CD4+ T cells was greater than 93%. Ten million CD4+ T cells of either genotype were injected intravenously into nu/nu mice.
Data are presented as means ± SEM. Comparisons of data among each experimental group were performed using an analysis of variance test with Scheffe's test. P values less than 0.05 were considered statistically significant.
First, we evaluated the effect of GATA-3 transgenes on the expression of GATA-3 in the lung tissue. Among saline-exposed control animals, the expression of GATA-3 mRNA was approximately 3.5 times higher in the lungs of GATA-3-tg mice than in that of wild-type mice (Figure 1A). After exposure to SR, pulmonary expression of GATA-3 mRNA was also significantly increased in GATA-3-tg mice compared with wild-type mice (Figure 1A). T-bet is known as a key transcription factor for differentiation of Th1 cells. We then evaluated the effect of GATA-3 transgenes on the expression of T-bet. In saline-exposed control mice, pulmonary expression of T-bet mRNA did not differ between wild-type mice and GATA-3-tg mice (Figure 1B). After exposure to SR, the expression of T-bet was markedly increased in the lungs of wild-type mice compared with saline-exposed control animals (Figure 1B). However, T-bet mRNA was not induced in the lungs of GATA-3-tg mice by SR exposure (Figure 1B). Pulmonary expression of T-bet was significantly lower in GATA-3-tg mice than in wild-type mice under the SR-exposed condition.
Figure 1. The expressions of GATA-3 (A) and T-bet (B) mRNA in the lungs of wild-type (WT) mice and GATA-3–overexpressing mice of the same background (GATA-3-tg) 4 days after the last exposure to Saccharopolyspora rectivirgula antigen (SR) or saline. Results are normalized to the expression of 18S ribosomal RNA and are expressed as fold increases over average T-bet and GATA-3 mRNA expression in saline-exposed WT mice. Data represent mean values ± SEM of four mice in each group. *P < 0.05 versus corresponding WT mice.
[More] [Minimize]Lung histopathology revealed that SR exposure resulted in marked inflammatory cell infiltration in peribronchial and perivascular areas with lymphoid follicle and granuloma formation in wild-type mice (Figure 2A, WT-SR). In GATA-3-tg mice, however, the inflammatory response was markedly lower, with minimal infiltration of inflammatory cells by SR exposure (Figure 2A, GATA-3-tg-SR). Saline-exposed wild-type and GATA-3-tg mice had no inflammation or granulomas (Figure 2A, WT-saline and GATA-3-tg-saline). The extent of pulmonary histologic abnormalities was then quantitatively assessed at light microscopic level. The histologic abnormalities after SR exposure were significantly attenuated in GATA-3-tg mice compared with those in wild-type mice (Figure 2B). In this murine HP model, increase in lung weight after SR exposure was shown to correlate with the histopathologic abnormality (26). We therefore evaluated the change in lung weight by calculating the lung index. The lung index after SR exposure was significantly lower in GATA-3-tg mice than in wild-type mice (Figure 2C).
Figure 2. (A) Histopathology examination of the lungs of wild-type (WT) mice and GATA-3–overexpressing mice (GATA-3-tg) 4 days after the last exposure to Saccharopolyspora rectivirgula antigen (SR) or saline. (B) Quantitation of inflammatory responses in WT and GATA-3-tg mice. **P < 0.01 versus corresponding WT mice. Data represent mean values ± SEM of four mice in each group. (C) Changes in lung weight calculated as lung indexes in WT and GATA-3-tg mice after exposure to SR. *P < 0.05 versus corresponding WT mice. Data represent mean values ± SEM of four mice in each group.
[More] [Minimize]To determine the inflammatory cell type in the lung tissue after SR exposure, the numbers of total cells, macrophages, lymphocytes, neutrophils, and eosinophils were counted in bronchoalveolar lavage (BAL) fluid of both genotypes of mice. SR exposure led to a significant increase in the numbers of total cells, macrophages, lymphocytes, and neutrophils in BAL fluid of both genotypes (Figures 3A–3D). In GATA-3-tg mice, however, the numbers of total cells, macrophages, and lymphocytes in BAL fluid were significantly lower than in wild-type mice (Figures 3A–3C). There was no significant difference in the number of lavagable neutrophils between the genotypes (Figure 3D). Eosinophils were not detectable in BAL fluid of either mouse genotype throughout the experiments (Figure 3E). These results suggest that the development of SR-induced HP, including macrophagic and lymphocytic inflammation, is significantly attenuated in mice overexpressing GATA-3.
