Morphine suppresses MHC-II expression on circulating B lymphocytes via activation of the HPA.

Morphine has been shown to alter gene expression of the major histocompatibility complex, class II (MHC-II) in circulating rat immunocytes. Here, we demonstrate that a single morphine injection (10 mg/kg) reduces basal MHC-II protein expression on circulating B lymphocytes by 33%, while also impairing the ability of B lymphocytes to increase MHC-II upon interleukin-4 induction. As these data implicate opioids in the regulation of antigen presentation, studies were undertaken to examine the potential mechanisms through which morphine exerts this suppressive effect. Central injection studies utilized Tyr-D-Ala-Gly-(me) Phe-Gly-ol (DAMGO), an opioid receptor agonist, which mimicked morphine's effect on MHC-II, while D-Phe-Cys_Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) pretreatment, prior to morphine, blocked the suppression of MHC-II. As central opioid receptor activation results in the activation of the hypothalamic-pituitary-adrenal axis, thereby, signaling increased circulating corticosterone levels, we examined whether MHC-II expression was suppressed after incubation with corticosterone at concentrations similar to those observed after morphine. Interestingly, corticosterone dramatically decreased basal MHC-II (88%) expression while completely preventing the induction of MHC-II. Additionally, MHC-II suppression was absent in morphine-treated adrenalectomized animals. Since prolonged morphine exposure has previously been shown to result in tolerance to both the steroidogenic and immunosuppressive effects of morphine, the effect of prolonged morphine exposure on MHC-II was also examined. Interestingly, MHC-II expression is no longer suppressed after chronic morphine, while morphine withdrawal results in both a renewed increase in circulating corticosterone levels and a renewed suppression of MHC-II in previously tolerant animals. Taken together, these data strongly implicate corticosterone in mediating the suppressive effects of morphine on circulating B-lymphocyte MHC-II expression.

Tec kinases, actin, and cell adhesion.

The Tec family non-receptor tyrosine kinases have been recognized for their roles in the regulation of phospholipase C-gamma and Ca(2+) mobilization downstream from antigen receptors on lymphocytes. Recent data, however, show that the Tec family kinase interleukin-2-inducible T-cell kinase (Itk) also participates in pathways regulating the actin cytoskeleton and 'inside-out' signaling to integrins downstream from the T-cell antigen receptor. Data suggest that Itk may function in a kinase-independent fashion to regulate proper recruitment of the Vav1 guanine nucleotide exchange factor. By enhancing actin cytoskeleton reorganization, recruitment of signaling molecules to the immune synapse, and integrin clustering in response to both antigen and chemokine receptors, the Tec kinases serve as modulators or amplifiers that can increase the duration of T-cell signaling and regulate T-cell functional responses.

Early phosphorylation kinetics of proteins involved in proximal TCR-mediated signaling pathways.

Activation of T cells via the stimulation of the TCR plays a central role in the adaptive immunological response. Although much is known about TCR-stimulated signaling pathways, there are still gaps in our knowledge about the kinetics and sequence of events during early activation and about the in vivo specificity of kinases involved in these proximal signaling pathways. This information is important not only for understanding the activation of signaling pathways important for T cell function but also for the development of drug targets and computer-based molecular models. In this study, phospho-specific Abs directed toward individual sites on signaling proteins were used to investigate the early phosphorylation kinetics of proteins involved in proximal TCR-induced pathways. These studies indicate that linker for activation of T cells' tyrosines have substantially different phosphorylation kinetics and that Src homology 2 domain-containing leukocyte protein of 76 kDa has rapid, transient phosphorylation kinetics compared to other proteins. In additions, we provide evidence that ZAP-70 is the primary in vivo kinase for LAT tyrosine 191 and that Itk plays a role in the phosphorylation of tyrosine 783 on phospholipase C-gamma1. In total, these studies give new insight into the sequence, kinetics and specificity of early TCR-mediated signaling events that are vital for T cell activation.

Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton.

The Tec family kinase Itk is an important regulator of Ca(2+) mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mutations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we find that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin polarization.

Rapid elevation of plasma interleukin-6 by morphine is dependent on autonomic stimulation of adrenal gland.

Several studies have demonstrated that opioids regulate a number of immune cell functions either through direct mechanisms or through the modulation of central nervous system outputs. It has been previously shown that morphine increases serum interleukin-6 (IL-6) levels; however, the mechanism by which this effect is produced is unknown. In the present study, experiments were designed to address the potential role of central opioid receptors, peripheral autonomic ganglia, and activation of the adrenals in the elevation of plasma IL-6 after morphine administration. A rapid and significant (2-fold) increase in plasma IL-6 was observed after morphine administration (10 mg/kg s.c.) to rats. This effect of morphine peaked within 30 min and remained elevated for at least 2 h. Central microinjection of morphine (10 microg/2 microl i.c.v.) mimicked the effects of peripherally administered morphine and was completely blocked by naltrexone (10 mg/kg s.c.) pretreatment. Pretreatment with a ganglionic blocker, chlorisondamine (0.5 mg/kg i.p.), also blocked the elevation of IL-6 by morphine, suggesting a role of the autonomic nervous system. In adrenalectomized animals, morphine administration did not increase IL-6 levels, whereas in adrenal demedullated animals, the effect of morphine remained intact. Thus, the adrenal cortex may be a potential source of IL-6, because IL-6 mRNA has been localized in the adrenal gland. Collectively, these data suggest a unique mechanism by which stimulation of central opioid receptors results in the elevation of plasma IL-6 through autonomic activation specifically of the adrenal cortex.

Differences in cytokine gene expression profile between acute and secondary injury in adult rat spinal cord.

It is likely that the environment within the injured spinal cord influences the capacity of fetal spinal cord transplants to support axonal growth. We have recently demonstrated that fetal spinal cord transplants and neurotrophin administration support axonal regeneration after spinal cord transection, and that the distance and amount of axonal growth is greater when these treatments are delayed by several weeks after injury. In this study, we sought to determine whether differences in inflammatory mediators exist between the acutely injured spinal cord and the spinal cord after a second injury and re-section, which could provide a more favorable environment for the axonal re-growth. The results of this study show a more rapid induction of transforming growth factor (TGF) beta1 mRNA expression in the re-injured spinal cord than the acutely injured spinal cord and an attenuation of proinflammatory cytokine mRNA expression. Furthermore, there was a rapid recruitment of activated microglia/macrophages in the degenerating white matter rostral and caudal to the injury but fewer within the lesion site itself. These findings suggest that the augmentation of TGFbeta-1 gene expression and the attenuation of pro-inflammatory cytokine gene expression combined with an altered distribution of activated microglia/macrophages in the re-injured spinal cord might create a more favorable milieu for transplants and axonal regrowth as compared to the acutely injured spinal cord.