Abstract
Rationale: Airway remodeling is an important feature of chronic asthma that causes irreversible airflow obstruction. Although asthma is considered to be a Th2 disease, the role of T-bet and GATA-3, the key transcription factors for differentiation toward Th1 and Th2 cells, in the pathogenesis of airway remodeling is poorly understood.
Objectives: We therefore examined the effects of GATA-3 or T-bet induction of Th1/Th2 bias on the development of airway remodeling in mice.
Methods: The development of airway remodeling after repeated allergen challenges was analyzed using transgenic mice overexpressing either GATA-3 or T-bet.
Main Results: The degrees of subepithelial fibrosis and airway smooth muscle hyperplasia after repeated allergen exposure were significantly enhanced in mice overexpressing GATA-3, compared with wild-type mice. Allergen-induced goblet cell hyperplasia and mucus hypersecretion were significantly lower in mice overexpressing T-bet than in wild-type mice. Eosinophilic airway inflammation increased in mice overexpressing GATA-3, but decreased in mice overexpressing T-bet after repeated allergen exposure. Cytokine analysis revealed that the Th1/Th2 cytokine balance shifted to Th2 in lung homogenates and lung T cells of mice overexpressing GATA-3, whereas this balance shifted to Th1 in those of mice overexpressing T-bet after allergen exposure. Lung transforming growth factor-β and eotaxin levels were associated with the degree of subepithelial fibrosis and eosinophilic airway inflammation, respectively.
Conclusions: Overall, the results indicate that development of airway remodeling is regulated by the lung Th1/Th2 bias induced by GATA-3 and T-bet.
Asthma is a chronic disease characterized by airway inflammation, airway hyperresponsiveness, and reversible airway obstruction. However, some patients with asthma also experience a gradual decline in lung function due to persistent airflow obstruction (1). This fixed obstruction may be related to structural changes in the airway, termed “remodeling,” that may occur as a result of imbalance in tissue regeneration and repair mechanisms (2, 3). Subepithelial fibrosis is a distinctive feature of airway remodeling and contributes to the thickened airway walls (4, 5), whereas increased airway smooth muscle mass and excessive mucus secretion from hyperplastic goblet cells are also features of airway remodeling (6, 7). Further understanding of the etiology of airway remodeling in patients with asthma is of importance for development of therapies that arrest or reverse this phenomenon.
It is now widely accepted that T cells, particularly Th2 cells, play a critical role in the pathogenesis of asthma. Although the contribution of Th2 cells to the pathogenesis of airway remodeling remains unclear, recent animal studies support the hypothesis that Th2 cytokines such as interleukin (IL)-4 and IL-13 play a key role in asthmatic airway remodeling (8). IL-4 and IL-13 have been shown to stimulate differentiation of lung fibroblasts to myofibroblasts with an accompanying increase in α-smooth muscle actin expression (9). Targeted overexpression of IL-13 generates a complex phenotype that includes eosinophilic and mononuclear inflammation, subepithelial airway fibrosis, airway obstruction, and airway hyperresponsiveness on methacholine challenge (10). Moreover, it has been demonstrated that IL-13 strongly enhances goblet cell hyperplasia, mucus hypersecretion, and the induction of MUC5AC expression (11). Conversely, IFN-γ, a Th1 cytokine, induces antifibrotic effects directly by suppressing fibroblast activity such as proliferation and collagen production (12, 13), or indirectly by attenuating the effects of IL-4 and IL-13 (14, 15). It is therefore possible to say that modulation of the Th1/Th2 balance may regulate the development of airway remodeling.
Th1 and Th2 cells are differentiated from common T precursor cells (16, 17), with differentiation requiring the activity of specific transcription factors. GATA-3, a member of the GATA family of zinc-finger transcription factors, has been identified as a key regulator of Th2 development (18). Blocking of GATA-3 with a dominant-negative construct or antisense DNA prevents Th2 cytokine activation and eosinophilia in allergen-challenge mouse models (19, 20). On the other hand, T-bet, a member of the T-box family of transcription factors, is a key controller of Th1 differentiation (21, 22). T-bet expression is strongly correlated with IFN-γ expression and is specifically up-regulated in primary Th cells that differentiate along the Th1 pathway (21). T-bet–deficient mice develop spontaneous airway changes consistent with major signs of human asthma (23), and an increased number of GATA-3–expressing cells, and fewer cells positive for T-bet have been observed in bronchial biopsy specimens from patients with asthma (23, 24). However, it remains unclear whether the Th1/Th2 balance controlled by these transcription factors regulates the development of airway remodeling.