Figure 3. The numbers of total cells (A), macrophages (B), lymphocytes (C), neutrophils (D), and eosinophils (E) in the bronchoalveolar lavage fluids of wild-type (WT) and GATA-3–overexpressing (GATA-3-tg) mice 4 days after the last exposure to Saccharopolyspora rectivirgula antigen (SR) or saline. *P < 0.05 versus corresponding WT mice. Data represent mean values ± SEM of four mice in each group.
[More] [Minimize]We then examined the expression of Th1/Th2 cytokine levels in the lungs of both wild-type and GATA-3-tg mice exposed to either saline or SR using quantitative real-time reverse transcription–polymerase chain reaction. In saline-exposed control animals, the expressions of Th1 cytokines IFN-γ and TNF-α were significantly lower in the lungs of GATA-3-tg mice than in wild-type mice (Figures 4A and 4B). The expressions of both IFN-γ and TNF-α were markedly increased after SR exposure in wild-type mice compared with corresponding saline-exposed control animals (Figures 4A and 4B). However, the level of IFN-γ and TNF-α was not up-regulated by SR exposure in the lungs of GATA-3-tg mice (Figures 4A and 4B). For Th2 cytokines, pulmonary expression of IL-4, IL-5, and IL-13 mRNA was not different between wild-type mice and GATA-3-tg mice in the saline-exposed condition (Figures 4C–4E). After SR exposure, pulmonary expression of IL-5 and IL-13 was increased significantly in GATA-3-tg mice but not in wild-type mice (Figures 4D and 4E). As a result, the expressions of IL-5 and IL-13 were significantly higher in GATA-3-tg mice than in wild-type mice (Figures 4D and 4E). The expression of IL-4 was not different between genotypes under either the saline- or SR-exposed condition (Figure 4C). These results indicate that SR exposure shifts the Th1/Th2 balance toward Th1 in wild-type mice. However, using the same antigen, the Th1/Th2 balance is shifted toward Th2 in mice overexpressing GATA-3.
Figure 4. The mRNA expressions of IFN-γ (A), tumor necrosis factor (TNF)-α (B), IL-4 (C), IL-5 (D), and IL-13 (E) in the lungs of wild-type (WT) and GATA-3–overexpressing (GATA-3-tg) mice 4 days after the last exposure to Saccharopolyspora rectivirgula antigen (SR) or saline. *P < 0.05 and **P < 0.01 versus corresponding WT mice. Results were normalized to expression of 18S ribosomal RNA. Data represent mean values ± SEM of four mice in each group.
[More] [Minimize]Previous studies and the above findings suggest that T-cell–mediated immunity is necessary for the development of HP. We therefore analyzed the proportions of lymphocyte subsets accumulated in the lung tissue. The proportion of T cells in lung lymphocytes was approximately 40% in saline-exposed wild-type mice (Figure 5A, T cell). Among saline-exposed mice, the proportion did not differ between GATA-3-tg and wild-type genotypes (Figure 5A, T cell). Although the proportion of lung T cells was increased by 50% after exposure to SR, the proportion was not different between wild-type and GATA-3-tg genotypes (Figure 5A, T cells). The proportion of natural killer (NK) cells and natural killer T (NKT) cells did not differ between the genotypes under either the saline- or SR-exposed condition (Figure 5A, NK cell and NKT cell).
Figure 5. (A) Lung lymphocyte subset in wild-type (WT) and GATA-3–overexpressing (GATA-3-tg) mice 4 days after the last exposure to Saccharopolyspora rectivirgula antigen (SR) or saline. Data represent mean values ± SEM of three mice in each group. (B) The ratio of CD4+ versus CD8+ T cells in the lungs of WT and GATA-3-tg mice after exposure to SR or saline. *P < 0.05 versus saline-exposed WT mice. Data represent mean values ± SEM of three mice in each group. (C) IFN-γ-production in CD4+ T cells (left panels), CD8+ T cells (center panels), and Ly6G-positive neutrophils of WT and GATA-3-tg mice after exposure to SR or saline. Data represent mean values of three mice in each group. NK = natural killer; NKT = natural killer T.
