Th2 cells and GATA-3 in asthma: new insights into the regulation of airway inflammation (original) (raw)
STAT6 and initiation of Th2 differentiation. The signal transducer and activator of transcription (STAT) proteins play an important role in the selective response of cells to particular cytokines (32). After stimulation with cytokines, STAT proteins undergo phosphorylation by the Janus family of kinases (Jak). This causes dimerization and nuclear translocation of the proteins, wherein they induce transcription of target genes. IL-4 engagement with its receptor leads to phosphorylation of STAT6 by Jak1 and Jak3, and results in the activation of IL-4–regulated genes such as IL-4R, IgE, FcR, and MHC class II molecules (32). An alternate pathway for STAT6, particularly in humans, is through IL-13 (33). Whereas IL-4–/– mice have residual Th2 responses, STAT6–/– mice have almost complete abolition of Th2 responses. In murine models of allergic inflammation, STAT6–/– mice showed marked attenuation of pulmonary eosinophilia, mucus production, AHR and serum IgE levels.
GATA-3: a key regulator of T-cell development, Th2 differentiation, and the Th1/Th2 balance. GATA-3 was first described as a transcription factor that interacted with the TCR-α gene enhancer (34). GATA-3 belongs to the GATA family of transcription factors, which bind to the WGATAR (W = A/T; R = A/G) DNA sequence through a highly conserved C4 zinc-finger domain. Six members (GATA-1 to GATA-6) of this family have been identified in avians, with homologues in mammals and avians. Based on their expression profile and structure, the GATA proteins may be classified as hematopoietic (GATA-1 to GATA-3) (35) or nonhematopoietic (GATA-4 to GATA-6).
Targeted disruption of the GATA-3 gene in mice results in embryonic death on day 12, with a failure of fetal liver hematopoiesis and defects in the central nervous system (36). The lethal effects of GATA-3 deficiency were bypassed by generating GATA-3–/– chimeras and by using antisense oligonucleotides for GATA-3 in fetal thymus organ cultures (37, 38). These elegant studies established an essential role for GATA-3 in the earliest steps of T-cell development. The targets of GATA-3 in the early T-cell progenitors are presently unknown, yet these initial studies clearly demonstrated that GATA-3 is not a functionally redundant GATA family protein.
In addition to its essential role in T-cell development, GATA-3 has also been identified as a Th2 differentiation factor. Naive CD4+ T cells express low levels of GATA-3 mRNA (39, 40). The expression of GATA-3 is, however, markedly upregulated in cells differentiating along the Th2 lineage, and is downregulated in cells differentiating along the Th1 pathway (39, 40). A role for GATA-3 in the expression of a Th2 cytokine gene was first established by Siegel et al., who demonstrated the critical importance of a GATA-3 binding site in the IL-5 promoter (41). Subsequently, by using established clones as well as Th1 or Th2 cells generated from naive CD4+ T cells (39), and by using representational differential analysis (42), Th2-specific expression of the GATA-3 gene was demonstrated. In further studies, GATA-3 was shown to be sufficient for activation of the IL-5, but not IL-4, promoter in a non-Th2 environment (43, 44). Other than the IL-5 gene, a functional GATA-3 binding site has yet to be identified in Th2-expressed cytokine genes. However, several regions around the IL-4/IL-13 locus have been shown to bind GATA-3 (44), the functional implications of which remain to be determined.
To investigate the functional significance of downregulation of GATA-3 gene expression in Th1 cells, GATA-3 was forcibly overexpressed in Th1 cells. In developing Th1 cells, overexpression of GATA-3 resulted in abolition of IL-12Rβ2 subunit expression with concomitant abrogation of IFN-γ production (40). In previously polarized Th1 effector cells, GATA-3 overexpression did not inhibit IFN-γ gene expression (40). This suggests that GATA-3 does not directly act on the IFN-γ promoter, but has indirect effects on activated CD4+ T cells, shifting their development away from Th1. Thus, GATA-3 controls Th2 activity by inducing Th2 cytokine gene expression and by biasing Th1/Th2 balance in vivo.
GATA-3 in asthma. In keeping with the crucial role of Th2 cells in asthma, a significant increase in GATA-3 expression was demonstrated in asthmatic airways compared with that in control subjects (45). The increase in GATA-3 expression in the asthmatic subjects correlated significantly with IL-5 expression and AHR (45).
