Morphine-6beta-glucuronide modulates the expression of inducible nitric oxide synthase.

The immunomodulatory effects of morphine are well established; however, suprisingly little is known about the immunomodulatory properties of the major metabolites of morphine. The present study tests the hypothesis that expression of inducible nitric oxide synthase (iNOS) is modulated by the administration of the morphine metabolite, morphine-6beta-glucuronide. The initial study using rats shows that morphine-6beta-glucuronide administration (0, 1.0, 3.163, 10 mg/kg s.c.) results in a pronounced reduction in lipopolysaccharide (LPS)-induced expression of iNOS (inducible nitricoxide synthease) in spleen, lung, and liver tissue as measured by western blotting. Morphine-6beta-glucuronide also produces a reduction in the level of plasma nitrite/nitrate, the more stable end-product of nitric oxide degradation. In a subsequent study, administration of the opioid receptor antagonist, naltrexone (0.1 mg/kg) prior to the injection of morphine-6beta-glucuronide (10 mg/kg) blocks the morphine-6beta-glucuronide induced reduction of iNOS expression and plasma nitrite/nitrite levels indicating that the effect is mediated via the opioid-receptor. This study provides the first evidence that morphine-6beta-glucuronide alters the expression of iNOS.

Morphine-6 beta-glucuronide induces potent immunomodulation.

The effect of morphine administration on immune parameters is well documented. However, there exists a limited knowledge of the effect of morphine's metabolites on immune status. The present study examines the immunomodulatory effects of the morphine metabolite, morphine-6 beta-glucuronide (M6G), in the rat and provides further evaluation of the antinociceptive effects of M6G. Animals were administered phosphate-buffered saline (PBS) or M6G in doses of 1.0, 3.16, or 10.0 mg/kg (subcutaneous (s.c.)) or 0.1, 0.316, or 1.0 microgram (intracerebroventricular (i.c.v.)). Animals were tested for antinociception in the warm water tail-withdrawal procedure. In a separate set of animals, assessments of splenic natural killer cell activity, lymphocyte proliferative responses to mitogenic stimulation, and production of interferon-gamma were made 1 h following the s.c. or i.c.v. administration of M6G. The results show that M6G induced potent antinociception that was evident for at least 120 min following administration. M6G also produced decreases in natural killer cell activity, lymphocyte proliferation, and interferon-gamma production 1 h following both routes of administration. The difference in potency between immune alterations induced by subcutaneous vs. intracerebroventricular administration suggest central mediation of the immunomodulatory properties of M6G. Thus, M6G produces significant antinociception and immunomodulation in the rat. These findings demonstrate potent immunomodulatory properties of a metabolite of morphine, 1M6G.

Involvement of substance P and central opioid receptors in morphine modulation of the CHS response.

Morphine administration prior to challenge with the antigen 2,4-dinitro-fluorobenzene increases the contact hypersensitivity (CHS) response in rats. The present study extended these findings by showing that central, but not systemic, administration of N-methylnaltrexone antagonized the morphine-induced enhancement of the CHS response. The importance of the neuroimmune mediator substance P was shown via the attenuation of the morphine-induced enhancement following both systemic and topical administration of the NK-1 antagonist WIN51,708. Taken together, the findings of the present study provide new data showing that central opioid receptors and peripheral substance P are involved in the morphine-induced enhancement of the CHS response.

Morphine modulation of the contact hypersensitivity response: characterization of immunological changes.

Previous investigations in our laboratory showed that systemic morphine administration 1 h prior to elicitation of the in vivo contact hypersensitivity (CHS) response produced a robust increase in inflammation at the site of antigen reexposure. The present study extended those findings by characterizing the effect of morphine on immunological processes important in the development of the CHS response. To induce contact hypersensitivity, the antigen 2,4-dinitrofluorobenzene was applied to the pinnae of previously sensitized rats. Morphine administration produced an increase in inducible nitric oxide synthase mRNA and the proinflammatory cytokine interleukin-6, at the site of antigen reexposure. In contrast, morphine did not alter expression of the anti-inflammatory cytokine interleukin-10. Morphine also produced an increase in the proliferation of lymphocytes from the peripheral (i.e., cervical) lymph nodes when assessed 72 h following challenge. These studies show that the morphine-induced increase in the in vivo CHS response involves immunologically specific alterations.