[More] [Minimize]We then evaluated the ratio of CD4+ T cells to CD8+ T cells. The numbers of CD4+ T cells were two times higher than those of CD8+ T cells in the lungs of saline-exposed wild-type mice. In the lungs of saline-exposed GATA-3-tg mice, the CD4/CD8 ratio was significantly elevated compared with the saline-exposed wild-type mice (Figure 5B). The proportion of CD4+ T cells was much increased in the lungs of both wild-type and GATA-3-tg mice after SR exposure, with the ratio of CD4+ to CD8+ cells increased up to 10-fold in both genotypes (Figure 5B). However, the CD4/CD8 ratio was not different between the genotypes after SR exposure. These findings indicate that CD4+ T cells predominantly increased after SR exposure. Several reports have demonstrated that IFN-γ plays an important role in the pathogenesis of murine HP. In the present study, we also found that the expression of IFN-γ was markedly increased after SR exposure in wild-type mice (Figure 4A). To clarify the cell source of IFN-γ, we assessed the production of IFN-γ in CD4+ T cells, CD8+ T cells, and neutrophils of both wild-type and GATA-3-tg mice after SR exposure. The proportion of IFN-γ–producing CD4+ T cells in lung lymphocytes was much higher in wild-type mice than in GATA-3-tg mice (10.2% in wild-type mice vs. 2.5% in GATA-3-tg mice; Figure 5C, left panels). The proportion of IFN-γ–producing CD8+ T cells in lung lymphocytes was low and there was no difference between mouse genotypes (2.2% in wild-type mice vs. 1.6% in GATA-3-tg mice; Figure 5C, center panels). We further assessed the production of IFN-γ in Ly6G-positive neutrophils of both wild-type and GATA-3-tg mice after SR exposure. Although the proportion of IFN-γ–producing neutrophils in lung inflammatory cells was higher in wild-type mice than in GATA-3-tg mice (1.1% in wild-type mice vs. 0.3% in GATA-3-tg mice), the proportion was much lower than that of IFN-γ–producing CD4+ T cells in both mouse genotypes (Figure 5C, right panels). SR exposure did not induce the production of IFN-γ in NK cells or NKT cells in either mouse genotype (data not shown). These results suggest that CD4+ T cells are the major cell source of IFN-γ after stimulation with SR. Overexpression of GATA-3 suppresses the production of IFN-γ predominantly in CD4+ T cells.
Several reports have demonstrated that IFN-γ plays an important role in the pathogenesis of murine HP. In the present study, SR induction of IFN-γ observed in wild-type mice was significantly diminished in GATA-3-tg mice. We therefore assessed the effect of IFN-γ supplementation on the development of HP in GATA-3-tg mice. Mouse recombinant IFN-γ or vehicle was administered intratracheally after every SR inoculation. Lung histopathology revealed that the inflammatory responses to SR were more severe in IFN-γ–treated GATA-3-tg mice, compared with untreated control animals (Figure 6A). The histology score after SR exposure was significantly increased in IFN-γ–treated GATA-3-tg mice, compared with untreated controls, reaching the level of SR-exposed wild-type mice (Figure 6B). The numbers of total cells, macrophages, and lymphocytes in BAL fluid after exposure to SR were also significantly increased in IFN-γ–treated GATA-3-tg mice, reaching the levels of SR-exposed wild-type mice (Figure 6B). These results indicate that the suppression of the development of HP in GATA-3-tg mice was at least in part due to a minimal induction of IFN-γ in response to the antigen.
Figure 6. (A) Histopathologic examination of the lungs of wild-type (WT) mice, GATA-3–overexpressing (GATA-3-tg) mice, and GATA-3-tg mice treated with IFN-γ (GATA-3-tg + IFN-γ) 4 days after the last exposure to Saccharopolyspora rectivirgula antigen (SR). (B) Quantitation of lung inflammatory responses in WT, GATA-3-tg, and GATA-3-tg + IFN-γ mice. *P < 0.05 versus corresponding GATA-3-tg control animals. Data represent mean values ± SEM of four mice in each group. (C) The numbers of total cells, macrophages, lymphocytes, and neutrophils in bronchoalveolar fluid from WT, GATA-3-tg, and GATA-3-tg + IFN-γ mice. *P < 0.05 versus corresponding GATA-3-tg control animals. Data represent mean values ± SEM of four mice in each group.
