Neutrophils provide an essential defense against infection, and play a major role in the tissue damage caused by inflammation. They also provide an excellent, simplified model for studying the modulation of mRNA levels in normal mammalian cells. With the emergence of public databases containing very large numbers of genes and expressed sequence tags, research in human genetics is now turning from investigation of single genes to the global analysis of gene expression. The principal investigators propose to apply this important new approach to human neutrophils. Using methods based on display of cDNA 3' end fragments, generated by specific restriction enzymes, the investigators will semi-quantitatively assess the levels of most mRNAs found in activated neutrophils. The proposed studies will test the central hypothesis that neutrophils show complex, stimulus-specific patterns of genetic regulatory responses, which can be explained, at least in part, by combinatorial use of """"""""subroutines"""""""" of coordinately regulated responses.
The specific aims are: 1) Determine the pattern of gene expression by neutrophils stimulated by a wide variety of well- defined agonists, as well as identify new genes involved in the neutrophil phagocytic response; 2) Compare the patterns of response to simple agonists versus complex physiological activators, including bacterial and sterile inflammatory stimuli, both in vitro and in vivo; 3) Dissect the components of the responses to bacterial and sterile inflammatory stimuli by inhibition of specific receptors and signal transduction pathways, exposure to clinically important anti-inflammatory drugs, and determination of the temporal and functional order of the complex response; 4) Determine the molecular mechanisms underlying the changes in gene transcript levels, including both transcriptional control mechanisms and regulation of mRNA stability. These studies may provide insight into the genetic repertoire of known and new genes that contribute to the neutrophil response and hence provide targets for intervention in augmentation of host defense or amelioration of inflammation. Insight into transcriptional and post-transcriptional mechanisms of gene expression in this system may also be applicable to other, more complex, cell types and tissues.
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