Joseph J. Meissler

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.

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.

Morphine Increases Susceptibility to Oral Salmonella typhimurium Infection

This study examined the effect of morphine on oral infection with virulent Salmonella typhimurium. Animals were treated with a 75-mg slow-release morphine pellet followed by inoculation with salmonellae. Morphine markedly sensitized mice to oral infection, as assessed by survival, mean survival time, and colony culture. By 24 h after Salmonella inoculation, morphine-treated mice had a 105-fold difference in number of organisms in the Peyers patches, compared with controls. The opioid antagonist naltrexone significantly blocked Salmonella colonization in Peyers patches and reduced Salmonella burden in other organs, indicating that morphine acts at least in part via an opioid receptor-mediated pathway. The data show that morphine markedly potentiates Salmonella infection at the gastrointestinal portal of entry and enhances subsequent dissemination of Salmonella organisms. The results have implications for potentiating gastrointestinal opportunistic infections in intravenous drug abusers and in opioid-medicated postsurgical patients.

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.

Innate immune responses to systemic Acinetobacter baumannii infection in mice: neutrophils, but not interleukin-17, mediate host resistance.

ABSTRACT Acinetobacter baumannii is a nosocomial pathogen with a high prevalence of multiple-drug-resistant strains, causing pneumonia and sepsis. The current studies further develop a systemic mouse model of this infection and characterize selected innate immune responses to the organism. Five clinical isolates, with various degrees of antibiotic resistance, were assessed for virulence in two mouse strains, and between male and female mice, using intraperitoneal infection. A nearly 1,000-fold difference in virulence was found between bacterial strains, but no significant differences between sexes or mouse strains were observed. It was found that microbes disseminated rapidly from the peritoneal cavity to the lung and spleen, where they replicated. A persistent septic state was observed. The infection progressed rapidly, with mortality between 36 and 48 h. Depletion of neutrophils with antibody to Ly-6G decreased mean time to death and increased mortality. Interleukin-17 (IL-17) promotes the response of neutrophils by inducing production of the chemokine keratinocyte-derived chemoattractant (KC/CXCL1), the mouse homolog of human IL-8. Acinetobacter infection resulted in biphasic increases in both IL-17 and KC/CXCL1. Depletion of neither IL-17 nor KC/CXCL1, using specific antibodies, resulted in a difference in bacterial burdens in organs of infected mice at 10 h postinfection. Comparison of bacterial burdens between IL-17a −/− and wild-type mice confirmed that the absence of this cytokine did not sensitize mice to Acinetobacter infection. These studies definitely demonstrate the importance of neutrophils in resistance to systemic Acinetobacter infection. However, neither IL-17 nor KC/CXCL1 alone is required for effective host defense to systemic infection with this organism.

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.

Decreases in macrophage mediated antitumor activity with aging

We have demonstrated that immunotherapy of young (6-10 weeks old), and aged, (greater than 24 months old), tumor bearing mice with biological response modifiers enhanced survival and inhibited tumor growth, while treatment of aged mice had little or no effect. We hypothesized that the antitumor activity in young mice was principally mediated by activated macrophages (M phi) and predicted that the change in aged mice was caused by an intrinsic M phi defect which develops with advancing age. To directly test our hypothesis, we examined the antitumor activity of resident peritoneal M phi, purified and activated in vitro with IFN gamma plus LPS. Paralleling the results seen in vivo, M phi from aged mice exhibited reduced antitumor activity in comparison with M phi from younger mice. Moreover, there was reduced capacity of in vitro activated M phi from aged mice to produce TNF, IL-1 and nitric oxide, which are critical monokines and effector molecules that have been established to either directly inhibit tumor growth or cause tumor cell destruction. These studies establish that peritoneal M phi from aged mice have an intrinsic defect which prevents them from fully expressing their antitumor potential.

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.

Immunosuppression to Tetanus Toxoid Induced by Implanted Morphine Pellets

There is considerable interest in the effects of drugs of abuse on immune responsiveness. Few studies are reported in the literature examining the immunomodulatory effect of opioids administered in vivo. Demonstration of in vivo alterations in immune competence by a drug in an animal model is the closest we can come, in a controlled experiment, to evaluating the effects of that drug on immune responses in humans. Given the fact that there is feedback between the neural, endocrine, and immune systems, in vivo drug administration allows all of the potential complex interactions of the products of these systems to occur. We have used the standard pharmacological technique of subcutaneous implantation of morphine pellets and examined the effects on serum antibody responses of mice to an important bacterial antigen, tetanus toxoid. Female mice of the C3HeB/FeJ strain (21 to 23 g) were obtained from Jackson Laboratories (Bar Harbor, ME). Animals were divided into two cohorts, each containing a group of 5 morphine-treated mice and a group of 5 placebo mice. Mice were bled from the retroorbital plexus to obtain “preimmune” serum, which was pooled by group. Drugs were administered S.C. by dorsal implantation of a single morphine pellet (75 mg) or a single placebo pellet under ether anesthesia. Animals were divided into two cohorts, each containing a group of morphine-treated and a group of placebo mice. One cohort was administered highly purified tetanus toxoid (10 pg) i.p. in a volume of 0.5 ml, 24 h after pellet implantation. The second cohort received the tetanus toxoid 72 h after drug or placebo pellet implantation. Each cohort was boosted 7 days after the respective primary immunization with a second i.p. injection of 10 pg of tetanus toxoid. Seven days after the booster injection, mice in each cohort were bled by cardiacpuncture under pentobarbital anesthesia and the anti-tetanus toxoid antibody titer determined for each individual serum by a solidphase ELISA (enzyme-linked immunosorbant assay). In this system, we measured the secondary response to tetanus toxoid, because the primary injection, even in control animals, does not result in a detectable antibody response to this antigen in its purified form. For the assay, tetanus toxoid (courtesy of Wyeth Laboratories, Marietta, PA) was coated onto 96-well plates at 1 pgiml following a modification of the procedure of