Dong-Hong Zhang

Transcription Factor GATA-3 Is Differentially Expressed in Murine Th1 and Th2 Cells and Controls Th2-specific Expression of the Interleukin-5 Gene

Interleukin-5 (IL-5), which is produced by CD4+ T helper 2 (Th2) cells, but not by Th1 cells, plays a key role in the development of eosinophilia in asthma. Despite increasing evidence that the outcome of many diseases is determined by the ratio of the two subsets of CD4+ T helper cells, Th1 and Th2, the molecular basis for Th1- and Th2-specific gene expression remains to be elucidated. We previously established a critical role for the transcription factor GATA-3 in IL-5 promoter activation in EL-4 cells, which express both Th1- and Th2-type cytokines. Our studies reported here demonstrate that GATA-3 is critical for expression of the IL-5 gene in bona fide Th2 cells. Whereas mutations in the GATA-3 site abolished antigen- or cAMP-stimulated IL-5 promoter activation in Th2 cells, ectopic expression of GATA-3 in Th1 cells or in a non-lymphoid, non-IL-5-producing cell line activated the IL-5 promoter. During the differentiation of naive CD4+ T cells isolated from T cell receptor transgenic mice, GATA-3 gene expression was up-regulated in developing Th2 cells, but was down-regulated in Th1 cells, and antigen- or cAMP-activated Th2 cells (but not Th1 cells) expressed the GATA-3 protein. Thus, GATA-3 may play an important role in the balance between Th1 and Th2 subsets in immune responses. Inhibition of GATA-3 activity has therapeutic potential in the treatment of asthma and other hypereosinophilic diseases.

Inhibition of allergic inflammation in a murine model of asthma by expression of a dominant-negative mutant of GATA-3.

The cytokines IL-4, IL-5, and IL-13, secreted by Th2 cells, have distinct functions in the pathogenesis of asthma. We have previously shown that the transcription factor GATA-3 is expressed in Th2 but not Th1 cells. However, it was unclear whether GATA-3 controls the expression of all Th2 cytokines. Expression of a dominant-negative mutant of GATA-3 in mice in a T cell-specific fashion led to a reduction in the levels of all the Th2 cytokines IL-4, IL-5, and IL-13. Airway eosinophilia, mucus production, and IgE synthesis, all key features of asthma, were severely attenuated in the transgenic mice. Thus, targeting GATA-3 activity alone is sufficient to blunt Th2 responses in vivo, thereby establishing GATA-3 as a potential therapeutic target in the treatment of asthma and allergic diseases.

Repression of interleukin-6 gene expression by 17β-estradiol:

The cytokine interleukin‐6 (IL‐6), a key mediator of immune and acute phase responses of the liver, has also been implicated in uterine functions. Estrogens are potent repressors of IL‐6 production by uterine stromal cells. In the endometrial adenocarcinoma cell line Ishikawa, phorbol ester‐induced activation of the IL‐6 promoter was inhibited to basal levels by 17β‐estradiol (E2) in a wild‐type receptor‐dependent fashion. Although tamoxifen has been shown to have estrogenic effects on the endometrium, it did not inhibit induction of the IL‐6 promoter. We previously showed that inhibition of IL‐6 gene expression by E2 does not involve high‐affinity binding of the estrogen receptor (ER) to IL‐6 DNA. We now report that the ER can directly interact with the transcription factors NF‐IL6 and NF‐κB and can inhibit their ability to bind DNA which might be the molecular basis for repression of IL‐6 gene expression by estrogens.

Gene expression of the GATA-3 transcription factor is increased in atopic asthma

BACKGROUND High expression of IL-5 by T cells in the airways of asthmatic individuals is believed to play a fundamental role in the eosinophilia associated with this disease. Recently, the transcription factor GATA-3 was shown to be critical for IL-5 gene expression in TH2 cells in vitro. OBJECTIVE Our aim was to examine the expression of GATA-3 mRNA and its colocalization within the airways of asthmatic and nonasthmatic individuals. METHODS We investigated the association between GATA-3 gene expression, airway inflammatory cells, and IL-5 gene expression in bronchoalveolar lavage fluid and bronchial biopsy specimens from atopic asthmatic subjects (n = 10) and normal control subjects (n = 10). RESULTS We report that GATA-3 mRNA expression is significantly increased in the airways of asthmatic subjects compared with those of normal control subjects (P <.001). Numbers of cells expressing GATA-3 transcripts correlated significantly with reduced airway caliber (P <.05) and airways hyperresponsiveness (P <.05) in asthmatic subjects. Colocalization studies showed that the majority (approximately 60% to 90%) of GATA-3 mRNA+ cells in asthmatic airways were CD3(+) T cells, with smaller contributions from major basic protein+ eosinophils and tryptase+ mast cells. The density of GATA-3 mRNA+ cells correlated significantly with the numbers of cells expressing IL-5 mRNA (P <.001, r = 0.879 for bronchoalveolar lavage fluid; P <. 05, r = 0.721 for biopsy specimens). Furthermore, double in situ hybridization demonstrated that approximately 76% of GATA-3 mRNA+ cells coexpressed IL-5 mRNA and that 91% of IL-5 mRNA+ cells coexpressed GATA-3 mRNA. CONCLUSION The results of this study provide the first evidence of increased GATA-3 gene expression in association with IL-5 mRNA+ cells in asthmatic airways. These findings support a causal association between augmented GATA-3 expression and dysregulated IL-5 expression in atopic asthma.