While the studies in humans clearly showed increased GATA-3 expression in asthma, the initial animal studies of GATA-3 in Th2 gene expression did not conclusively indicate that GATA-3 was sufficient for the expression of all Th2 cytokine genes. Thus, it was unclear whether GATA-3 inhibition would block the production of all the key Th2 cytokines that have been implicated in asthma, i.e., IL-4, IL-5, and IL-13. Because deficiency of the GATA-3 gene causes embryonic lethality, transgenic mice expressing a dominant-negative mutant of GATA-3 in an inducible and T cell–specific fashion were developed to address this question. These studies demonstrated that inhibition of GATA-3 activity causes a severe blunting of Th2 effects, both locally in the lung (eosinophil influx and mucus production) and systemically (IgE production) (46). Although the precise mechanisms by which GATA-3 controls expression of all the Th2 cytokine genes is presently unknown, it is possible that in addition to directly transactivating the IL-5 promoter, it also acts as a chromatin remodeling factor, allowing the transcription of IL-4 and IL-13. Taken together, studies of GATA-3 in mice and humans provide a basis for targeting this important regulator of Th2 gene expression in therapies of asthma and allergic diseases.
The role of c-Maf (Th2-specific), NF-AT, AP-1, C/EBPβ, and NF-κB in Th2 gene expression. Because IL-4 is a potent stimulus and a critical factor for Th2 cell generation, IL-4 gene regulation has been intensely studied in many laboratories as a model system for Th2 gene expression. The inability of GATA-3 to activate the IL-4 promoter and support IL-4 production in a non-Th2 environment suggests that there is a requirement for additional transcription factors that probably act in concert with GATA-3 to activate IL-4 gene expression. In a study by Ho et al., the transcription factor c-Maf was identified as a Th2-specific factor and was shown to transactivate the IL-4 promoter (47). Studies of c-Maf–/– mice recently reported by Kim et al. also demonstrate a crucial role for c-Maf in IL-4 gene expression (48). Whereas c-Maf–/– mice displayed impaired IL-4 production by CD4+ T cells, production of IL-5 and IL-13 was normal in these animals (48). Also, upon immunization, c-Maf–/– mice expressed normal IgE levels, which was probably due to unimpaired IL-13 production. Therefore, unlike GATA-3, c-Maf directly activates the IL-4 promoter, but does not appear to regulate the expression of all Th2 cytokine genes (48).
NF-AT has also been shown to directly activate the IL-4 promoter. NF-AT proteins contain a cytoplasmic subunit and an inducible nuclear component. Four related genes encoding the cytoplasmic subunit are currently known: NF-ATp (NF-AT1), NF-ATc (NF-AT2), NF-AT3 and NF-AT4. NF-AT proteins are present in both Th1 and Th2 cells, and have been implicated in the expression of both Th1 and Th2 genes in in vitro studies (49). However, studies of mice genetically deficient in specific NF-AT members have revealed a Th2-selective role of these proteins in CD4+ T-cell responses. While NF-ATp–deficient mice are viable (50), NF-ATc deficiency causes embryonic lethality (50). In studies of immune responses in NF-ATc–/– chimeric mice, severe T-cell defects in IL-4 production and Th2 differentiation were observed (50).
Other transcription factors that are more generally expressed, such as NF-κB, C/EBPβ, and AP-1, are also important for Th2 gene expression. The transcription factor NF-κB belongs to the Rel family of proteins, and the classic NF-κB complex is a heterodimer composed of 2 polypeptide subunits: p50 and RelA (p65). Studies of mice with targeted disruptions of specific Rel proteins show that these proteins are not functionally redundant. For example, while p65 deficiency caused embryonic lethality in mice, p50–/– mice developed normally. However, when p50–/– mice were tested in a murine model of allergic airway inflammation, no airway inflammation was induced in these mice. Strikingly, the p50–/– mice failed to express IL-5, which is important for the proliferation and differentiation of eosinophils, or eotaxin, which is important for the recruitment of eosinophils into the asthmatic airway (51). The transcription factors C/EBPβ and AP-1, which are widely expressed in many cell types, have been also shown to be important for the expression of Th2 cytokine genes (41, 50). Collectively, these studies emphasize that while the Th2-specific factors, such as GATA-3 and c-Maf, are essential and determine the tissue specificity of gene expression, they are not sufficient for optimal expression of the cytokine genes. The concomitant activation of more general factors, such as NF-κB, AP-1, NF-AT and C/EBPβ, is required for high-level expression of the Th2 cytokine genes.