In the present study, we generated transgenic mice overexpressing either GATA-3 (GATA-3-tg mice) or T-bet (T-bet-tg mice). The development of airway remodeling after repeated allergen challenges in these mice was analyzed to clarify the role of GATA-3 and T-bet in the pathogenesis of airway remodeling in asthma.
To generate GATA-3-tg mice, a 2.0-kb murine, full-length GATA-3 cDNA was inserted into the VA vector (25). The VA vector contains a CD2 transgene cassette including the upstream gene regulatory region and locus control region of the human CD2 gene. T-bet-tg mice were also generated by the same procedure as for the GATA-3-tg mice, using a 2.5-kb murine T-bet cDNA. These constructs were injected into BDF1-fertilized eggs. Both GATA-3-tg mice and T-bet-tg mice were backcrossed with Balb/c mice for eight generations. Mice (6–8 wk old) were intraperitoneally sensitized by 100 μg ovalbumin (OVA; Sigma Chemical Co., St. Louis, MO) on Days 0 and 14. Starting on Day 21, mice were challenged intranasally with 10 μg of OVA on 5 d each week for 8 consecutive wk. Control mice were injected and challenged with saline. All animal studies were approved by the institutional review board.
Lung paraffin sections were stained with Masson's Trichrome to demonstrate the presence of extracellular matrix, and with periodic acid Schiff to demonstrate the presence of mucin within goblet cells. The sections were also stained immunohistochemically using anti–α-smooth muscle actin antibody (Sigma) to identify contractile elements. The percentages of extracellular matrix, contractile elements in the 20-μm region beneath the epithelium, and mucin in the epithelial portion of the airway wall were morphometrically analyzed using a previously described method (26).
The concentration of MUC5AC protein in the first bronchoalveolar lavage (BAL) sample was measured by enzyme-linked immunosorbent assay, as previously described (27).
After extraction of total RNA from the lung tissues, the expression levels of GATA-3 and T-bet genes were determined by quantitative reverse transcription–polymerase chain reaction (RT-PCR) using ready-made fluorogenic probes and primers (Applied Biosystems, Foster City, CA). Expression levels for each amplicon were quantified using the ΔΔCT method according to the manufacturer's protocols. RT-PCR for pro-α1(I) procollagen mRNA was performed using primers of 5′-ATTGGTAATGTTGGTGCT-3′ and 5′-GTGACCCTTTATGC CTCTGT-3′, and the expression level was quantified using the National Institutes of Health Image software (Bethesda, MD). The expression levels were normalized against GAPDH mRNA.
Serum levels of total IgE, total IgG, IgG1, and IgG2a were determined by enzyme-linked immunosorbent assay, as described previously (28).
The concentrations of IL-4, IL-5, IL-13, IFN-γ, eotaxin, and the active form of transforming growth factor-β (TGF-β) in lung homogenates were determined by enzyme-linked immunosorbent assay (IL-4, IL-5, IFN-γ, and eotaxin; BioSource International, Camarillo, CA: IL-13 and TGF-β; R&D Systems, Minneapolis, MN).
The lungs were digested with 75 U/ml collagenase (type 1; Sigma) at 37°C for 90 min and the isolated cells were filtered through 20-μm nylon mesh. The cells were then stained with anti–T cell receptor β (TCRβ) antibodies (BD PharMingen, San Diego, CA), and analyzed by flow cytometry. Intracellular production of IFN-γ or IL-4 in these cells was also determined, as previously described (29).
Data are expressed as means ± SEM. Data were evaluated by analysis of variance and Tukey's multiple comparison test; p values less than 0.05 were considered to be significant.
First, to confirm the effect of the transgene, we evaluated the pulmonary expression level of GATA-3 and T-bet genes in wild-type (WT) and transgenic mice using quantitative PCR. Expression of GATA-3 mRNA was significantly higher in the lungs of GATA-3-tg mice than in the lungs of WT mice under both saline- and OVA-challenged conditions (Figure 1A). Expression of T-bet mRNA was also significantly higher in the lungs of T-bet-tg mice than in the lungs of WT mice under both saline- and OVA-challenged conditions (Figure 1B). It is of note that expression of both GATA-3 and T-bet mRNAs decreased in the lungs of WT mice with repeated OVA challenges (Figures 1A and 1B).