[More] [Minimize]To delineate the role of CD4+ T cells in the development of HP and to confirm that GATA-3 exerts its effect on HP through CD4+ T cells, CD4+ T cells purified from spleens of either wild-type or GATA-3-tg mice were transferred to nu/nu mice which were subsequently exposed to SR. CD4+ T cells that were taken from GATA-3-tg mice were equivalent to those from wild-type mice in their ability to reconstitute after the transfer (Figure 7A). In line with previous reports, nu/nu mice without transfer of CD4+ T cells showed only minimal inflammatory responses after SR exposure (Figure 7B, left panel). Inflammatory cell infiltration was observed in the peribronchial and perivascular regions of nu/nu mice with CD4+ T cells transferred from either wild-type or GATA-3-tg mice. However, the degree of inflammation after exposure to SR was much lower in nu/nu mice with CD4+ T cells from GATA-3-tg mice than in nu/nu mice with CD4+ T cells from wild-type mice (Figure 7B, center and right panels). Correspondingly, both the lung histology score and pulmonary expression of IFN-γ were significantly lower in nu/nu mice with GATA-3-tg CD4+ T cells than in those with wild-type CD4+ T cells (Figures 7C and 7D). These results indicate that CD4+ T cells play a role in the pathogenesis of HP by mediating IFN-γ production. Overexpression of GATA-3 may suppress CD4+ T-cell–mediated development of HP.
Figure 7. (A) The appearance of CD4+ T cells in the spleens from wild-type (WT) mice, nu/nu mice of the same background (B6 nude), nu/nu mice transferred with CD4+ T cells of WT mice (WT CD4+ reconstituted), and nu/nu mice transferred with CD4+ T cells of GATA-3–overexpressing mice (GATA-3-tg CD4+ reconstituted). (B) Histopathology examination of the lungs from nu/nu mice not reconstituted (not reconstituted) and nu/nu mice reconstituted with CD4+ T cells from WT mice (WT CD4+ reconstituted) or from GATA-3-tg mice (GATA-3-tg CD4+ T reconstituted) 4 days after the last exposure to Saccharopolyspora rectivirgula antigen. (C) Quantitation of lung inflammatory responses in nu/nu mice reconstituted with CD4+ T cells from WT mice (WT CD4+ reconstituted) or from GATA-3-tg mice (GATA-3-tg CD4+ T reconstituted). *P < 0.05 versus WT CD4+ T reconstituted. (D) The expression of IFN-γ mRNA in the lungs of nu/nu mice reconstituted with CD4+ T cells from WT mice (WT CD4+ reconstituted) or from GATA-3-tg mice (GATA-3-tg CD4+ T reconstituted). *P < 0.05 versus WT CD4+ T reconstituted. Data represent mean values ± SEM of four mice in each group.
[More] [Minimize]This study illustrates the pivotal regulatory role of GATA-3 in the SR-induced murine HP model. Lung inflammatory responses to SR were much less severe in GATA-3-tg mice than in wild-type mice of the same background. Analysis of cytokines revealed that overexpression of GATA-3 shifts the Th1/Th2 balance toward Th2 after SR exposure, whereas the balance was shifted to Th1 in wild-type mice by the same antigen exposure.
In line with previous reports (8, 27), the expression of IFN-γ was significantly enhanced in wild-type C57BL/6 mice after SR exposure. However, the induction of IFN-γ mRNA did not occur in GATA-3-tg mice after exposure to SR. These findings suggest that overexpression of GATA-3 not only transactivates Th2 cytokine genes but also inhibits Th1 cytokine expression. This observation is consistent with previous findings using GATA-3-tg mice of C57BL/6 background. We recently demonstrated that production of lung IFN-γ was significantly hampered in GATA-3-tg mice in bleomycin-induced pulmonary fibrosis (28). Ozawa and coworkers also demonstrated, in purified protein derivative–induced delayed-type hypersensitivity, that IFN-γ production in lymph node cells of sensitized mice was significantly lower in GATA-3-tg mice (29). In contrast, in another report from our laboratory using Balb/c mice, suppression of IFN-γ production was not observed in either saline- or ovalbumin-treated GATA-3-tg mice (30). Although the effect of GATA-3 on the suppression of Th1 bias might be dependent on such factors as genetic background or antigenic stimuli, GATA-3 might represent a potential therapeutic target for diseases in which IFN-γ plays crucial roles.
