Intramuscular delivery of heterodimeric IL-15 DNA in macaques produces systemic levels of bioactive cytokine inducing proliferation of NK and T cells.

Interleukin-15 (IL-15) is a common γ-chain cytokine that has a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting infection and cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha (IL-15Rα) chain, which together form the IL-15 heterodimer. We have generated DNA vectors expressing the heterodimeric IL-15 by optimizing mRNA expression and protein trafficking. Repeated administration of these DNA plasmids by intramuscular injection followed by in vivo electroporation in rhesus macaques resulted in sustained high levels of IL-15 in plasma, with no significant toxicity. Administration of DNAs expressing heterodimeric IL-15 also resulted in an increased frequency of NK and T cells undergoing proliferation in peripheral blood. Heterodimeric IL-15 led to preferential expansion of CD8(+)NK cells, all memory CD8(+) T-cell subsets and effector memory CD4(+) T cells. Expression of heterodimeric IL-15 by DNA delivery to the muscle is an efficient procedure to obtain high systemic levels of bioactive cytokine, without the toxicity linked to the high transient cytokine peak associated with protein injection.

Inhibition of primary murine macrophage cytokine production in vitro following treatment with the kappa-opioid agonist U50,488H.

Previous work in our laboratory has shown that both mu- and kappa-opioid agonists exhibit immunosuppressive activity for antibody responses in vitro. Our earlier work has suggested that both accessory cells and T cells may be altered following treatment with the kappa-opioid agonist U50,488H. We intend to further determine the identity of the immune cell population(s) which are affected by opioid treatment, and to determine the nature of the opioid receptor type expressed on these cells. In this study, non- elicited peritoneal macrophages were treated simultaneously with the kappa-agonist U50,488H and lipopolysaccharide (LPS), and the levels of the cytokines interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF)-alpha were determined. The results show that U50,488H had a suppressive effect on the production of TNF-alpha and IL-1 at concentrations as low as 1 nM, while IL-6 was suppressed at concentrations as low as 10 nM. Additional experiments utilizing the opiate antagonist naloxone and the kappa-selective antagonist norbinaltorphimine (norBNI) were performed in order to further characterize the opioid receptor involved in the cytokine suppression produced by treatment with U50,488H. Results showed that naloxone was able to partially block U50,488H suppression while norBNI was able to completely reverse the suppression of IL-6 production. These results suggest that macrophage/monocyte function is significantly modulated following activation of the kappa-opioid receptor.

Characterization of kappa-opioid receptor transcripts expressed by T cells and macrophages.

We have found that the immature T cell lines R1.1 and DPK and the macrophage lines P388D1 and WEHI-3 also express kappa-opioid receptor (KOR) mRNA. Characterization of the KOR transcripts in both brain tissue and these T cells has revealed both the normal full-length as well as a truncated form of the mRNA. Our results show that the truncated transcript lacks the second exon. Primary macrophages express this truncated form of the transcript in the absence of detectable levels of the full-length form. These results suggest a degree of heterogeneity in the expression of the opioid receptors which has not previously been reported.

Inhibition of interleukin-1 and tumor necrosis factor-alpha synthesis following treatment of macrophages with the kappa opioid agonist U50, 488H.

Previous reports from this laboratory, and others, have shown that exogenous mu and kappa opioids modulate both cellular and humoral immune responses. Our earlier work has suggested that accessory cells may serve as a target for the direct effects of kappa opioid compounds. In the present study, the function of the macrophage cell line P388D1 was modulated by the kappa-selective opioid agoinst U50,488H (trans-3,4-dichloro-N-methyl-N-[7-(1- pyrrolidinyl)cyclohexyl]benzene-acetamide methanesulfonate). Lipopolysaccharide-induced interleukin (IL)-1 and tumor necrosis factor-alpha production were inhibited after the administration of nanomolar concentrations of U50,488H. Furthermore, inhibition of IL-1 produced by the P388D1 cell line was reversed by both the classical opioid antagonist naloxone and by the kappa opioid receptor antagonist norbinaltorphimine. Examination of IL-1 mRNA levels in P388D1 by northern blot analysis showed that the inhibition mediated by U50, 488H apparently occurred at the level of transcription. On the other hand, U50,488H failed to modulate the production of IL-6 by this macrophage-like cell line. In addition, U50,488H failed to modulate the production of either IL-1 or tumor necrosis factor-alpha from the macrophage-like cell line RAW 264.7, an indication that subpopulations of macrophages exist with different sensitivities to opioids. These results are consistent with a growing body of data which suggests that a component of the inhibition mediated by opioid compounds involves a reduction in the production of cytokines.