Figure 1. (A) Expression of GATA-3 mRNA in the lungs of Balb/c wild-type mice (WT) and GATA-3–overexpressing mice of the same background (GATA-3-tg) 1 d after final exposure to saline or ovalbumin (OVA). (B) Expression of T-bet mRNA in the lungs of WT and T-bet–overexpressing mice of the same background (T-bet-tg) 1 d after final exposure to saline or OVA. All signals are expressed relative to the signal obtained from corresponding saline-exposed WT mice. Data are expressed as the mean ± SEM of four mice in each group.
[More] [Minimize]Then, we evaluated the degree of subepithelial fibrosis, one of the characteristic features of airway remodeling, in WT, GATA-3-tg, and T-bet-tg mice after repeated challenges with OVA or saline. In saline-challenged control mice, subepithelial regions occurred infrequently and little deposition of extracellular matrix was observed in the subepithelium in all genotypes of mice (Figures 2A–2C). Although subepithelial deposition of extracellular matrix increased in all mouse genotypes after repeated OVA challenges, the extent of deposition was much higher in GATA-3-tg mice than in other genotypes (Figures 2D–2F).
Figure 2. Photomicrographs of the airways of WT mice (A, D), mice overexpressing GATA-3 (GATA-3-tg; B, E), and mice overexpressing T-bet (T-bet-tg; C, F) 1 d after final exposure to saline (A–C) or OVA (D–F). Masson's Trichrome stain. (G) Percentage of extracellular matrix in the 20-μm region beneath the epithelium in the airways of WT, GATA-3-tg, and T-bet-tg mice 1 d after final exposure to saline or OVA. Data are expressed as the mean ± SEM of eight mice in each group. * Significant difference (p < 0.05) between WT mice and GATA-3-tg mice. (H) Expression of pro-α1(I) procollagen mRNA in lung homogenates of WT, GATA-3-tg, and T-bet-tg mice 1 d after final exposure to saline or OVA. Data are expressed as the mean ± SEM of eight mice in each group. * Significant difference (p < 0.05) between WT mice and GATA-3-tg mice.
[More] [Minimize]Morphometric analysis also revealed that the proportion of extracellular matrix in the 20-μm region beneath the epithelium significantly increased in all mouse genotypes after repeated OVA challenges, compared with corresponding saline-challenged control animals (Figure 2G). The proportion of subepithelial extracellular matrix in GATA-3-tg mice was significantly higher than that in WT mice after repeated OVA challenges, whereas these values were similar in T-bet-tg and WT mice (Figure 2G).
To clarify the effects of GATA-3 and T-bet on collagen synthesis, we also evaluated the expression of procollagen mRNA in the lungs of WT, GATA-3-tg, and T-bet-tg mice after repeated challenges with OVA or saline. Expression of the procollagen gene significantly increased in the lungs of GATA-3-tg mice after repeated OVA challenges, compared with that after saline challenge (Figure 2H). On the other hand, procollagen gene expression was similar in saline- and OVA-challenged WT and T-bet-tg mice. Among the mouse genotypes, procollagen gene expression after repeated OVA challenges was significantly higher in the lungs of GATA-3-tg mice than in WT mice or T-bet-tg mice (Figure 2H). These findings indicate that overexpression of GATA-3 enhances development of subepithelial fibrosis, whereas overexpression of T-bet dose not affect this process.
We next evaluated the roles of overexpression of GATA-3 and T-bet in mucin production and secretion. Few epithelial cells were positive for periodic acid Schiff staining in the airway of all mouse genotypes after saline challenge (Figures 3A–3C). After repeated OVA challenges, goblet cell hyperplasia and mucus hyperproduction were observed in the airways of WT and GATA-3-tg mice, compared with the corresponding saline-challenged control groups (Figures 3D and 3E). However, goblet cell hyperplasia was not observed and only a few periodic acid Schiff–positive cells were present in the airways of T-bet-tg mice after repeated OVA challenges (Figure 3F).
