Rahil T. Rahim

Effects of Opioid Tolerance and Withdrawal on the Immune System

Review of the robust literature using acute drug injection paradigms points clearly to the conclusion that morphine is immunosuppressive. In contrast, studies of the effect of subacute or chronic administration of morphine on immune function is limited, with variable results. In some cases tolerance to the immunosuppressive effects of the drug is clearly demonstrated, but in other cases, selected immune parameters do not demonstrate tolerance. Discrepancies in findings may result from differences in species or route and manner of drug administration. Even fewer studies (total of 10) have been published on the effects of withdrawal on immune function. Most immune parameters tested are suppressed following drug withdrawal. Recovery time to baseline response levels varies in the studies. In the single report of withdrawal in humans, immune function was suppressed for up to 3 years. It is clearly established that withdrawal suppresses capacity of murine spleen cells to make an ex vivo antibody response, which contrasts with evidence of polarization of the lymphocytes towards a Th2 phenotype. Several laboratories have shown that subacute and chronic exposure to morphine, as well as drug withdrawal, sensitize to the lethal effects of bacterial lipopolysaccharide. Underlying sepsis, combined with morphine-induced hypofunction of the hypothalamic-pituitaryadrenal (HPA) axis, may be occult variables modulating immune responses during opioid administration and withdrawal. As episodes of withdrawal are common among drug abusers, more intensive investigation is warranted on the effects of withdrawal on immune function, on mechanisms of immune modulation, and on sensitization to infection.

Abrupt or precipitated withdrawal from morphine induces immunosuppression

The present studies tested the effect of withdrawal from morphine by two different paradigms, abrupt withdrawal (AW) or precipitated withdrawal (PW), on the capacity of murine spleen cells to mount an in vitro antibody response. Mice were made dependent by chronic treatment using s.c. implanted morphine slow-release pellets. Splenocytes were harvested at various time points after withdrawal and the number of antibody-forming cells determined using a plaque-forming cell (PFC) assay. The results indicate that induction of abstinence from morphine in dependent mice by either paradigm caused marked immunosuppression between 24 and 48 h post-withdrawal. However, the kinetics of onset and recovery from immunosuppression were different in AW and PW.

Administration of mu-, kappa- or delta2-receptor agonists via osmotic minipumps suppresses murine splenic antibody responses.

Previously, our laboratory has shown that morphine given by implantation of a 75-mg slow-release pellet for 48 h suppresses murine splenic antibody responses to sheep red blood cells (SRBCs) in a plaque-forming cell (PFC) assay. However, the use of slow-release pellets for such studies is limited, as these pellets are only available in fixed doses and similar pellets for kappa and delta agonists have not been developed. In the present study, we investigated the feasibility of administering opioids via Alzet osmotic minipumps to assess their immunomodulatory effects. Groups of mice received minipumps dispensing morphine sulfate, which has primary activity at the mu opioid receptor; U50,488H, which is a kappa-selective agonist; deltorphin II, which is a delta2-selective agonist; or DPDPE, which has greater selectivity for delta1 than delta, receptors. Morphine, U50,488H and deltorphin II were all immunosuppressive, with biphasic dose-response curves exhibiting maximal (approximately 50%) suppression of the PFC response at doses of 0.5 to 2 mg/kg/day 48 h after pump implantation. Further, immunosuppression by morphine sulfate, U50,488H or deltorphin II was blocked by simultaneous implantation of a minipump administering the opioid receptor-selective antagonists CTAP (1 mg/kg/day), nor-binaltorphimine (5 mg/kg/day), or naltriben (3 mg/kg/day), respectively. DPDPE was inactive at doses lower than 10 mg/kg/day. We conclude that osmotic minipumps are a practical and useful way of administering opioids to study their effects on the immune system, and give further evidence that immunosuppression induced in vivo by opioid agonists is mediated not only via mu, but also via kappa and delta2 opioid receptors.

Withdrawal from morphine in mice suppresses splenic macrophage function, cytokine production, and costimulatory molecules.

We have previously shown that abstinence from morphine by either abrupt (AW) or precipitated (PW) withdrawal induces greater than 80% suppression in the capacity to mount an in vitro plaque-forming cell (PFC) response to sheep red blood cells at 24-h post withdrawal. Present studies on the mechanisms of immunosuppression showed that addition of normal unfractionated spleen cells, macrophage-enriched adherent cells, or CD11b(+) purified macrophages, to spleen cells taken from withdrawn mice, restored immune responses. Spleen cells from mice undergoing withdrawal also had decreased splenic mRNA and/or protein levels of IL-1beta, IL-1Ra, TNF-alpha, IL-12, and IFN-gamma. Addition of IL-1beta or IFN-gamma to AW cultures was able to reverse their immunosuppression. These results strongly suggest that morphine withdrawal results in a deficit of macrophage function.

