Toby K. Eisenstein

Opioids, opioid receptors, and the immune response

It is now clear that opioid receptors participate in the function of the cells of the immune system, and evidence suggests that opioids modulate both innate and acquired immune responses. We review literature here which establishes that mu-, kappa-, and delta-opioid compounds alter resistance to a variety of infectious agents, including the Human Immunodeficiency Virus (HIV). The nature of the immunomodulatory activity of the opioids has been the subject of a great deal of research over the last ten years. There is increasing evidence that effects of opioids on the immune response are mediated at several levels. Modulation of the inflammatory response appears to be a target of these compounds, including effects on phagocytic activity, as well as the response of cells to various chemoattractant molecules. Moreover, findings from several laboratories have demonstrated the impact of opioid treatment on antibody responses, and the molecular basis for this effect is likely due, at least in part, to the modulation of both cytokine and cytokine receptor expression. Future research should provide a clearer understanding of the cellular and molecular targets of opioid action within the immune system.

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 treatment in vitro or in vivo decreases phagocytic functions of murine macrophages

Studies were performed to compare in vitro and in vivo effects of morphine on the phagocytic function of murine peritoneal macrophages. Macrophage monolayers were incubated with Candida albicans for 30 min in the absence of autologous serum. Morphine added in vitro was found to decrease both the phagocytic activity (percent of phagocytic cells) and the phagocytic index (average number of ingested yeasts per cell) in a concentration-dependent manner, with maximal effects of 26% and 41%, respectively, at 10(-6) M. When morphine was administered in vivo via an implanted 75-mg pellet, there was a 22% decrease in phagocytic activity and a 40% decrease in the phagocytic index. Naltrexone completely blocked the effects of morphine both in vitro and in vivo. The results suggest that morphine is capable of interacting directly with opioid receptors on macrophages, resulting in a decrease in phagocytic function.

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.

Win 55212-2, a cannabinoid receptor agonist, attenuates leukocyte/endothelial interactions in an experimental autoimmune encephalomyelitis model

Multiple sclerosis (MS) is the most common of the immune demyelinating disorders of the central nervous system (C NS). Leukocyte/endothelial interactions are important steps in the progression of the disease and substances that interfere with these activities have been evaluated as potential therapeutic agents. C annabinoid receptor agonists have been shown to downregulate immune responses and there is preliminary evidence that they may slow the progress of MS. The purpo se of this investigation was to determine how cannabinoid recepto r agonists interfere with leukocyte rolling and adhesion. This was investigated in an experimental autoimmune encephalo myelitis (EAE) model using six to eight week old C 57BL/6 mice. Mouse myelin oligodendrocyte protein and pertussis toxin were used to induce EAE. WIN 55212-2, C B1 and C B2 antagonist were given. By use of in vivo intravital microscopy, leukocyte/endothelial interactio ns were evaluated via a cranial window implanted two days before. The results demonstrated that EAE increases leukocyte rolling and firm adhesion in the brain, and that this increased leukocyte/endothelial interactio n can be attenuated by administration of WIN 55212-2. Furthermore, use of the selective antagonists for the C B1 recepto r (SR 141716A) and the C B2 receptor (SR144528) in this study demonstrated that the cannabinoid’s inhibitory effects on leukocyte/endothelial interactions can be mediated by activating C B2 receptor.

Differential Effects of Morphine and Naltrexone on the Antibody Response in Various Mouse Strains

Morphine treatment has been shown to suppress several immunologic parameters. In this study, we examined the effects of morphine pellet implantation in vivo on the primary antibody response measured in vitro in various mouse strains. Effects of mouse strain and sex on morphine-induced suppression of the plaque-forming cell response, as well as spleen weight and mortality were determined. Morphine suppressed the primary antibody response in C3HeB/FeJ, C3H/HeJ and C57Bl/6 mice, while Balb/cByJ and the mu-receptor-deficient strain CxBk/ByJ mice were not affected. There was no difference in the response to morphine between male and female C3HeB/FeJ mice. Naltrexone reversed the morphine-induced suppression in the C3H strains, but not in C57Bl/6 mice. In addition, naltrexone caused significant mortality in Balb/cByJ mice. Spleen weight was decreased by morphine treatment in all the strains, but only the C3H strains were sensitive to the lethal effects of morphine. Thus, immune suppression did not correlate with splenic atrophy or mortality. The strain differences in response to chronic morphine and naltrexone treatment suggest that morphine may be acting through both opioid and non-classical opioid (e.g., not blocked by naltrexone) mechanisms.