Negative regulators of Th2 gene expression: BCL-6 and NF-ATp. Studies of NF-ATp–/– and NF-ATc–/– mice have revealed an interesting reciprocal relationship between the 2 proteins in the regulation of Th2 responses. Whereas NF-ATc–/– chimeric mice displayed impaired IL-4 production, NF-ATp–/– mice showed exaggerated and prolonged IL-4 production and constitutive nuclear localization of NF-ATc (50). The mice displayed increased Th2 and decreased Th1 responses both in vivo and in vitro. Thus, unlike NF-ATc, NF-ATp can behave as a negative regulator of Th2 responses in vivo.
Another molecule that appears to be a repressor of Th2 responses in vivo is BCL-6, which was originally identified as a proto-oncogene that is frequently translocated in diffuse large-cell lymphoma. BCL-6 is a potent transcriptional repressor, and has been shown to bind the STAT6 DNA-binding sequence in the CD23b promoter and to repress IL-4–induced activation of CD23 expression (52). Interestingly, targeted deletion of the BCL-6 gene led to a massive inflammatory response in many organs (including the lung), a response characterized by eosinophilic infiltration (52, 53). IgE responses were also elevated in immunized BCL-6–deficient mice (52, 53). Although the precise mechanism by which BCL-6 represses Th2 responses is unclear, dysregulation of STAT-responsive gene expression most likely underlies the aberrant inflammatory response in BCL-6–deficient mice. Interestingly, mice lacking STAT6 and BCL-6, or mice lacking both BCL-6 and IL-4, were shown to mount a Th2 response upon antigen provocation in vivo (54). Therefore, Th2 cell generation can be independent of IL-4 and STAT6. The factors that control this alternate pathway have not yet been identified.
The hierarchy of transcription factors and differential control in Th2 gene expression. The pattern of expression of the various transcription factors during Th2 gene expression suggests a hierarchy of control that determines coordinate or differential expression of the Th2 cytokine genes. The precise relationship between STAT6, GATA-3, NF-AT, and c-Maf is not clear. However, the initiation of Th2 differentiation in antigen- and cytokine-stimulated naive CD4+ T cells appears to be orchestrated by STAT6, which preexists in the cell cytoplasm. NF-ATp and NF-ATc also exist in the cytoplasm of naive CD4+ T cells, and undergo rapid nuclear translocation upon stimulation of the cells. However, their precise positions in the activation cascade are undefined. The expression of both GATA-3 and c-Maf is clearly induced in developing Th2 cells, albeit with different kinetics. While GATA-3 expression peaks at 48 hours after stimulation (39, 40), the expression of c-Maf peaks between 5 and 7 days after antigenic stimulation (47). Are both factors required for the expression of all the Th2 cytokines? As discussed above, neither GATA-3 nor c-Maf is sufficient, but both are critical for IL-4 gene expression in vivo. However, GATA-3 plays an essential role in the control of production of all key Th2 cytokines implicated in asthma pathogenesis (46). Analysis of genetically manipulated mice has also revealed how the transcription of individual Th2 cytokine genes is differentially controlled. For example, NF-κB appears to be more important for antigen-induced IL-5 gene expression than for IL-4 gene expression (51). In contrast, c-Maf is a critical regulator of IL-4, but not IL-5 or IL-13, gene expression (48). GATA-3, on the other hand, regulates the expression of all of these genes (46). These different levels of molecular control might explain differential expression of Th2 cytokines in particular situations. There is increasing interest in the dissociation of IL-4 and IL-5 gene expression in T cells in various disease situations and in T-cell lineages. In intrinsic asthmatics, increased IL-5, but not IL-4, production has been reported (5). In contrast, in leukemic Sézary cells, IL-4 production is upregulated but IL-5 production is downregulated (55). Although there is no evidence to support different Th cell lineages for IL-4 and IL-5 production, it is reasonable to speculate that once cells have been committed to the Th2 lineage, different microenvironmental factors might activate different sets of transcription factors, such as GATA-3, c-Maf, NF-AT, and NF-κB, to cause differential expression of cytokine genes.