Figure 3. Photomicrographs of the airways of WT mice (A, D), mice overexpressing GATA-3 (GATA-3-tg; B, E), and mice overexpressing T-bet (T-bet-tg; C, F) 1 d after final exposure to saline (A–C) or OVA (D–F). Periodic acid Schiff (PAS) stain. (G) Percentage of PAS-positive areas in the airway epithelium of WT, GATA-3-tg, and T-bet-tg mice 1 and 7 d after final exposure to saline or OVA. Data are expressed as the mean ± SEM of eight mice in each group. * Significant difference (p < 0.05) between WT and T-bet-tg mice. (H) Level of MUC5AC protein in the bronchoalveolar lavage fluid of WT, GATA-3-tg, and T-bet-tg mice 1 and 7 d after final exposure to saline or OVA. The level of MUC5AC protein is represented as the percentage change from the value in saline-treated WT mice. Data are expressed as the mean ± SEM of eight mice in each group. ** Significant difference (p < 0.01) between WT and T-bet-tg mice.
[More] [Minimize]Morphometric analysis showed that the proportion of mucin in the epithelial portion of the airway wall increased in all mouse genotypes after repeated OVA challenges, compared with the corresponding saline-challenged control animals (Figure 3G). However, among the mouse genotypes, the proportion of mucin after repeated OVA challenges was significantly lower in T-bet-tg mice than in WT and GATA-3-tg mice (Figure 3G).
We also evaluated mucus secretion in the airways of WT, GATA-3-tg, and T-bet-tg mice after repeated challenges with OVA or saline. The concentration of MUC5AC, the major component of airway mucin, in the BAL fluids significantly increased in all mouse genotypes after repeated OVA challenges, compared with the corresponding saline-challenged control animals (Figure 3H). However, among the mouse genotypes, the concentration of MUC5AC after repeated OVA challenges was significantly lower in the BAL fluids of T-bet-tg mice than in those of WT and GATA-3-tg mice (Figure 3H). These findings indicate that allergen-induced mucus hyperproduction and hypersecretion were attenuated by T-bet overexpression.
We next evaluated the degree of smooth muscle cell hypertrophy in the airways of WT, GATA-3-tg, and T-bet-tg mice after repeated challenges with OVA or saline. A thin smooth muscle cell layer that was positive for α-smooth muscle actin was observed in the airways of all genotypes of saline-challenged control mice (Figures 4A–4C). The thickness of the airway smooth muscle cell layer increased in all mouse genotypes after repeated OVA challenges, but especially in GATA-3-tg mice (Figures 4D–4F).
Figure 4. Immunohistochemical staining of α-smooth muscle actin (α-SMA) in the airways of WT mice (A, D), mice overexpressing GATA-3 (GATA-3-tg; B, E), and mice overexpressing T-bet (T-bet-tg; C, F) 1 d after final exposure to saline (A–C) or OVA (D–F). (G) Percentage of airway smooth muscle in the 20-μm region beneath the epithelium in the airways of WT, GATA-3-tg, and T-bet-tg mice 1 d after final exposure to saline or OVA. Data are expressed as the mean ± SEM of eight mice in each group. ** Significant difference (p < 0.01) between WT mice and GATA-3-tg mice.
[More] [Minimize]Morphometric analysis also revealed that the proportion of α-smooth muscle actin–positive cells in the 20-μm region beneath the epithelium significantly increased in all mouse genotypes after repeated OVA challenges, compared with the corresponding saline-challenged control animals (Figure 4G). Among the mouse genotypes, the proportion of α-smooth muscle actin–positive cells after repeated OVA challenges was significantly higher in GATA-3-tg mice than in WT mice, whereas the proportion of such cells in T-bet-tg mice was similar to that in WT mice (Figure 4G). These results indicate that airway smooth muscle cell hypertrophy is enhanced by overexpression of GATA-3.
To clarify the roles of overexpression of GATA-3 and T-bet in the development of eosinophilic airway inflammation, the number of eosinophils in BAL fluids was determined in WT, GATA-3-tg, and T-bet-tg mice after repeated challenges with OVA or saline. Eosinophils in BAL fluids were not detectable after repeated saline challenges, but were detectable after repeated OVA challenges in all mouse genotypes (Figure 5). Among the mouse genotypes, the number of eosinophils 1 d after the final OVA challenge significantly increased in BAL fluids of GATA-3-tg mice, compared with WT mice (Figure 5). In contrast, the number of BAL eosinophils was lower in T-bet-tg mice than in WT mice at the same time point (Figure 5). In T-bet-tg mice, the number of lavagable eosinophils returned to the levels in saline-challenged control animals 7 d after the final OVA challenge, whereas the number of eosinophils was still higher than the respective control levels in the BAL fluids of WT and GATA-3-tg mice (Figure 5).
