This is a revised application of a proposal to study the intracellular signal transduction pathway which leads to expression of pro-inflammatory chemokines when macrophages encounter particles in the lung. Alveolar regions of the lung are continuously exposed to inhaled particles, including both inert particulates and aerosolized pathogens. Alveolar macrophages (AMs) are primarily responsible for clearance of these particles from the lung, and for initiating and propagating a """"""""proper"""""""" inflammatory response. Responses to particle binding and/or phagocytosis of AMs is dependent upon characteristics of the particles encountered. Binding of opsonized particles by cell surface receptors of macrophages results in a substantial respiratory burst and subsequently increases in mRNA transcripts for pro-inflammatory cytokines including the Platelet Factor-4 family chemokines, macrophage inflammatory protein-2 (MIP-2) and KC. We hypothesize that pulmonary inflammation is induced when inhaled particles are opsonized and taken up by alveolar macrophages using specific surface receptors which transduce intracellular signals to activate transcription of the MIP-2 and KC genes. The nature of the intracellular signal(s) that mediate the induction of MIP-2/KC gene expression is unknown, however, we present substantial preliminary data implicating reactive oxygen species (ROS) as the primary stimulus for induction of these mRNAs. We propose to use molecular and biochemical techniques to define this transduction pathway in a bi-directional fashion starting from both the stimulus at the macrophage cell membrane and the response, MIP-2/KC mRNA synthesis.
The specific aims of the studies we propose are: (l) To determine the regulatory mechanisms operative when macrophage binding of opsonized particles increases MIP-2 and KC mRNA levels; (2) To clone and characterize the 5'-flanking regions of the MIP-2 and KC genes; (3) To identify putative cis elements and establish their role in controlling transcription of the MIP-2 and KC genes; and (4) To establish that reactive oxygen species are a key component of the signal transduction pathway inducing MIP-2 and KC gene expression. Elucidation of the pathway by which opsonized particles induce chemokine synthesis will add significantly to our understanding of pulmonary inflammation and associated diseases.
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