Cell-intrinsic mechanisms and probabilistic gene expression in CD4+ T cells. The studies noted above describe the processes that regulate Th2 gene transcription, which may influence the amount of cytokine produced on a per cell basis. Recent studies have shown that the intensity of a Th2 response can also be regulated by the number of cells making Th2 cytokines (IL-4), without influencing the amount produced by individual CD4+ T cells. This alternate genetically controlled pathway of probabilistic gene activation has been proposed to explain the greater Th2 bias in BALB/c mice compared with C57BL/6 mice. As a result, a greater number of CD4+ T cells are committed to IL-4 production in the BALB/c animals. Interestingly, this regulatory event occurs upstream of cytokine-mediated effects that induce expression of Th2-specific factors such as GATA-3 and c-Maf (56). Another unexpected finding in cytokine gene expression that is consistent with probabilistic gene expression is monoallelic expression of cytokine genes (for IL-2, IL-4, and probably others), which may be due to differential chromatin remodeling at independent alleles. The potential importance of this regulation for human diseases such as asthma and atopy is obvious but remains to be explored in the future.
Thus, the key intracellular events that orchestrate Th2-specific gene expression upon antigen and cytokine stimulation of naive CD4+ T cells include chromatin remodeling accompanied by induction of Th2-specific factors such as GATA-3 and c-Maf (Figure 2). These initial events help maintain a stable Th2 phenotype in the committed cells. Th2-specific factors synergize with other, more generally expressed factors (NF-AT, NF-κB, AP-1) that are transiently induced during this process, resulting in cytokine (IL-4, IL-5, IL-13) gene transcription. Superimposed on this process is a genetically controlled mechanism that appears to determine the fraction of CD4+ T cells that are committed to the Th2 lineage and the specific allele that is expressed in these cells.
Treatment of asthma. The impressive data implicating Th2 cell activation in the pathogenesis of asthma support a range of new therapeutic strategies that would reduce Th2-induced inflammation and its consequences in the airways. These interventions can be aimed at inhibiting Th2 cell effects at 3 different points in the inflammatory cascade in asthma (Figure 3): (a) Th2 cell differentiation can be blocked, thus inhibiting the generation of Th2 cells; (b) Th2 activation can be blocked and cytokine secretion inhibited; and (c) Th2 cytokine effects can be blocked at the target tissue.
Potential strategies for inhibiting Th2 function in asthma. (a) Block Th2 cell differentiation. The cytokine environment that is present during CD4+ T-cell differentiation affects the development of new Th2 cells. Blocking IL-4 and/or IL-13 or the Th2-specific transcription factor GATA-3 leads to inhibition of Th2 cell induction. Alternatively, increasing levels of IL-12 and IFN-γ in the environment may shift the Th1/Th2 balance toward Th1 and reduce the number of Th2 cells in the respiratory tract. This can be accomplished by stimulating a Th1 response to certain infectious agents, such as Mycobacterium, or by the administration of CpG oligodeoxynucleotides. All of these interventions may have an effect in asthma by reducing the number of Th2 cells in the airways and by inhibiting the generation of new Th2 cells. (b) Block Th2 cell activation. Glucocorticoids are standard therapy in asthma and cause nonselective immunosuppression. Th2 cell activation may be selectively blocked using inhibitors of GATA-3, a T cell–specific factor that controls the production of key Th2 cytokines. (c) Block Th2 cytokine effects. Blocking IL-4, IL-5, or IL-13 may inhibit the effects of these cytokines on target tissues. In addition, Th1 cells have been shown to inhibit Th2 cytokine effects on eosinophils and airway epithelial mucus production. Allergy immunotherapy and CpG oligodeoxynucleotides both induce Th1 cells and lead to a reduction in allergic airway pathology.
Blockade of Th2 cell differentiation. Altering the cytokine environment that is present during CD4+ T-cell differentiation can inhibit the development of new Th2 cells. Blocking IL-4, IL-13, and the molecular signaling mechanisms activated by engagement of IL-4Rα (STAT6) has been shown to inhibit the generation of Th2 cells in animals (10). As discussed in this review, GATA-3 blockade by overexpression of a dominant-negative mutant inhibited Th2 cell induction (46). In many cases, blockade of Th2 cell differentiation results in the generation of a Th1 cell population. An alternative method to reduce Th2 cell generation in the respiratory tract is to increase levels of IL-12 and IFN-γ in the environment, thus shifting the Th1/Th2 balance toward Th1. Recent epidemiologic evidence suggests that prior infection with M. tuberculosis or measles is associated with a reduction in atopy and asthma (57). In mice infected with Bacille Calmette-Guérin (BCG) prior to antigen sensitization, there was a reduction in eosinophilic airway inflammation and AHR (58). These studies imply that altering the cytokine milieu in the respiratory tract by stimulating a Th1 response to certain infectious agents can reduce symptoms in asthma. The administration of CpG oligonucleotides to mice at the time of antigen sensitization also resulted in skewing of the inflammatory response toward Th1, possibly by stimulating sustained IL-12 production (59). This led to a reduction in eosinophilia and AHR. All of these interventions may have an effect in asthma by reducing the number of Th2 cells in the airways. Even when Th2 cells are already established in the airway, altering the cytokine environment by Th2 cytokine blockade or by stimulating Th1 cell induction may inhibit the generation of new Th2 cells.