Figure 5. The number of eosinophils in the bronchoalveolar lavage fluids of WT mice, mice overexpressing GATA-3 (GATA-3-tg), and mice overexpressing T-bet (T-bet-tg) 1 and 7 d after final exposure to saline or OVA. Data are expressed as the mean ± SEM of eight mice in each group. * Significant difference (p < 0.05) between WT and T-bet-tg mice. ** Significant difference (p < 0.01) between WT and GATA-3-tg mice.
[More] [Minimize]To evaluate the Th1/Th2 shift in each mouse genotype, serum levels of IgE and IgG, including the subclasses of IgG1 and IgG2a, were determined in each genotype after repeated challenges with OVA or saline. The serum IgE level was slightly lower in T-bet-tg mice than in WT and GATA-3-tg mice after saline challenge. However, the levels of total IgG and IgG subclasses did not differ among the mouse genotypes after saline challenge (Table 1).
Saline | OVA | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Measurement | WT | GATA-3-Tg | T-bet-Tg | WT | GATA-3-Tg | T-bet-Tg | ||||
| IgE, ng/dl | 1,433.9 ± 269.0 | 1,447.7 ± 427.6 | 426.7 ± 121.9 | 3,005.9 ± 187.5 | 3,723.2 ± 140.3 | 1,188.3 ± 241.1* | ||||
| IgG, mg/dl | 288.6 ± 12.0 | 273.5 ± 11.4 | 295.3 ± 11.2 | 706.0 ± 42.4 | 777.1 ± 59.5 | 465.7 ± 9.6* | ||||
| IgG1, mg/dl | 52.0 ± 6.0 | 57.8 ± 7.1 | 101.5 ± 26.4 | 683.2 ± 86.0 | 810.5 ± 185.9 | 177.6 ± 10.1* | ||||
| IgG2a, mg/dl | 140.7 ± 19.2 | 60.1 ± 14.4 | 120.3 ± 20.4 | 152.8 ± 29.1 | 153.6 ± 29.2 | 177.3 ± 42.0 | ||||
| IgG1/IgG2a ratio | 0.37 ± 0.02 | 1.08 ± 0.14 | 0.83 ± 0.11 | 5.66 ± 1.55 | 7.17 ± 2.38 | 1.34 ± 0.38 | ||||
Serum levels of IgE, total IgG, and IgG1 increased in all genotypes after repeated challenges with OVA, compared with the corresponding saline-challenged control animals (Table 1). Among the mouse genotypes, the serum levels of IgE and IgG1 after OVA challenges were significantly lower in T-bet-tg mice but slightly higher in GATA-3-tg mice, compared with WT mice (Table 1). Thus, the IgG1/IgG2a and IgE/IgG2a ratios increased in GATA-3-tg mice and decreased in T-bet-tg mice after repeated allergen challenges. These results indicate that Th2 bias occurs in GATA-3-tg mice and Th1 bias occurs in T-bet-tg mice under these conditions.
We next analyzed the levels of Th1 and Th2 cytokines in the lungs of WT, GATA-3-tg, and T-bet-tg mice after repeated challenges with OVA or saline. The concentration of IL-4 increased in the lungs of all genotypes of mice after repeated OVA challenges, compared with saline-challenged control animals (Figure 6A). However, the IL-4 concentration was significantly higher in the lungs of GATA-3-tg mice than in those of WT mice, whereas the pulmonary IL-4 level in T-bet-tg mice was similar to that in WT mice (Figure 6A).
Figure 6. The concentrations of interleukin (IL)-4 (A), IL-5 (B), IL-13 (C), and IFN-γ (D) in the lungs of WT mice, mice overexpressing GATA-3 (GATA-3-tg), and mice overexpressing T-bet (T-bet-tg) 1 d after final exposure to saline or OVA. Data are expressed as the mean ± SEM of eight mice in each group. * Significant difference (p < 0.05) in comparison with corresponding WT mice. ** Significant difference (p < 0.01) in comparison with corresponding WT mice.
[More] [Minimize]The concentration of IL-5 also increased in the lungs of all genotypes of mice after repeated OVA challenges, compared with saline-challenged control animals (Figure 6B). After the OVA challenges, the IL-5 concentration was significantly lower in the lungs of T-bet-tg mice than in those of WT mice (Figure 6B), whereas the pulmonary IL-5 levels were similar in GATA-3-tg and WT mice (Figure 6B).