Splenic macrophages and B cells mediate immunosuppression following abrupt withdrawal from morphine

We have previously shown that abrupt withdrawal (AW) from morphine induces greater than 80% immunosuppression in murine spleen cells, as assessed by the capacity to mount an in vitro plaque‐forming cell response to sheep red blood cells. Present studies about the mechanisms of immunosuppression following AW showed that addition of highly enriched (CD11b+) splenic macrophages (obtained by cell sorting or magnetic separation) from AW mice to cultures of normal, unfractionated spleen cells suppressed immune responses. Further, addition of highly enriched (CD19+) B cells (but not T cells) from AW mice to normal cells was also immunosuppressive. B cells from AW mice were also able to inhibit the proliferative response of normal spleen cells to concanavalin A but not to lipopolysaccharide. Overall, the data suggest that immunosuppression by AW spleen cells is a result of active suppression by macrophages and B cells.

Paradoxes of immunosuppression in mouse models of withdrawal

Previously, our laboratory showed that either abrupt (AW) or precipitated withdrawal (PW) from morphine led to profound suppression of murine splenic antibody responses to sheep red blood cells at 24 h post-withdrawal. In the present studies, we examined the immune mechanisms mediating suppression at that time point. A co-culture method was used to examine whether cells from withdrawn mice had (1) a deficit in function and/or (2) contained populations of suppressor cells. To examine the first hypothesis, cells from normal mice were co-cultured with cells from withdrawn mice in a 1:3 ratio (normal/withdrawn). To test the second hypothesis, the ratio was reversed. The results were paradoxical. Co-culture of cells in a 1:3 ratio showed that spleen cells from withdrawn mice had a deficit in macrophage function. Spleen cells from withdrawn mice also showed decreased mRNA levels of IL-1beta, IL-1-Ra, and TNF-alpha and a suppression of co-stimulatory molecule expression. To examine the second hypothesis, cells were co-cultured in a 3:1 ratio (normal/withdrawn). In this paradigm, spleen cells from abrupt withdrawn mice were shown to contain populations of both suppressor macrophages and B-cells. In vivo experiments carried out on mice 24 h post-withdrawal showed increased sensitivity to the lethal effects of LPS and increased production of TNF-alpha, implying a state of macrophage activation. Thus evidence for both suppressed and activated macrophages has been obtained in mice 24 h after abrupt withdrawal from morphine.

Protein Kinase Cζ Mediates μ-Opioid Receptor-induced Cross-desensitization of Chemokine Receptor CCR5

We have previously shown that the μ-opioid receptor (MOR) is capable of mediating cross-desensitization of several chemokine receptors including CCR5, but the biochemical mechanism of this process has not been fully elucidated. We have carried out a series of functional and biochemical studies and found that the mechanism of MOR-induced cross-desensitization of CCR5 involves the activation of PKCζ. Inhibition of PKCζ by its pseudosubstrate inhibitor, or its siRNA, or dominant negative mutants suppresses the cross-desensitization of CCR5. Our results further indicate that the activation of PKCζ is mediated through a pathway involving phosphoinositol-dependent kinase-1 (PDK1). In addition, activation of MOR elevates the phosphorylation level and kinase activity of PKCζ. The phosphorylation of PKCζ can be suppressed by a dominant negative mutant of PDK1. We observed that following MOR activation, the interaction between PKCζ and PDK1 is immediately increased based on the analysis of fluorescent resonance energy transfer in cells with the expression of PKCζ-YFP and PDK1-CFP. In addition, cells expressing PKCζ kinase motif mutants (Lys-281, Thr-410, Thr-560) fail to exhibit full MOR-induced desensitization of CCR5 activity. Taken together, we propose that upon DAMGO treatment, MOR activates PKCζ through a PDK1-dependent signaling pathway to induce CCR5 phosphorylation and desensitization. Because CCR5 is a highly proinflammatory receptor, and a critical coreceptor for HIV-1, these results may provide a novel approach for the development of specific therapeutic agents to treat patients with certain inflammatory diseases or AIDS.