Anuradha 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.

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.

Production of IL-6 by Keratinocytes: Implications for Epidermal Inflammation and Immunity

Recent evidence clearly indicates that the keratinocyte, the predominant cell of epidermal epithelium, is capable of producing protein molecules called cytokines that are best understood in the context of immune and inflammatory responses. These cytokines include interleukins (1L)1 alpha and beta; IL-3 (under defined circumstances); and granulocyte (G), macrophage (M), and granulocyte/macrophage (GM) colony stimulating factors (CSF, references 1 and 2). With the exception of IL-1, which appears to reside in normal uninduced epidermis (as well as uninduced keratinocytes in vitro), all of the above cytokines require an inductive signal. The inductive signal may come from mobile cells such as monocytes (via IL-1 or T N F alpha) or T cells (gamma IFN); alternatively, because keratinocytes both produce and respond to IL-1, induction of these proinflammatory cytokines in mouse skin can be entirely autocrine. The biological significance and the potential role of keratinocyte cytokines in cutaneous immunity have been the subject of several recent reviews.)-5 Epidermis is the primary interface between the host and the environment and, as such, it contains cells that can induce and amplify immune and inflammatory res p o n s e ~ . ~ Langerhans’ cells, bone marrow-derived epidermal symbionts that are capable of presenting antigen to T lymphocytes in an immunogenic form, reside within epidermis as do dendritic populations of ‘r lymphocytes. Keratinocytes can produce factors that enhance the efficiency with which Langerhans’ cells present antigen (GMCSF, reference 6), as well as factors that costimulate T cells (IL-1, references 1 and 7). In the present study, it is shown that keratinocytes are capable of producing IL6 bioactivity, protein, and mRNA in vitro. Because IL-6 has diverse effects upon T

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.

T-helper type 2 cell-directed therapy for asthma

Asthma is a chronic inflammatory disease of the bronchial airways. Current research in humans and animals suggests that T-helper type 2 (Th2) cells and the cytokines they elaborate cause many of the pathophysiologic abnormalities characteristic of the disease. We review the evidence implicating Th2 cells in asthma and discuss the cellular and molecular mechanisms that control Th2 cell differentiation in the respiratory tract. Based on the steps in Th cell development, we discuss how traditional therapies can modulate Th2 cell function. Furthermore, we explore newer immunomodulatory strategies to inhibit Th2 cell effects, including therapies that may block Th2 cell differentiation, neutralize cytokines, and redirect immune responses towards Th1 and away from Th2.

The Classic Steroid Hormone Receptors and ERβ, the Novel Estrogen Receptor

Steroid hormones control diverse biological functions, such as growth and development, regulation of immune functions, and cell death (1). The regulation of gene transcription by steroid hormones is mediated by intracellular receptors that belong to a complex superfamily of ligand-binding transcription factors (2,3). Members of this superfamily include the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), the estrogen receptor (ER), the progesterone receptor (PR), and the thyroid hormone receptor (TR). These receptors may be found in both the cytosolic and nuclear fractions of target tissues. Immunocytochemical studies with monoclonal antibodies against the various receptors indicate that most steroid hormone receptors (e.g., ER and PR) are nuclear even in the absence of hormone (4,5). GR and MR appear to be exceptions to this general rule. In the absence of hormone, these latter receptors are predominantly found in the cytoplasm complexed with other proteins (e.g., hsp 90) and concentrate in the nucleus only after hormone addition (6,7).