Polymorphonuclear leukocytes (PMNs) are an essential arm of the host defense against periodontal disease and other bacterial infections. Their antimicrobial activity can be """"""""primed"""""""" by exposure to TNF or GM-CSF at inflammatory sites. These cytokines enhance the oxidative and secretory response to other stimuli (e.g., fMLP) and directly induce adhesion, degranulation, and expression of immunoregulatory cytokines (e.g., IL- 1beta) by PMNs. While priming is a useful response that facilitates full PMN activation at infection sites, """"""""hyperactivation"""""""" by priming agents can lead to deleterious tissue damage. Polyamines appear to play an important role in PMN priming. TNF induces rapid increases in PMN putrescine and spermine content. Selective inhibition of ornithine decarboxylase (ODC), the rate limiting enzyme of polyamine biosynthesis, inhibits these increases and blunts the enhancement of oxidative activity normally associated with priming by TNF. However, inhibition of ODC does not alter TNF receptor expression, block activation of the PMN NADPH oxidase by phorbol esters, or inhibit induction of PMN adhesion or IL-1beta mRNA expression by TNF, suggesting that it selectively antagonizes PMN priming by TNF. ODC inhibition is associated with attenuation of Ca2+ signaling in fMLP-stimulated PMNs, while exogenous polyamines found at diseased periodontal sites potentiate Ca2+ signaling. Elevated Ca2+ levels promote activation of phospholipase D (PLD), which plays an essential role in priming by TNF and GM-CSF. Thus, modulation of the PLD pathway is a plausible mechanism by which polyamines could influence priming. This proposal will test the hypothesis that polyamines enhance PMN priming by TNF and GM-CSF. Its long-term objective is to provide a better understanding of the molecular basis of PMN priming.
Specific aim 1 is to characterize the effects of TNF and GM-CSF on polyamine biosynthesis in PMNs by examining: a) induction of ODC mRNA, b) activation of ODC and accumulation of polyamines.
Specific aim 2 is to characterize the influence of polyamines on the PMN response to TNF and GM-CSF by examining the effects of ODC inhibitors and exogenous polyamines on: a) enhancement of oxidative and secretory activities in PMNs primed with TNF and GM-CSF, b) induction of adhesion, degranulation, and IL-1beta expression, and c) activation of signaling mechanisms associated with phospholipase D up- regulation in PMNs primed with TNF-alpha or GM-CSF. These studies will provide a better understanding of how PMNs are up-regulated at diseased periodontal sites, and provide insight into the role of PMN priming in the pathogenesis of periodontal disease and other inflammatory disorders. They may also lead to new approaches for blunting the deleterious effects of PMN priming seen in rapidly progressive periodontitis and in life- threatening disorders like septic shock and adult respiratory distress syndrome.
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