In the present study, expression of another Th1 cytokine, TNF-α, was also induced markedly by exposure to SR in wild-type mice but not in GATA-3-tg mice. Previous reports have demonstrated that SR exposure induces lung TNF-α expression in both mRNA and protein (27, 31). Mice treated with anti–TNF-α antibody decreased cellular recruitment into the lungs after SR exposure and showed normal lung histology (32). Ouyang and coworkers have demonstrated that, in addition to IFN-γ, the expressions of other Th1 cytokines, including TNF-α, TNF-β, and lymphotoxin-β, were significantly inhibited in GATA-3–expressing Th1 cells (17). Nance and coworkers have demonstrated that the expression of TNF-α after exposure to SR was significantly decreased in IFN-γ–knockout mice (27). Although the regulatory mechanisms of TNF-α expression by GATA-3 are not fully understood, it is likely that the impaired induction of TNF-α mRNA expression in GATA-3-tg mice is at least in part attributable to the suppression of IFN-γ in this model.
It is widely accepted 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, pulmonary expression of IL-5 and IL-13, but not IL-4, was significantly up-regulated in GATA-3-tg mice after SR exposure. This observation can be explained from the findings that GATA-3 binds and transactivates the IL-5 (32–34) and the IL-13 (35) promoters, whereas GATA-3 alone weakly transactivates the IL-4 gene and an additional transcription factor, c-Maf, is required for IL-4 production (16, 19, 32, 36, 37). Although IL-4 mRNA stability was shown to correlate with the susceptibility to HP (6), the contribution of Th2 cytokines to the development of HP is not well understood. In the present study, however, no phenotypic changes related to these cytokines, such as eosinophilia or mucus hypersecretion, were observed in GATA-3-tg mice after exposure to SR. Thus, the effect of Th2 cytokine induction on the phenotypic changes of HP might be relatively limited in this model, in contrast with the effect of Th1 cytokine suppression.
In the lungs of wild-type mice, a significant increase in T-bet expression was observed after SR exposure. T-bet is a major Th1 commitment factor and transcription factor of Th1 cytokines, including IFN-γ in CD4+ T cells (38). Increased expression of the T-bet gene suggests that Th1 polarization and Th1 cytokine production in CD4+ T cells are still maintained at the later stage of HP. It was also demonstrated that IFN-γ markedly enhances T-bet mRNA expression in Th cells, then drives Th1 commitment and IFN-γ mRNA expression in CD4+ T cells (39). Concomitant induction of IFN-γ and T-bet genes in SR-exposed wild-type mice may result in the augmentation of Th1 responses by this positive feedback loop between T-bet and IFN-γ. In contrast, the expression of T-bet was not induced in the lungs of GATA-3-tg mice after exposure to SR, implying that overexpression of GATA-3 inhibits T-bet expression. The interaction between GATA-3 and T-bet is somewhat complicated and is not fully understood. Usui and coworkers have reported that retroviral T-bet expression in developing and established Th2 cells leads to down-regulation of GATA-3 levels (40). In contrast, retroviral GATA-3 expression in developing Th1 cells does not block Th1 development in cells coexpressing Stat4 but does so in cells coexpressing T-bet, suggesting that GATA-3 suppresses Th1 development by down-regulation of Stat4 but not through effects on T-bet (18). Thus, inhibition of T-bet expression in GATA-3-tg mice observed in this study might not be a direct effect of GATA-3 but an indirect effect via mediated suppression of the IFN-γ level.
Supplementation with IFN-γ aggravated the inflammatory response to SR in GATA-3-tg mice to the level of wild-type mice, implying that IFN-γ plays a crucial role in determining susceptibility to HP. Several reports have also disclosed its importance in the development of HP. Treatment with the neutralizing antibody against IFN-γ attenuated the severity of HP (41). IFN-γ–knockout mice exhibited minimal inflammatory responses after SR exposure, and IFN-γ replacement to these mice resulted in the aggravation of HP (8). Although the cellular mechanisms of IFN-γ–mediated development of HP are not fully understood, IFN-γ may be produced predominantly by innate immune cells such as neutrophils during the early stages of HP (42). Hwang and coworkers have also demonstrated that NKT cells producing IL-4 play a protective role in SR-induced HP by suppressing IFN-γ–producing neutrophils, which induce the activation and proliferation of CD8+ T cells (43). Subsequently, IFN-γ mediates the recruitment of CXCR3+CD4+ T cells into the lung through production of chemokines such as interferon-inducible protein-10 (IP-10), monokine induced by interferon γ (Mig), and interferon-inducible T cell α chemoattractant (I-TAC). (29). In addition to these chemokines, T-bet itself also plays crucial roles in controlling Th1 cell migration to inflammatory sites. T-bet can modulate the binding of CD4+ T cells to P-selectin by regulating the expression of CXCR3 (44). Thus, CD4+ T cells recruited to the inflamed site by these mechanisms are believed to be the major source of IFN-γ in the late stage of HP, in which enhanced production of IFN-γ occurs, possibly through the positive feedback loop between T-bet and IFN-γ mentioned above. In the present study, SR-induced IFN-γ production predominantly occurred in lung CD4+ T cells of wild-type mice. Overexpression of GATA-3 clearly decreased IFN-γ production in CD4+ T cells after SR exposure to the level of saline-exposed control animals without the alteration of the lung CD4/CD8 T-cell ratio and neutrophil influx. Moreover, the proportion of NKT cells in lung lymphocytes was quite low, both before and after SR exposure in GATA-3-tg mice. These results suggest that overexpression of GATA-3 affects IFN-γ production in CD4+ T cells in the late stage of HP, rather than innate immune cells during the early stage of HP.
