Bacterial lipopolysaccharide (LPS) is a potent stimulator of the cells of the immune system, including B cells and macrophages. The responses of these cells are likely to represent an evolved mechanism for rapidly recognizing and responding to infection by Gram-negative bacteria. Although generally beneficial, the responses to UPS become life threatening when induced systemically during bacterial sepsis. The proposed experiments are concerned with understanding intracellular signal transduction reactions that are elicited in the macrophage upon contact with LPs. We have recently found that LPS rapidly induces tyrosine phosphorylation of several macrophage proteins. As tyrosine phosphorylation has recently emerged as a major receptor signaling mechanism, we propose to study these reactions further with the hope of gaining insight into the processes that mediate the macrophage responses to LPS. We have just identified two of the LPS-induced tyrosine phosphoproteins in the RAW264.7 macrophage cell line as two isoforms of mitogen-activated protein (MAP) kinase. These serine/threonine kinases are activated by tyrosine kinase growth factor receptors in many cell types, and may be involved in the regulation of a variety of cellular processes, including transcription and translation. The propose experiments will first examine the mechanism by which LPS activates MAP kinase. The possible roles of the GTP-binding protein Ras, and the protein kinases Raf-1 and MAP kinase in the activation of MAP kinase by LPS will be examined. If some or all of these signaling components play a role in mediating LPS action, that would bring us closer to understanding the initial signal transduction events triggered by the putative LPS receptor. We shall determine whether LPS activation of MAP kinases and upstream events is seen in macrophages and macrophage cell lines of varied differentiation or activation status, or whether it is restricted to certain macrophage subpopulations. The molecular identity of the two isoforms of MAP kinase expressed in macrophages will be determined, and then these molecules will be overexpressed by introducing the appropriate cDNAs encoding the MAP kinase isoforms into the RAW264.7 macrophage cell line. The effects of overexpression on MAP kinase activation and LPS- induced biological responses will be examined. These experiments may provide evidence for a role of MAP kinases in the responses of macrophages to LPS.
