Activated macrophages are a common hallmark of many inflammatory disease and play a significant role in the outcome of endotoxic shock. This proposal studies the down-regulation of macrophages by the Mer receptor tyrosine kinase using molecular and cellular methods, both in vivo and in vitro. The purpose of this proposal is to determine mechanisms by which Mer receptor tyrosine kinase inhibit macrophages and the consequence of the macrophage down-regulation on inflammation. The finding of this proposal would have broad implications toward attenuating inflammation associated with macrophages.
The Specific Aims are fourfold: 1). We will assess the molecular mechanism by which Mer inhibits LPS signal transduction. We will assess alteration in IkBalpha and IkBbeta in CD14 expressing CHO cell lines transfected with mer/kd and mer/WT constructs. We will also assess the role of Mer in JNK, p38 and ERK signaling pathways activated by LPS. Finally, we will assess whether Mer is interacting or affecting the cell surface expression of CD14. 2). We will determine how Mer modulates macrophages during inflammation. We will use mer/kd and mer/WT mice undergoing inflammation and assess severity of histology, cytokine profiles and macrophage activation. In addition, we propose a transgenic animal under the control of an inducible system to directly determine the consequence of Mer expression during inflammation. 3). We will determine whether the regulation of TNF-alpha-induced apoptosis by Mer is at a control point upstream of NF-kappaB. We will assess whether Mer alters levels of TRAFs to induce NF-kappaB. Also we will assess in vivo apoptosis in sites of inflammation mer/kd and mer/WT mice. 4). We will determine whether Mer activity can be correlated to macrophage activation from patients with chronic inflammation. Protein and phosphorylation assays of Mer will be assessed in macrophages isolated from patients. In addition, cytokine profiles and NF-kappaB will be quantified to determine correlation with inflammation.
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