Schuyler and coworkers have established a model of experimental HP by the adoptive transfer method (45). Using this model, they demonstrated that depletion of CD4+ T cells, but not CD8+ T cells, ablated the ability of recipient animals to express adoptively transferred HP (46, 47). They also demonstrated that the Th1 CD4+ T-cell line that produces a high level of IFN-γ, but not the Th2 CD4+ T-cell line that produces a low level of IFN-γ, can adoptively transfer HP (11). Because GATA-3 is a key regulator of Th2 development (15–18), it is likely that the difference in the development of HP between wild-type and GATA-3-tg mice might be ascribed to differences in the CD4+ T-cell characteristics of the two genotypes. To evaluate the overall contribution of CD4+ T cells to the development of HP, CD4+ T cells isolated from naive wild-type or GATA-3-tg mice were transferred to nu/nu mice, and SR was repeatedly introduced to induce HP. Lung inflammatory responses and IFN-γ levels after SR exposure were significantly lower in nu/nu mice reconstituted with CD4+ T cells from GATA-3-tg mice compared with those with wild-type CD4+ T cells. Thus, using a different method from Schuyler and coworkers, we clearly demonstrate that CD4+ T cells play a critical role in the development of HP. By contrast, Nance and coworkers have reported that IFN-γ production by T cells is not necessary in the development of HP (42). In their study, recombination activating gene-1–knockout mice reconstituted with spleen cells from IFN-γ–knockout mice developed granulomas to a similar degree as those reconstituted with wild-type spleen cells. Because they evaluated the presence or absence of granuloma formation as an endpoint, the contribution of T-cell IFN-γ production to the total inflammatory responses to SR, as investigated in our study, still remains unclear. One of the possible ways to explain the discrepancy is that GATA-3–overexpressing CD4+ T cells act on other cells to modulate IFN-γ production indirectly by secreting several cytokines that are under the influence of GATA-3. In this regard, GATA-3 has an advantage over the targeting of single T-cell cytokines in therapeutic strategies against the development of HP.
In conclusion, overexpression of GATA-3 attenuated HP by shifting the lung Th1/Th2 balance toward Th2, and especially by suppressing IFN-γ production. The degree of inflammatory response to SR was much less severe in the nude mice reconstituted with CD4+ T cells from GATA-3-tg mice, compared with those reconstituted with wild-type CD4+ T cells, with decreased expression of IFN-γ. It appears that modulation of the characteristics of CD4+ T cells can regulate the development of HP.