Activation of the Interleukin-5 Promoter by cAMP in Murine EL-4 Cells Requires the GATA-3 and CLE0 Elements

Interleukin-5 (IL-5) plays a central role in the growth and differentiation of eosinophils and contributes to several disease states including asthma. Accumulating evidence suggests a role for cAMP as an immunomodulator; agents that increase intracellular cAMP levels have been shown to inhibit production of cytokines predominantly produced by T helper (Th) 1 cells such as IL-2 and interferon (IFN-). In contrast, the production of IL-5, predominantly produced by Th2 cells, is actually enhanced by these agents. In this report, we have performed transient transfection experiments with IL-5 promoter-reporter gene constructs, DNase I footprinting assays, and electrophoretic mobility shift assays to investigate the key regulatory regions necessary for activation of the IL-5 promoter by dibutyryl cAMP and phorbol esters in the mouse thymoma line EL-4. Taken together, our data demonstrate the critical importance of two sequences within the IL-5 5′-flanking region for activation by these agents in EL-4 cells: one, a highly conserved 15-base pair element present in genes expressed by Th2 cells, called the conserved lymphokine element 0 (CLE0; located between −53 and −39 in the IL-5 promoter), and the other, two overlapping binding sites for the transcription factor GATA-3 (but not GATA-4) between −70 and −59. Taken together, our data suggest that activation via the unique sequence combination GATA/CLE0 results in selective expression of the IL-5 gene in response to elevated levels of intracellular cAMP.

Cyclic AMP activates p38 mitogen-activated protein kinase in Th2 cells: phosphorylation of GATA-3 and stimulation of Th2 cytokine gene expression.

cAMP is an important second messenger with immunomodulatory properties. Elevation of intracellular cAMP in T cells, induced by agents such as IL-1α or PGs, inhibits T cell activation. In effector T cells, an increase in the level of intracellular cAMP inhibits cytokine production in Th1 cells but stimulates cytokine production in Th2 cells. Here we report that cAMP-induced effects in Th2 cells occur independently of the protein kinase A pathway, which is the major mediator of cAMP-induced signaling events in most cell types. Instead, cAMP stimulates activation of p38 mitogen-activated protein kinase in Th2 cells. This appears to be a Th2-selective event because cAMP barely increased p38 phosphorylation in Th1 cells. We show that in Th2 cells, cAMP promotes the production of both IL-5 and IL-13, which play distinct but critical roles in asthma pathogenesis. Our data also show that cAMP causes increased phosphorylation of the transcription factor GATA-3, which we have shown is a critical regulator of Th2 cytokine gene expression and, in turn, of airway inflammation in mice. Thus, Th2-specific GATA-3 expression and p38 mitogen-activated protein kinase activation together provide a molecular basis for the differential effects of cAMP in the two T helper cell subsets.

Dominance of IL-12 Over IL-4 in γδ T Cell Differentiation Leads to Default Production of IFN-γ: Failure to Down-Regulate IL-12 Receptor β2-Chain Expression

γδ T cells secrete Th1- and Th2-like cytokines that help mediate innate and acquired immunity. We have addressed the mechanism whereby murine γδ T cells acquire the capacity to differentially produce such cytokines. Splenic γδ T cells could be polarized into IFN-γ- or IL-4-secreting cells in vitro; however, in contrast to CD4+ αβ T cells, γδ T cells predominantly produced IFN-γ, even in the presence of IL-4, a finding independent of genetic background. Like CD4+ Th1 cells, IFN-γ-producing cells expressed the IL-12 receptor β2-chain after activation in the presence of IL-12; however, unlike Th2 cells, IL-4-primed γδ T cells also expressed this receptor, even in the absence of IFN-γ and despite the presence of the transcription factor GATA-3. IL-12 also induced IL-4-primed γδ T cells to proliferate and to translocate Stat3/Stat4, indicating signaling through the IL-12 receptor. These molecular events can account for the predominant production of IFN-γ by γδ T cells in the presence of IL-12, despite the availability of IL-4. Early and predominant production of IFN-γ by γδ T cells likely is critical for the roles that these cells play in protection against intracellular pathogens and in tumor immunity.

Selective Up-regulation of Cytokine-induced RANTES Gene Expression in Lung Epithelial Cells by Overexpression of IκBR

We previously reported the cloning of a cDNA for IκBR (for IκB-related) from human lung alveolar epithelial cells. IκBR is related to the IκB proteins that function as regulators of the NF-κB family of transcription factors. Here, we investigated the consequence of IκBR overexpression on the expression of NF-κB-regulated chemokine genes in lung alveolar epithelial cells. Chemokines play an important role in many inflammatory diseases such as asthma and AIDS. Overexpression of IκBR in the lung cells resulted in a rapid 50–100-fold greater production of the RANTES (regulated upon activation, normalT expressed and presumablysecreted) protein upon cytokine-induction compared with control cells. IκBR overexpression, however, did not enhance interleukin-8 or MIP-1α gene expression, despite the fact that the expression of all three chemokine genes are regulated by NF-κB. The up-regulation of RANTES gene expression resulting from overexpression of IκBR correlated with increased amounts of a unique RANTES-κB binding activity and decreased binding of p50 homodimers. Previous studies have shown that p50 homodimers function as repressors of certain κB sites. Our studies suggest that IκBR can aid activation of select NF-κB-regulated genes by sequestering p50 homodimers.