IL-13 was not detectable in the lungs in any genotypes of saline-challenged mice (Figure 6C). The pulmonary level of IL-13 markedly increased in WT and GATA-3-tg mice after repeated OVA challenge, but IL-13 remained low in the lungs of T-bet-tg mice (Figure 6C). Among the mouse genotypes, the pulmonary IL-13 level was significantly lower in T-bet-tg mice after OVA challenges, compared with other genotypes (Figure 6C).
The concentration of IFN-γ was elevated in the lungs of all genotypes of mice after repeated OVA challenges, compared with saline-challenged control animals (Figure 6D). Among the mouse genotypes, the IFN-γ level significantly increased in the lungs of T-bet-tg mice after OVA challenges, compared with WT and GATA-3-tg mice (Figure 6D). These findings clearly indicate that the pulmonary Th1/Th2 cytokine balance is shifted to Th1 in T-bet-tg mice and to Th2 in GATA-3-tg mice after repeated OVA challenges.
To clarify the contribution of T cells to Th1/Th2 cytokine production in the mice models, we analyzed the intracellular production of IFN-γ and IL-4 in lymphocytes obtained from lung tissues. In WT mice, 5.56 ± 1.18% of T cells in the total population of T cells recovered from the lung were positive for IFN-γ after saline challenge (Figure 7A; WT-saline). The proportion of IFN-γ–producing T cells was higher in the lungs of T-bet-tg mice than in the lungs of WT mice, even under saline-challenged conditions (Figure 7A; T-bet-Tg–saline). The proportion of IFN-γ–producing T cells increased significantly in the lungs of WT and T-bet-tg mice but not in those of GATA-3-tg mice on repeated OVA challenge, compared with the values of corresponding saline-challenged control animals (Figure 7A, lower panels). Among the mouse genotypes, the proportion of IFN-γ–positive T cells was significantly higher in the lungs of T-bet-tg mice and significantly lower in the lungs of GATA-3-tg mice after repeated OVA challenges, compared with WT mice (Figure 7A, lower panels).
Figure 7. Intracellular production of IFN-γ (A) and IL-4 (B) in T cells obtained from lungs of WT mice, mice overexpressing GATA-3 (GATA-3-tg), and mice overexpressing T-bet (T-bet-tg) after exposure to saline or OVA. TCRβ = T cell receptor β. Data are expressed as the mean ± SEM of three mice in each group.
[More] [Minimize]The proportion of IL-4–producing cells in the total population of T cells recovered from the lung was less than 5%, and did not differ among the genotypes after saline challenge (Figure 7B, upper panels). However, after repeated OVA challenges, the proportion of IL-4–producing T cells increased significantly in the lungs of GATA-3-tg mice, whereas the proportion of such cells did not differ in the lungs of WT and T-bet-tg, compared with the corresponding saline-challenged control animals (Figure 7B). Among the mouse genotypes, the proportion of IL-4–positive T cells was significantly higher in the lungs of GATA-3-tg mice than in WT and T-bet-tg mice after repeated OVA challenges (Figure 7B, lower panels).
TGF-β is known to play an important role in the development of fibrosis. We therefore assessed the concentration of the active form of TGF-β in the lungs of WT, GATA-3-tg, and T-bet-tg mice after repeated challenges with OVA or saline. Among the mouse genotypes, the concentration of TGF-β significantly increased in the lungs of GATA-3-tg mice and significantly decreased in the lungs of T-bet-tg mice after repeated OVA challenges, compared with WT mice (Figure 8A).
Figure 8. The concentrations of the active form of transforming growth factor-β1 (TGF-β1; A) and eotaxin (B) in the lungs of WT mice, mice overexpressing GATA-3 (GATA-3-tg), and mice overexpressing T-bet- (T-bet-tg) 1 d after final exposure to saline or OVA. Data are expressed as the mean ± SEM of eight mice in each group. * Significant difference (p < 0.05) between WT and GATA-3-tg mice. † Significant difference (p < 0.05) between WT and T-bet-tg mice.
[More] [Minimize]Eotaxin is known as a potent and selective chemoattractant of eosinophils. Hence, we assessed the concentration of eotaxin in the lungs of WT, GATA-3-tg, and T-bet-tg mice after repeated challenges with OVA or saline. Among the mouse genotypes, the concentration of eotaxin was significantly reduced in the lungs of T-bet-tg mice after repeated OVA challenges, compared with WT and GATA-3-tg mice (Figure 8B).