| 1. | Girard M. Israel-Assayag, Cormier Y. Pathogenesis of hypersensitivity pneumonitis. Curr Opin Allergy Clin Immunol 2004;4:93–98. |
| 2. | Mohr LC. Hypersensitive pneumonitis. Curr Opin Pulm Med 2004;10:401–411. |
| 3. | Denis M, Cormier Y, Fournier M, Tardiff J, Laviolette M. Tumor necrosis factor plays an essential role in determining hypersensitivity pneumonitis in a mouse model. Am J Respir Cell Mol Biol 1991;5:477–483. |
| 4. | Denis M, Cormier Y, Laviolette M. Murine hypersensitivity pneumonitis: a study of cellular infiltrates and cytokine production and its modulation by cyclosporine A. Am J Respir Cell Mol Biol 1992;6:68–74. |
| 5. | Takizawa H, Ohta K, Horiuchi T, Suzuki N, Ueda T, Yamaguchi M, Yamashita N, Ishii A, Suko M, Okudaira H, et al. Hypersensitivity pneumonitis in athymic nude mice: additional evidence of T cell dependency. Am Rev Respir Dis 1992;146:479–484. |
| 6. | Butler NS, Monick MM, Yarovinsky TO, Powers LS, Hunninghake GW. Altered IL-4 mRNA stability correlates with Th1 and Th2 bias and susceptibility to hypersensitivity pneumonitis in two inbred strains of mice. J Immunol 2002;169:3700–3709. |
| 7. | Gudmundsson G, Monick MM, Hunninghake GW. IL-12 modulates expression of hypersensitivity pneumonitis. J Immunol 1988;161:991–999. |
| 8. | Gudmundsson G, Hunninghake GW. Interferon-gamma is necessary for the expression of hypersensitivity pneumonitis. J Clin Invest 1997;99:2386–2390. |
| 9. | Ghadirian E, Denis M. Murine hypersensitivity pneumonitis: interleukin-4 administration partially abrogates the disease process. Microb Pathog 1992;12:377–382. |
| 10. | Gudmundsson G, Bosch A, Davidson L, Berg DJ, Hunninghake GW. Interleukin-10 modulate the severity of hypersensitivity pneumonitis. Am J Respir Cell Mol Biol 1998;19:812–818. |
| 11. | Schuyer M, Gott K, Cherne A, Edwards B. Th1 CD4+ cells adoptively transfer experimental hypersensitivity pneumonitis. Cell Immunol 1997;177:169–175. |
| 12. | Irifune K, Yokoyama A, Kohno N, Sakai K, Hiwada K. T-helper 1 cells induce alveolitis but do not lead to pulmonary fibrosis in mice. Eur Respir J 2003;21:11–18. |
| 13. | Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996;383:787–793. |
| 14. | Liblau RS, Singer SM, McDevitt HO. Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol Today 1995;16:34–38. |
| 15. | Zhang 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;272:21597–21603. |
| 16. | Zheng DH, Flavell RA. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 1997;89:587–596. |
| 17. | Ouyang 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;9:745–755. |
| 18. | Usui 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;18:415–428. |
| 19. | Zhang DH, Yang L, Ray A. Differential responsiveness of the IL-5 and IL-4 genes to transcription factor GATA-3. J Immunol 1998;161:3817–3821. |
| 20. | Ferber 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;91:134–144. |
| 21. | Pai 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;101:1993–1998. |
| 22. | Matsuno Y, Ishii Y, Kikuchi N, Haraguchi N, Morishima Y. Suppression of hypersensitivity pneumonitis in GATA-3-overexpressing mice [abstract]. Am J Respir Crit Care Med 2007;175:A537. |
| 23. | Yoh K, Shibuya K, Morito N, Nakano T, Ishizaki K, Shimohata H, Nose M, Izui S, Shibuya A, Koyama A, et al. 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;14:2494–2502. |
| 24. | Wilson BD, Sternick JL, Yoshizawa Y, Katzenstein AL, Moore VL. Experimental murine hypersensitivity pneumonitis: multigenic control and influence by genes within the I-B subregion of the H-2 complex. J Immunol 1982;129:2160–2163. |
| 25. | Murphy 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. |
| 26. | Takizawa H, Suko M, Kobayashi N, Shoji S, Ohta K, Nogami M, Okudaira H, Miyamoto T, Shiga J. Experimental hypersensitivity pneumonitis in the mouse: histologic and immunologic features and their modulation with cyclosporine A. J Allergy Clin Immunol 1988;81:391–400. |