Inhibition of primary murine macrophage cytokine production in vitro following treatment with the K-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.

μ-Opioid Induction of Monocyte Chemoattractant Protein-1, RANTES, and IFN-γ-Inducible Protein-10 Expression in Human Peripheral Blood Mononuclear Cells

Strong evidence for the direct modulation of the immune system by opioids is well documented. μ-Opioids have been shown to alter the release of cytokines important for both host defense and the inflammatory response. Proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-γ-inducible protein-10 (IP-10) play crucial roles in cell-mediated immune responses, proinflammatory reactions, and viral infections. In this report, we show that [d-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO), a μ-opioid-selective agonist, augments the expression in human PBMCs of MCP-1, RANTES, and IP-10 at both the mRNA and protein levels. Because of the proposed relationship between opioid abuse and HIV-1 infection, we also examined the impact of DAMGO on chemokine expression in HIV-infected cells. Our results show that DAMGO administration induces a significant increase in RANTES and IP-10 expression, while MCP-1 protein levels remain unaffected in PBMCs infected with the HIV-1 strain. In contrast, we show a dichotomous effect of DAMGO treatment on IP-10 protein levels expressed by T- and M-tropic HIV-infected PBMCs. The differential modulation of chemokine expression in T- and M-tropic HIV-1-infected PBMCs by opioids supports a detrimental role for opioids during HIV-1 infection. Modulation of chemokine expression may enhance trafficking of potential noninfected target cells to the site of active infection, thus directly contributing to HIV-1 replication and disease progression to AIDS.

Morphine enhances interleukin-12 and the production of other pro-inflammatory cytokines in mouse peritoneal macrophages

In this study we investigated the capacity of morphine to modulate expression of cytokines in peritoneal macrophages. Mice were implanted subcutaneously with a 75‐mg morphine slow‐release pellet, and 48 h later resident peritoneal macrophages were harvested. Control groups received placebo pellets, naltrexone pellets, or morphine plus naltrexone pellets. Adherent cells were stimulated with lipopolysaccharide (LPS: 10 μg/mL) plus interferon‐γ (IFN‐γ: 100 units/mL) to induce cytokine production. After 24 h RNA was extracted for analysis of cytokine mRNA levels by reverse transcriptase‐polymerase chain reaction, or supernatants were collected after 48 h for determination of cytokine production by enzyme‐linked immunosorbent assay (ELISA). Morphine enhanced mRNA expression of interleukin (IL)‐12 p40 and tumor necrosis factor α (TNF‐α) compared with controls, whereas IL‐10 levels were unchanged by drug treatment. ELISA data showed that both IL‐12 p40 and p70 were increased by morphine. The enhancement of IL‐12 at both the mRNA and protein levels was antagonized by naltrexone, indicating that the modulation of this cytokine by morphine is via a classic opioid receptor. These results are particularly interesting in light of our previous observation that 48 h after morphine pellet implantation, the peritoneal cavity is colonized with gram‐negative and other enteric bacteria. The enhancement of IL‐12 by morphine might be related to morphine‐induced sepsis.

Immunity to Salmonella Infections

The foregoing literature review and data presentation have been set forth in the hope of clarifying some complex and confusing issues in regard to Salmonella infection. From a practical point of view, the information presented has implications for the direction to take with regard to improving the current typhoid vaccine, as the presently used acetone-killed cell preparation has considerable toxicity. The issues are important from a theoretical standpoint, because they have bearing on the nature of the concepts researchers and clinicians carry as working hypothesis with regard to the mechanisms of immunity to Salmonella infection. An incomplete appreciation of the literature seems to have led many scientists to believe that only cellular immunity can protect a mouse, and by analogy a human, against Salmonella. The logical deduction from such a premise is that only live vaccines will be effective in humans againsT S. typhi. Such a conclusion would appear unfounded, as documented in this review, for killed vaccines have been shown to be highly effective in vaccinating many mouse strains, as well as humans, against enteric fever.