T-bet and GATA-3 are believed to be master regulators of Th1/Th2 differentiation that specifically function in helper T cells (18–22). To generate T-bet-tg and GATA-3-tg mice, full-length murine T-bet and GATA-3 cDNAs were inserted into a VA CD2 transgene cassette (25). The VA vector has been reported to express the inserted cDNA directly in all single-positive mature T cells of transgenic mice (30). These transgenic mice models are therefore concluded to be adequate for evaluation of various disorders that are due to Th1/Th2 imbalance. Using these models, we first demonstrated that overexpression of T-bet and GATA-3 regulates the development of allergen-induced airway remodeling. Serum immunoglobulin levels and cytokine analysis showed that the Th1/Th2 balance shifted toward Th1 in T-bet-tg mice and toward Th2 in GATA-3-tg mice after chronic allergen exposure. Consistent with this, the Th1/Th2 cytokine balance shifted to Th2 in both lung homogenates and lung T cells of GATA-3-tg mice, whereas this balance shifted to Th1 in those of T-bet-tg mice after chronic allergen exposure. Interestingly, however, the phenotypes of airway remodeling were not completely opposite in T-bet-tg mice and GATA-3-tg mice.
Subepithelial fibrosis is a characteristic feature of the asthmatic bronchus. It appears to consist of a plexiform deposition of collagen I and III, tenascin, and fibronectin proteins that are mainly produced by activated myofibroblasts (31). In the present study, both the degree of subepithelial fibrosis and expression of the procollagen gene were significantly enhanced in GATA-3-tg mice after repeated allergen challenges. However, overexpression of T-bet did not affect the degree of subepithelial fibrosis after allergen challenge. Lung cytokine analysis revealed that the levels of IL-4 and TGF-β paralleled the degree of subepithelial fibrosis in these animals. IL-4 and TGF-β are known to enhance the fibrotic process by augmenting fibroblast growth and collagen production, as well as promoting differentiation of fibroblasts into myofibroblasts that secrete collagen and other extracellular matrix components (32). Moreover, IL-4 also enhances the release of TGF-β (14). It is therefore likely that IL-4 and TGF-β, increased as a result of GATA-3 overexpression, contribute to enhanced development of subepithelial fibrosis.
Our data show that goblet cell hyperplasia and mucus hyperproduction induced by chronic allergen challenge were significantly attenuated in T-bet-tg mice. However, overexpression of GATA-3 did not affect allergen-induced mucus hyperproduction. Goblet cell hyperplasia and mucus hyperproduction are important features of chronic asthma and contribute substantially to morbidity and mortality. Goblet cells are the major source of mucin glycoproteins, which are the major constituents of airway mucus and the major determinants of its viscoelastic and adhesive properties (33). It has been demonstrated in a mouse model that overexpression of Th2 cytokines, such as IL-5 and IL-13, induces goblet cell hyperplasia independently of IL-4 (34–36). In particular, IL-13 may be a central mediator for mucus hyperproduction by inducing MUC5AC expression via activation of the epidermal growth factor (EGF) receptor (37). On the other hand, it has also been reported that the Th1 cytokine IFN-γ inhibits airway mucus production induced by Th2 and non-Th2 inflammatory responses (38). Consistent with this, our present study showed a marked reduction in the IL-13 level and a significant increase in the IFN-γ level in the lungs of T-bet-tg mice after repeated allergen challenges. Thus, a Th1 shift induced by overexpression of T-bet can inhibit allergen-induced goblet hyperplasia, mucus hyperproduction, and mucus hypersecretion. It is unclear why goblet cell hyperplasia and mucus production were not enhanced in mice overexpressing GATA-3, but it is possible that an increase in IL-4 alone may not be sufficient for mucus hyperproduction.
We also showed that airway smooth muscle hyperplasia is enhanced in GATA-3-tg mice after repeated allergen challenges, with accompanying elevation of the levels of IL-4 and TGF-β. An increase in the airway smooth muscle mass is also an important feature of airway remodeling, and in vivo animal studies have demonstrated that prolonged allergen exposure can increase the amount of smooth muscle (39). Although the mechanisms that regulate the proliferation of smooth muscle cells are still uncertain, several factors, including growth factors and leukotrienes, may affect the airway smooth muscle mass (40). Among growth factors, EGF-mediated signal transduction may play a role in proliferation of these cells. The effect of TGF-β on airway smooth muscle cell proliferation is controversial: TGF-β can enhance proliferation of airway smooth muscle cells by up-regulating the cysteinyl leukotriene-1 receptor (41), but TGF-β also inhibits EGF-mediated smooth muscle cell proliferation (42). Previous data suggest that Th2 cytokines can act directly on airway smooth muscle cells, leading to changes in contractile and relaxant responses, proliferation, and the ability of smooth muscle cells to generate chemokines such as eotaxin and TARC (43), but further studies are required to elucidate the relationship between overexpression of GATA-3 and allergen-induced smooth muscle hyperplasia.
