The overall goal of the proposed research is to understand the cellular and molecular mechanisms of immune and inflammatory responses within the central nervous system (CNS). The expression of class II major histocompatibility complex (MHC) antigens on professional antigen presenting cells of the immune system has a critical role in regulating the immune response by presenting foreign antigens to CD4+ T lymphocytes, leading to their activation and differentiation. Aberrant expression of class II MHC by glial cells in the CNS (i.e., astrocytes and microglia), however, may allow the presentation of self antigens that are responsible for certain autoimmune diseases such as multiple sclerosis (MS). The hypothesis that drives this proposal is based on the observation that regulation of class II MHC genes occurs primarily at the transcriptional level, dependent on a non-DNA binding protein, Class II transactivator (CIITA). Thus, it is proposed that aberrant CIITA expression in glial cells results in inappropriate expression of class II MHC antigens, and detrimental immunological activities in the CNS. Furthermore, the anti-inflammatory, disease-ameliorating properties of TGF( (and perhaps IL-4 as well) are due to the ability of TGF( to down-regulate CIITA expression. The three Specific Aims described in this application are as follows:
Specific Aim 1. Delineate the molecular mechanism(s) of CIITA expression. Since it is known that IFN( is able to induce the expression of class II MHC on glial cells, studies are to be performed intended to determine if the same cytokine stimulus induces CIITA gene activation. Astroglioma cell lines (i.e., CRT, CH235-MG), microglial SV40 T antigen transfectants, as well as primary human astrocytes and microglia are to be examined and compared for induced CIITA gene transcriptional activity, mRNA production, and protein production.
Specific Aim 2. Elucidate the signal transduction pathways that mediate IFN( induced CIITA expression in glial cells. Studies in this Specific Aim are intended to examine the involvement of tyrosine kinases and serine/threonine kinases in IFN( induced CIITA gene activation, CIITA mRNA expression and stability, and CIITA protein expression. In addition, the absolute requirement of the STAT-1( protein in IFN( induced CIITA and class II expression is to be assessed in primary cultures of astrocytes and microglia from STAT1 deficient mice.
Specific Aim 3. Determine the mechanism(s) of TGF( and IL-4 mediated repression of class II MHC expression in glial cells. Studies are to be performed designed to examine the effect of TGF( and IL-4 on CIITA gene transcription, CIITA mRNA expression, CIITA protein production, and CIITA function. In addition, promoter associated regulatory elements are to be sought as possible targets of the inhibition. It is also to be determined if TGF( and IL-4 inhibit IFN( induction of CIITA and class II MHC by modulating IFN( receptor expression, or by interfering with the IFN(-induced intracellular signaling events necessary for CIITA/class II MHC expression. Class II MHC antigens have a critical role in regulating the immune response by presenting antigen to CD4+ T cells, leading to their activation. Regulation of class II MHC genes occurs at the transcriptional level, and recently, a non-DNA binding protein, termed class II transactivator (CIITA), has been shown to be the """"""""master control factor"""""""" for class II MHC transcription. Aberrant expression of class II MHC antigens has been correlated with autoimmune diseases, including multiple sclerosis (MS). In the inflamed CNS, class II MHC antigens are expressed by astrocytes and microglia, allowing them to function as antigen-presenting cells. This leads to activation of autoreactive CD4+ T cells, and subsequent inflammation and demyelination. IFN-gamma is the most potent inducer of class II MHC expression on glial cells. Dr Benveniste has shown that IFN-gamma acts at the transcriptional level to regulate class II MHC expression in astrocytes and microglia, and that the transcription factor STAT-1a is involved in IFN-gamma induced CIITA and class II MHC expression. TGF-beta, an anti- inflammatory cytokine, suppresses IFN-gamma induced class II MHC expression in astrocytes by inhibiting the expression of CIITA. We hypothesize that aberrant CIITA expression in glial cells results in the subsequent expression of class II MHC antigens, leading to detrimental immunologic activities within the CNS. Furthermore, downregulation of CIITA by TGF-beta results in a reduction of immune responsiveness and inflammation in the CNS, events that are beneficial in diseases such as MS. In this application, Dr Benveniste will delineate the molecular basis by which IFN-gamma induces CIITA gene expression in human astrocytes and microglia (Aim #1). This information on CIITA regulation is essential for understanding the IFN-gamma-induced signal transduction pathways involved in CIITA expression (Aim #2), particularly the involvement of tyrosine kinase and serine/threonine kinase activity in IFN-gamma induction of CIITA. In addition, the obligatory role of STAT-la in IFN-gamma induction of CIITA and class II expression will be investigated using glial cells from STATI deficient mice.
In Aim #3, she will determine the molecular mechanisms by which TGF-beta inhibits CIITA expression. Dr Benveniste has preliminary data that IL-4 also inhibits IFN-gamma- induced class II MHC expression. She will elucidate whether TGF-beta and IL-4 utilize similar or disparate mechanisms for suppression of class II expression in glial cells. The combinatorial use of inhibitory cytokines (TGF-beta, IL-4) holds promise in modifying demyelinating diseases. To develop strategies for implementation of combinatorial cytokine therapy, Dr Benveniste must first understand how these cytokines act in isolation, and their mechanisms of action in the microenvironment of the diseased CNS.
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