Determining the molecular basis of opiate and opioid modulation of immune function is critical to understanding such apparently diverse phenomena as the immunosuppressive effects of drug abuse and stress, their correlations with progression of HIV infection to clinical AIDS, and the possibilities for therapeutic intervention. T cell functions are critical to both cellular and humoral immunity. The immunodeficiency of AIDS is associated with specific declines in CD4+ T cell dependent functions and impaired T cell receptor signal transduction. CD8+ T cell derived factors suppress HIV replication. There is evidence that the opioid peptide met-enkephalin ameliorates HIV associated T cell defects in vivo. Although some opiate immunomodulatory effects may be mediated through the CNS there is abundant evidence for immunomodulation effected by direct opioid immunocyte interactions. And, cells of the immune system express opioid receptors thought to be similar to those in neural tissue. The objective of this proposal is to identify the opioid receptors responsible for the apparently opposite effects of methionine enkephalin in vitro on activation of purified CD4 (helper type) and CD8 (suppressor type) positive T cells and to initiate an investigation of the underlying biochemical mechanisms. Immobilized monoclonal antibody against the T cell receptor associated CD3 complex (anti-CD3 mAb) elicits specific polyclonal activation of T cells in the absence of accessory cells. The cascade of signal transduction, intracellular second messengers and induction of specific gene transcription elicited by anti- CD3 mAb is well characterized and essentially identical to that elicited by physiologic T cell activation. This model will be used to identify specific receptors in CD4+ and CD8+ T cells through which opioid peptide modulation of T cell activation is mediated, and should provide an excellent system in which to identify the specific T cell activation event(s) affected. In the first aim, RT-PCR will be used to determine if delta and mu receptor messenger RNAs identical or closely related to those recently determined in neural tissues are expressed in CD4+ and CD8+ T cells purified from murine spleen. Although Met-enkephalin is primarily considered a delta opioid receptor agonist there is evidence to suggest that both delta and mu opioid receptors may be involved in its effects on T cells. In the second aim, radioreceptor assays will be used to quantify and characterize the putative delta and mu opioid receptor binding sites in CD4+ and CD8+ T cells, and agonist and antagonist binding to these sites will be correlated with modulation of anti-CD3 stimulated T cell proliferation. The results of Aim 2 will confirm that expression of appropriate binding sites parallels expression of specific opioid receptor messenger RNA and may be informative as to whether the ligand specificity of expressed receptors is dependent in part on cell background.
The third aim i s to identify the early signal transduction events of T cell activation which are postulated to be modulated by activity of delta and mu opioid receptors and result in the observed opioid peptide effects on T cell proliferation. Opioid peptide effects on anti-CD3 mAb stimulated phospholipase C dependent phosphoinositide hydrolysis, specific tyrosine kinase phosphorylation, and elevation of intracellular free calcium will be investigated.