Blockade of Th2 cell activation. Glucocorticoids are currently the mainstay of therapy in asthma, and lead to effective control of symptoms by reducing airway inflammation. Steroid treatment leads to a reduction in cytokines, chemokines, cell adhesion molecules, and DCs in the respiratory tract. All of these effects are believed to contribute to the suppression of the inflammatory response in asthma. Therapy with glucocorticoids results in a reduction in total CD4+ T cells in the airways and a decrease in mRNA for IL-4, IL-5, and IL-13, yet also results in increased IFN-γ expression (60). These effects likely indicate an increased sensitivity to steroid treatment of IL-4, IL-5, and IL-13 genes compared with IFN-γ. It is unclear what role this shift from Th2 to Th1 plays in the therapeutic effects of steroids, as this is only one of many possible mechanisms by which glucocorticoids can influence inflammation in asthma.
What is clear is that systemic glucocorticoids cause general immunosuppression and many well-described side effects. These effects can be reduced by local steroid treatment, but the suppression of airway inflammation is only temporary. Thus, selective approaches that block Th2 cell activation are needed. If selective inhibition of Th2 cell activation can be achieved, it will reduce Th2 cytokine secretion, without compromising Th1 cell function. By blocking cytokine secretion, not only will the effects on target tissues be reduced, but the cytokine environment may be altered, thus reducing the generation of new Th2 cells. GATA-3 is T cell–specific and, at present, the only identified Th2-specific factor that has the potential to inhibit the production of all the key Th2 cytokines (46). Blockade of GATA-3 may therefore inhibit Th2 cytokine secretion and also block the generation of new Th2 cells, as described above. Although some studies have shown that Th1 cells, through the production of IFN-γ, can inhibit Th2 cell function in vitro, these specific effects have not been demonstrated in vivo in animals.
Blockade of Th2 cytokine effects. Blocking the effects of Th2 cytokines at the target tissue is another potential option for reducing the inflammatory effects of Th2 cells. The effects of cytokine blockade may not only have local influence on airway epithelial cells and on the vascular endothelium, but also may reduce systemic effects on the bone marrow eosinophilopoiesis and on B-cell immunoglobin production. We have recently shown that when both Th1 and Th2 cells are transferred into recipient mice and activated in the respiratory tract with inhaled antigen, Th1 effects predominate, resulting in decreased airway eosinophilia and mucus production (61). These effects are mediated by IFN-γ acting on the target tissue in recipient mice, and occur while Th2 cells are actively secreting cytokines. In ragweed-sensitized mice, administration of CpG oligodeoxynucleotides, potent inducers of Th1 cells, led to a reduction in ragweed-induced airway eosinophilia, IgE, and AHR (62). This may also be a mechanism by which immunotherapy results in improvement of symptoms, as a reduction in eosinophilia in some allergic patients is associated with an increase in IFN-γ levels (63). Thus, Th1 cells, through secretion of IFN-γ, may block the effects of Th2 cells on target tissues, leading to a reduction in airway pathology.
If our ultimate goal is the long-term reduction in the number and activity of Th2 cells in the respiratory tract, then we must focus on new modes of therapy in asthma. One approach could be to increase the population of activated Th1 cells at sites of allergic inflammation, which may suppress ongoing Th2 cytokine effects. An alternate approach is to selectively target Th2 cells. Targeting Th2-specific factors such as GATA-3 by using pharmacologic or antisense approaches will inhibit Th2 responses while sparing Th1 cells and avoiding general immunosuppression. Over time, it is possible that these effects will be sustained, as there is evidence that potent, long-term stimulation with Th1 cytokines may shift a Th2-predominant population toward Th1 (24). These immunomodulatory approaches have the potential of disturbing the highly regulated immune defense system in the respiratory tract, including increasing unwanted Th1 effects such as autoimmune diseases, delayed-type hypersensitivity reactions, and excessive inflammation (64). The potential value of immunomodulation in controlling asthma, however, demands that these therapies be further developed and tested.