Prabir Ray

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.

A critical role for NF-kappa B in GATA3 expression and TH2 differentiation in allergic airway inflammation.

The transcription factor GATA-3 is expressed in T helper 2 (TH2) but not TH1 cells and plays a critical role in TH2 differentiation and allergic airway inflammation in vivo. Mice that lack the p50 subunit of nuclear factor κB (NF-κB) are unable to mount airway eosinophilic inflammation. We show here that this is not due to defects in TH2 cell recruitment but due to the inability of the p50−/− mice to produce interleukin 4 (IL-4), IL-5 and IL-13: cytokines that play distinct roles in asthma pathogenesis. CD4+ T cells from p50−/− mice failed to induce Gata3 expression under TH2-differentiating conditions but showed unimpaired T-bet expression and interferon γ (IFN-γ) production under TH1-differentiating conditions. Inhibition of NF-κB activity prevented GATA-3 expression and TH2 cytokine production in developing, but not committed, TH2 cells. Our studies provide a molecular basis for the need for both T cell receptor and cytokine signaling for GATA-3 expression and, in turn, TH2 differentiation.

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.

Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodeling, and airways obstruction.

Interleukin-11 is a pleotropic cytokine produced by lung stromal cells in response to respiratory viruses, cytokines, and histamine. To further define its potential effector functions, the Clara cell 10-kD protein promoter was used to express IL-11 and the airways of the resulting transgene mice were characterized. In contrast to transgene (-) littermates, the airways of IL-11 transgene (+) animals manifest nodular peribronchiolar mononuclear cell infiltrates and impressive airways remodeling with subepithelial fibrosis. The inflammatory foci contained large numbers of B220(+) and MHC Class II(+) cells and lesser numbers of CD3(+), CD4(+), and CD8(+) cells. The fibrotic response contained increased amounts of types III and I collagen, increased numbers of alpha smooth muscle actin and desmin-containing cells and a spectrum of stromal elements including fibroblasts, myofibroblasts, and smooth muscle cells. Physiologic evaluation also demonstrated that 2-mo-old transgene (+) mice had increased airways resistance and non-specific airways hyperresponsiveness to methacholine when compared with their transgene (-) littermates. These studies demonstrate that the targeted expression of IL-11 in the mouse airway causes a B and T cell-predominant inflammatory response, airway remodeling with increased types III and I collagen, the local accumulation of fibroblasts, myofibroblasts, and myocytes, and obstructive physiologic dysregulation. IL-11 may play an important role in the inflammatory and fibrotic responses in viral and/or nonviral human airway disorders.

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.

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.