Eosinophilic airway inflammation is the most characteristic feature of asthma. As well as the contribution of eosinophils to acute responses, recent reports support a potential role for eosinophils in development of airway remodeling (44–46). Eosinophils are also known as major secretors of TGF-β and cysteinyl leukotrienes (47), which further supports a role for these cells in airway remodeling. It is widely accepted that differentiation, maturation, and activation of eosinophils are regulated by Th2 cytokines, and especially by IL-5. The recruitment of eosinophils at the inflamed site is regulated by CC chemokines, which are small inducible proteins that possess four cysteines, of which the first two are adjacent. Eotaxin, a CC chemokine, is produced in the lungs of patients with asthma and is a potent and selective chemoattractant for eosinophils. In the present study, reduction of allergen-induced eosinophilic inflammation was associated with decreased levels of IL-5 and eotaxin in the lungs of T-bet-tg mice. On the other hand, exacerbation of eosinophilic airway inflammation in GATA-3-tg mice occurred independently of IL-5 and eotaxin expression. Although the precise mechanisms are unclear, this observation is consistent with previous findings that eosinophilic airway inflammation is reduced by antisense-induced local blockade of GATA-3 expression (20).
The mechanisms of Th1/Th2 differentiation induced by T-bet and GATA-3 have been identified at the molecular level. T-bet specifies Th1 lineage commitment by inducing lineage-restricted target genes, such as IFN-γ and IL-12 receptor β2 genes, and GATA-3 specifies Th2 lineage commitment by inducing genes for IL-4, IL-13, and IL-5 (18, 21, 22). T-bet and GATA-3 regulate activation of their respective target genes both transcriptionally and epigenetically. Interestingly, the signature gene activity of helper T cells is initially flexible but later becomes fixed during helper T cell differentiation (48). In this process, T-bet and GATA-3 may be essential for the initial establishment but not necessarily the maintenance of cytokine gene activity. The interaction between T-bet and GATA-3 has also been investigated. Hwang and colleagues have reported that T-bet represses Th2 lineage commitment through tyrosine kinase-mediated interaction with GATA-3, which interferes with the binding of GATA-3 to its target DNA (49), and observed that induction and phosphorylation of T-bet occur in early Th1 differentiation. Thus, T-bet and GATA-3 are essential for gene activation and gene silencing during Th1/Th2 differentiation, but may not be critical factors in mature Th1 and Th2 cells. These findings are not in contradiction with the observation in the current study that expression of both T-bet and GATA-3 mRNAs decreased in the lungs of WT mice after repeated antigen challenges. Moreover, the production of Th1/Th2 cytokines in mature Th1/Th2 cells may not be directly regulated by T-bet or GATA-3, because there was a discrepancy between the expression of T-bet and GATA-3 genes and Th1/Th2 cytokine levels in the lungs of WT mice after repeated antigen challenges.
A critical role for T cells, and especially Th2 cells, in humans with asthma is now widely accepted. However, studies with antibodies or receptor fusion proteins to block individual T-cell–derived cytokines have been disappointing (50). Because T-bet and GATA-3 regulate the plurality of T-cell cytokines by mediating Th1/Th2 differentiation, activation of T-bet or inactivation of GATA-3 has an advantage over the targeting of single T-cell cytokines in therapeutic strategies against airway remodeling. Moreover, transactivation of the target proteins occurs specifically in T cells, and systemic side effects can therefore be reduced. Although the precise effects of T-bet and GATA-3 in the pathogenesis of airway remodeling in humans with asthma are still not fully understood, the finding of overexpression of GATA-3 and underexpression of T-bet in the airways of patients with chronic asthma suggests that these proteins have a significant role in this process (23, 24). Transcription factor regulation therapy cannot currently be used for treatment of chronic asthma. However, we believe that the results in this study may provide the basis for development of new therapies for treatment of airway remodeling in asthma.
The authors thank Dr. Laurie H. Glimcher for providing mouse T-bet cDNA.
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