Autoimmunity (AI) exemplifies the potent and destructive activity expressed by the immune system when normal constraints against self- reactivity are lost or compromised. This proposal has developed from our discovery of a dramatic and intrinsic defect in cytokine expression in macrophages from young AI-prone mice. This defect is among the earliest known immunological problems in these animals, being apparent during the first week of life, and therefore cannot be a consequence of the disease process. The defect characterizes most strains that express multigenic lupus-like AI disease, and is not found in any normal strains.
The specific aims of this proposal are directed towards understanding the molecular mechanisms that underlie the defect and its potential relationship to disease. A defect in the cytokine network has the potential to disrupt normal regulation of self-reactivity, and thus lead to AI. Moreover, systemic multigenic AI is likely to be based on a global defect, such as the cytokine network. The finding that defective cytokine expression appears to be controlled primarily, if not exclusively, at the level of gene transcription leads to the hypothesis that aberrant cytokine production by AI-prone macrophage is likely to result from defective production or function of transcriptional regulatory factors (TRFs) or from a mutation within DNA sequences that bind the TRFs.
The specific aims of this proposal are to: (1a) identify regulatory DNA sequences that are potentially involved in interleukin 1beta (IL-1beta) gene dysregulation in AI-prone (MRL/+) macrophage. In vivo DNA footprinting of BALB/c and MRL/+ macrophage will be performed in order to identify relevant TRF-binding sites within the IL-1beta gene, and subsequently assess the functional importance of these TRF binding sites using transient transfection analysis. Subsequently, (1b) those TRFs implicated in dysregulated IL- 1beta gene transcription will be characterized. The possibility that any observed differences in TRF binding to DNA might result from specific mutations within the AI-prone macrophage DNA will also be tested. In parallel with these molecular approaches, the role of aberrant macrophage function on the development of AI disease will be explored. Aberrant expression of macrophage-derived cytokines may play a significant role in determining the specificity and magnitude of the immune response. Specifically, it will be (2) determined whether normal myeloid cells, and macrophage in particular, can prevent the development of AI. Disease signs will be monitored in adult irradiation chimeras constructed by the adoptive transfer of total bone marrow (BM) and of BM-derived macrophage from SCID or RAG-2 mutant mice (that do not express the defect) into AI-prone recipients. In situ hybridization analysis of cytokine transcript levels will be performed to determine if the transfer of BM or macrophage from mutant mice is accompanied by a normalized pattern of cytokine expression in AI-prone recipients. As a long range goal, these studies have the potential to develop critical insights into the pathogenesis of lupus and other systemic AI diseases, leading to the identification of potential targets for therapeutic intervention.
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