The goals of this project are to define factors which affect T lymphocyte motility and migration. Our studies show that the naturally occurring opioids, Leuenkephalin (LEU-ENK) and Metenkephalin (MET-ENK) stimulate T cell migration. Because MET-and LEU-ENK bind to opioid receptors (OR) on neurons, as do opiates and other families of neurohormones, these findings suggest that opiates, certain other related analgesics, and other neuropeptides may affect lymphocyte motility. We will measure the effects of neurohormones, including naturally occurring enkephalins, endorphins, and dynorphins, of synthetic enkephalin analogs and of opiates on migration of human T lymphocytes. Migration will be assayed using a microchemotaxis chamber and lymphocyte responses will be quantified by counting cells which have moved into nitrocellulose filters. Using highly enriched populations of CD4 and CD8 lymphocytes isolated by magnetic bead immunoabsorption, the lymphocyte populations which respond to the opioids/opiates will be identified. Opioid binding sites on CD4 and CD8 cells will be identified using radiolabelled opioids. Specific binding will be measured and the dissociation constant for the ligand-receptor interaction will be determined. Competition assays will be used to define the specificity of the lymphocyte receptors for the endorphins, enkephalins, enkephalin analogs, and opiates. Migration of activated and nonactivated human peripheral blood lymphocytes will be measured in response to neurohormones, including naturally occurring enkephalins, endorphins, and dynorphins, synthetic enkephalin analogs and opiates. From these studies we expect to determine the effects of activation on lymphocyte migration to the opioids and opiates. Migration of virus specific and alloreactive murine T lymphocyte and T clones will be measured to the neurohormones, enkephalin analogs, and opiates. From these studies we expect to determine whether opioids and/or opiates stimulate migration of allo- and antigen-reactive T cells. Biochemical pathways involved in ligand receptor-signalling will be studied. Intracellular levels of cAMP, Ca2+ flux, [Ca2+]i, and generation of inositol triphosphates will be monitored in lymphocytes challenged with opioids and opiates. Adenyl cyclase activity will be measured by the formation of cAMP using a radioimmunoassay. Changes in [Ca2+]i will be measured as shifts in the emission spectra of Indo 1 fluorescent dye and Ca+ flux will be measured by uptake of 45Ca C1+. Phospholipid catabolism will be monitored by 32P labeling of inositol triphosphates. From these studies we expect to identify specific opioids and/or opiates which stimulate T cell motility, measure binding of these agents to T cell receptor sites, and identify second messengers involved in opioid-receptor signalling systems. The effects of lymphocyte activation and antigen reactivity on the opioid response will be studied. This series of experiments will allow us to determine how these neurohormones regulate T cell motility.
| Heagy, W; Teng, E; Lopez, P et al. (1999) Enkephalin receptors and receptor-mediated signal transduction in cultured human lymphocytes. Cell Immunol 191:34-48 |
