The primary goal of this research is to elucidate the interactions and effects of the morphine-immune axis. We have shown that chronic exposure to morphine in vivo desensitizes interleukin-1 beta (IL- 1 B)-induced neuronal activation, as shown by FOS expression, in the hypothalamic paraventricular nucleus (PVN), a critical component of the hypothalamic-pituitary-adrenal (HPA) axis. We also reported that chronic morphine suppresses IL-1 B-induction of corticotropin releasing factor (CRF) mRNA in the hypothalamus and plasma corticosterone levels. In addition, we have shown that, by desensitizing the HPA response to IL-1 B, chronic morphine potentiates leukocyte-endothelial adhesion (LEA) during IL- 1 B-induced inflammation. Thus, it appears that chronic morphine exposure can promote a potentially damaging inflammatory reaction by disrupting the balance between IL- 1 P-mediated local inflammation, as measured by LEA, and the anti-inflammatory effects of the HPA axis. Based on these data, Specific Aims 1 and 2 will analyze the mechanisms involved in systemic morphine- immunity interactions with a focus on opioid receptor pathways.
In Specific Aim 1, we propose to extend our research on the opiate-immune axis by investigating how chronic exposure to morphine may affect the inflammatory process induced by a pathogen in vivo. Using intravital microscopy, ELISA, and RIA, we will examine morphine's effects on various indicators of inflammation in the rat model using lipopolysaccharide (LPS), a well-characterized bacterial endotoxin, as a stimulus for inflammation. We propose to examine, in Specific Aim 2, how LPS and the pro-inflammatory cytokines, IL-1 0, TNF-a, and IL- 6, that are stimulated by LPS affect opiate-dependent pathways by examining the expression of the mu opioid receptor (MOR) using quantitative competitive reverse transcriptase polymerase chain reaction (QC-RT-PCR) and immunocytochemistry, and determining the levels of endogenous opiate peptides with high affinity for the MOR using RIA.
In Specific Aim 3, the convergence of opiate-mediated systemic effects and the actions of LPS and related cytokines will be examined in vitro at the molecular level in a stably transfected cell model, using QC-RT-PCR, gel mobility shift assay, supershift assay, and ligand binding analysis. In summary, the studies in this revised proposal are designed to link the findings from our previous in vivo studies showing that chronic exposure to morphine abolishes morphine- and IL-1 B-induced FOS activation in the PVN, to those in Specific Aims 1 and 2 which will define the process by which the MOR-dependent pathways can be modulated by bacterial endotoxins, and how such modulation may be related to the progression of endotoxin shock, to the in vitro studies in Specific Aim 3 using transfected MDCK-MOR1 cells to elucidate the molecular mechanisms involved in the convergence of the morphine- immune axis.
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