The long-term objective of this proposal is to understand the mechanisms responsible for the termination of proinflammatory cytokine biosynthesis in macrophages. Proinflammatory cytokines, TNF-alpha and IL-1 in particular, play an important role in the pathogenesis of a variety of human diseases, including rheumatoid arthritis, Crohn's disease, and septic shock. Biosyntheses of both TNF-a and IL-1 in LPS-stimulated macrophages are regulated by complex signal transduction pathways involving MAP kinases and NF-kappaB. Preliminary studies in our laboratory with RAW264.7 cells provide strong evidence to support the hypothesis that MKP-1 plays a critical role in the feedback control of p38 and JNK MAP kinases and is responsible for the termination of pro-inflammatory cytokine production in LPS-stimulated macrophages. Moreover, MKP-1 is potently induced by glucocorticoids. The First Specific Aim of the present proposal is to test the hypothesis that MKP-1 plays critical roles in restraining the LPS-induced biosynthesis of proinflarnmatory cytokines in both peritoneal and alveolar macrophages after LPS stimulation. The Second Specific Aim is to test the hypothesis that MKP-1 also acts as a critical negative regulator in the restraint of macrophage responses to Gram-positive bacteria. The Third Specific Aim is to test the hypothesis that the responses to LPS stimulation of primary peritoneal macrophages from Mkp-1-/- mice differ from the responses of macrophages isolated from Mkp-l+/+ mice. The cryopreserved embryos derived from the Mkp-1 knockout mice are obtained from Bristol-Myers Squibb Pharmaceutical Research Institute, through a materials transfer agreement. Regeneration of these embryos into mice has been accomplished in The Jackson Laboratory. We will isolate peritoneal macrophages from the Mkp-l+/+ and Mkp-1-/- mice and use these macrophages to determine the role of MKP- 1 in the responses to LPS, with respect to MAP kinase inactivation and control of inflammatory cytokine production. We will also examine whether the lack of Mkp-1 gene will compromise the suppressant effects of glucocorticoids on TNF-alpha production induced by LPS. These studies are designed to answer pivotal questions regarding the regulatory mechanisms responsible for terminating proinflammatory cytokine production in macrophages during bacterial infections, and to reveal novel targets for developing new antiinflammatory/ anti-rheumatic drugs.
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