| 27. | Nance S, Cross R, Fitzpatrick E. Chemokine production during hypersensitivity pneumonitis. Eur J Immunol 2004;34:677–685. |
| 28. | Kimura T, Ishii Y, Yoh K, Morishima Y, Iizuka T, Kiwamoto T, Matsuno Y, Homma S, Nomura A, Sakamoto T, et al. Overexpression of the transcription factor GATA-3 enhances the development of pulmonary fibrosis. Am J Pathol 2006;169:96–104. |
| 29. | Ozawa H, Tamauchi H, Ito M, Terashima M, Inoue M, Hozumi K, Habu S, Watanabe N. Immune responses to Nippostrongylus brasiliensis and tuberculin protein in GATA-3-transgenic mice. Immunol Lett 2005;99:228–235. |
| 30. | Kiwamoto T, Ishii Y, Morishima Y, Yoh K, Maeda A, Ishizaki K, Iizuka T, Hegab AE, Matsuno Y, Homma S, et al. Transcription factors T-bet and GATA-3 regulate development of airway remodeling. Am J Respir Crit Care Med 2006;174:142–151. |
| 31. | Denis M, Cormier Y, Fournier M, Tardiff J, Laviolette M. Tumor necrosis factor plays an essential role in determining hypersensitivity pneumonitis in a mouse model. Am J Respir Cell Mol Biol 1991;5:477–483. |
| 32. | Lee HJ, Takemoto N, Kurata H, Kamogawa S, Miyatake S, O'Garra A, Arai N. GATA-3 induces T helper cell type 2 (Th2) cytokine expression and chromatin remodeling in committed Th1 cells. J Exp Med 2000;192:105–115. |
| 33. | Siegel MD, Zhang DH, Ray P, Ray A. Activation of the interleukin-5 promoter by cAMP in murine EL-4 cells requires the GATA-3 and CLE0 elements. J Biol Chem 1995;270:24548–24555. |
| 34. | Lee HJ, O'Garra A, Arai K, Arai N. Characterization of cis-regulatory elements and nuclear factors conferring Th2-specific expression of the IL-5 gene: a role for a GATA-binding protein. J Immunol 1998;160:2343–2352. |
| 35. | Kishikawa H, Sun J, Choi A, Miaw SC, Ho IC. The cell type-specific expression of the murine IL-13 gene is regulated by GATA-3. J Immunol 2001;167:4414–4420. |
| 36. | Ranganath S, Ouyang W, Bhattacharya D, Sha WC, Grupe A, Peltz G, Murphy KM. GATA-3-dependent enhancer activity in IL-4 gene regulation. J Immunol 1998;161:3822. |
| 37. | Kim JI, Ho C, Grusby MJ, Glimcher LH. The transcription factor c-Maf controls the production of interleukin-4 but not other Th2 cytokines. Immunity 1999;10:745–751. |
| 38. | Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman GC, Glimcher LH. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 2000;100:655–669. |
| 39. | Lighvani AA, Frucht D, Jancobic D, Yamane H. Aliberti J, Hissong BD, Nguyen BV, Gadina M, Sher A, Paul WE, et al. T-bet is rapidly induced by interferon-gamma in lymphoid and myeloid cells. Proc Natl Acad Sci USA 2001;98:15137–15142. |
| 40. | Usui T, Preiss JC, Kanno Y, Yao ZJ, Bream JH, O'Shea JJ, Strober W. T-bet regulates Th1 responses through essential effects on GATA-3 function rather than on IFNG gene acetylation and transcription. J Exp Med 2006;203:755–766. |
| 41. | Denis M, Ghadirian E. Murine hypersensitivity pneumonitis: bidirectional role of interferon-gamma. Clin Exp Allergy 1992;22:783–792. |
| 42. | Nance S, Cross R, Yi A, Fitzpatrick EA. IFN-γ production by innate immune cells is sufficient for development of hypersensitivity pneumonitis. Eur J Immunol 2005;35:1928–1938. |
| 43. | Hwang SJ, Kim S, Park WS, Chung DH. IL-4-secreting NKT cells prevent hypersensitivity pneumonitis by suppressing IFN-gamma-producing neutrophils. J Immunol 2006;177:5258–5268. |
| 44. | Lord GM, Rao RM, Choe H, Sullivan BM, Lichtman AH, Luscinskas FW, Glimcher LH. T-bet is required for optimal proinflammatory CD4+ T-cell trafficking. Blood 2005;106:3432–3439. |
| 45. | Schuyler M, Gott K, Haley P. Experimental murine hypersensitivity pneumonitis. Cell Immunol 1991;136:303–317. |
| 46. | Schuyler M, Gott K, Edwards B, Nikula KJ. Experimental hypersensitivity pneumonitis: effect of CD4 cell depletion. Am J Respir Crit Care Med 1994;149:1286–1294. |
| 47. | Schuyler M, Gott K, Edwards B, Nikula KJ. Experimental hypersensitivity pneumonitis: effect of Thy1.2+ and CD8+ cell depletion. Am J Respir Crit Care Med 1995;151:1834–1842. |
