Pharmacology and intracellular signaling mechanisms of the native human orphan receptor BRS-3 in lung cancer cells.

Neither the native ligand nor the cell biology of the bombesin (Bn)-related orphan receptor subtype 3 (BRS-3) is known. In this study, we used RT-PCR to identify two human lung cancer lines that contain sufficient numbers of native hBRS-3 to allow study: NCI-N417 and NCI-H720. In both cell lines, [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) stimulates [3H]inositol phosphate. In NCI-N417 cells, binding of 125I-[DTyr6,betaAla11,Phe13,Nle14]Bn(6-14) was saturable and high-affinity. [DPhe6,betaAla11,Phe13,Nle14]Bn(6-14) stimulated phospholipase D activity and a concentration-dependent release of [3H]inositol phosphate (EC50 = 25 nM) and intracellular calcium (EC50 = 14 nM); the increases in intracellular calcium were primarily from intracellular stores. hBRS-3 activation was not coupled to changes in adenylate cyclase activity, [3H]-thymidine incorporation or cell proliferation. No naturally occurring Bn-related peptides bound or activated the hBRS-3 with high affinity. Four different bombesin receptor antagonists inhibited increases in [3H]inositol phosphate. Using cytosensor microphysiometry, we found that [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) caused concentration-dependent acidification. The results show that native hBRS-3 receptors couple to phospholipases C and D but not to adenylate cyclase and that they stimulate mobilization of intracellular calcium and increase metabolism but not growth. The discovery of human cell lines with native, functional BRS-3 receptors, of new leads for a more hBRS-3-specific antagonist and of the validity of microphysiometry as an assay has yielded important tools that can be used for the identification of a native ligand for hBRS-3 and for the characterization of BRS-3-mediated biological responses.

Opioid modulation of immune responses: effects on phagocyte and lymphoid cell populations.

The literature describing effects of morphine on cells of the immune system points to the clear conclusion that morphine given in vivo suppresses a variety of immune responses that involve the major cell types in the immune system, including natural killer (NK) cells, T cells, B cells, macrophages and polymorphonuclear leukocytes (PMNs). Depression of NK cell activity has been reported in humans, monkeys and rodents. Similarly, responses of T cells are depressed by morphine, as assessed by inhibition of induction of delayed-type hypersensitivity reactions and cytotoxic T-cell activity, modulation of T-cell antigen expression, and depression of responses to T-cell mitogens. Effects on T cells have been reported in humans, monkeys and rodents. Effects of morphine on B-cell activity have mainly been tested in rodents using assays of antibody formation, which also require macrophages and T cells, preventing a conclusion as to the cell type being affected. Consistent effects on phagocytic cell function have been reported in rodents given morphine. In contrast, studies on immunomodulatory effects of morphine added to cells of the immune system in vitro have shown robust effects on some of these cell types, but not others. There is a rich literature demonstrating downregulation of phagocytic cell function by morphine, particularly for human peripheral blood mononuclear cells (PBMCs) and PMNs. Phagocytosis, chemotactic responses, interleukin production, and generation of activated oxygen intermediates and arachidonic acid products have all been reported to be inhibited. On the contrary, the literature does not support direct effects of morphine on NK cell function, is inconclusive concerning effects on B cells, and provides limited evidence for effects on T cells. The divergence between the in vivo and in vitro data suggests that effects on some cells in the immune system observed after in vivo morphine are probably not direct, but mediated. In aggregate, the literature supports the existence of an in vivo neural-immune circuit through which morphine acts to depress the function of all cells of the immune system. Further, there is strong evidence that morphine can directly depress the function of macrophages and PMNs, and modulate expression of one type of T-cell surface marker. There is, however, little evidence for direct effects of morphine on NK cells and B cells. A further complication emerges from reports of immunopotentiation of immune function in in vitro assays using endogenous opioids. The possibility of different receptors for endogenous and exogenous opioids or of interactions among the activated opioid receptors may account for these opposing effects.

Morphine alters macrophage and lymphocyte populations in the spleen and peritoneal cavity.

We have previously shown that subcutaneous implantation of a 75 mg morphine pellet results in suppression of the ability of murine splenocytes to mount an antibody response to sheep red blood cells, due in part to a reduction of macrophage function. The present studies used flow cytometry to examine whether the decrement in macrophage function in the spleens of morphine-treated mice results from a reduction in macrophage numbers. Parallel analysis was carried out on non-elicited peritoneal cells. In the spleen, morphine resulted in a reduction in the relative proportion of macrophages and B-cells, with a concomitant increase in the proportion of T-cells. Alteration in the ratio of CD4+ to CD8+ T-cells was not observed. In contrast, in the peritoneal cavity, morphine increased the number of macrophages and reduced the number of B-cells. Naltrexone blocked all of the changes in cellular composition. These results support the conclusion that an important mechanism in the immunosuppression seen in the spleens of mice implanted with morphine pellets is a differential reduction in the number of macrophages and B-cells as compared with T-cells. Further, these studies show that subsets of cells of the immune system are differentially affected by morphine in different anatomical compartments.

Morphine induces sepsis in mice.

Gram-negative sepsis and subsequent endotoxic shock remain major health problems in the United States. The present study examined the role of morphine in inducing sepsis. Mice administered morphine by the subcutaneous implantation of a slow-release pellet developed colonization of the liver, spleen, and peritoneal cavity with gram-negative and other enteric bacteria. In addition, the mice became hypersusceptible to sublethal endotoxin challenge. The effects were blocked by the simultaneous implantation of a pellet containing the opioid antagonist naltrexone. These findings show that morphine pellet implantation in mice results in the escape of gram-negative organisms from the gastrointestinal tract, leading to the hypothesis that morphine used postoperatively or chronically for analgesia may serve as a cofactor in the precipitation of sepsis and shock. In addition, morphine-induced sepsis may provide a physiologically relevant model of gram-negative sepsis and endotoxic shock.

Antigen presentation by macrophages from bacille Calmette-Guérin (BCG)-resistant and -susceptible mice.

We have compared the antigen-presenting capacity of macrophages from congenic BALB/c.Bcgr and BALB/c.Bcgs mice that differentially express MHC class II glycoproteins. Several different criteria were used to evaluate the presentation of a protein antigen, ovalbumin (OVA), including limiting the concentration of antigen or the numbers of macrophages, and using both native OVA and OVA peptide 323-339. No differences in the capacity of macrophages from Bcgr and Bcgs mice to present antigen to a OVA-specific T cell hybridoma were found. Splenic macrophages from BCG-infected congenic mice also induced an equivalent amount of IL-2 production by the T cell hybridoma. The relationship of these findings to other differences that have been attributed to Bcg are discussed.