Naltrexone administration attenuates surgery-induced immune alterations in rats.

Surgery is a commonly performed procedure which produces substantial alterations in immune function in both humans and animals. To better understand the mechanism of surgery-induced immunomodulation, the present study investigated the effect of the opioid antagonist naltrexone on surgery-induced immune alterations in rats. Based on previous investigations in our laboratory, rats underwent a 6-cm laparotomy with no internal manipulation and immunological assessments were completed 24 h following the surgical procedure. Naltrexone was administered at the time of surgery and every 4 h thereafter until immune assessment. Results showed that naltrexone attenuated the surgery-induced decrease in natural killer cell cytotoxicity, B-cell proliferation, T-cell proliferation, and production of the cytokine IFN-gamma. These results are among the first to show that pharmacological antagonism of opioid receptors can prevent deleterious immune changes in the postoperative state, suggesting a detrimental role of the endogenous opioids in surgical procedures.

Endomorphin-1 induces antinociception without immunomodulatory effects in the rat.

RATIONALE: Although there is evidence that central opioid receptors are involved in immunomodulation, it has been only recently that an endogenous agonist, designated endomorphin-1, possessing high selectivity and affinity for the mu opioid receptor has been identified. OBJECTIVE: The present study assesses the immunomodulatory effects of endomorphin- in the rat and provides further evaluation of the antinociceptive effects of endomorphin-1. METHODS: Rats were surgically implanted with cannulae directed at the lateral cerebral ventricle. Animals received vehicle or endomorphin-1 at doses of 31.63 or 56.23 microg (ICV) and were tested for antinociception in two different assays, the warm water tail withdrawal procedure and the hotplate assay. Additional studies assessed the effect of naltrexone on the antinociception produced by endomorphin-1 in both antinociceptive assessments. Assessments of immune status following endomorphin-1 treatment included measurements of splenic natural killer cell activity, production of interferon-y, and lymphocyte proliferative responses to mitogenic stimulation by Con-A, LPS, and the microbial superantigen, TSST-1. RESULTS: Endomorphin-1 induced significant and naltrexone reversible antinociception 30 and 60 min following drug administration, as measured by the hotplate assay and warm water tail withdrawal procedure. In marked contrast, endomorphin-1 did not produce immunomodulatory effects up to 120 min following ICV administration. CONCLUSIONS: Endomorphin-1 produces antinociception but does not induce immunomodulatory effects in the rat. These findings suggest that it is possible to develop therapeutic strategies for separating antinociception and immunomodulatory properties through the mu opioid receptor.

Involvement of central mu- but not delta- or kappa-opioid receptors in immunomodulation.

Studies completed in both humans and animals have shown that opioids have significant effects on the immune system via pharmacological interactions with the opioid receptor. However, the type of opioid receptor at which morphine binding produces changes in immune status has not been well characterized. To determine the type of opioid receptor involved in opioid-induced immune alterations, the present study assessed the effects of agonists selective for the mu-, delta-, and kappa-opioid receptors. The site of action (i.e., peripheral vs central) at which opioids produce immune changes was investigated by injecting the agonists directly into the left lateral ventricle of the brain. Specifically, Lewis rats received an intracerebroventricular administration of [d-Ala(2),N-Me-Phe(4), Gly-ol(5)]enkephalin (DAMGO), a mu-receptor selective agonist, [D-Pen(2,5)]enkephalin (DPDPE), a delta-opioid receptor agonist, or U69,593, a kappa-receptor agonist. Immune assessments completed 1 h following drug administration showed that the mu-receptor selective agonist DAMGO produced a dose-dependent decrease in natural killer cell activity and T-lymphocyte proliferation to the mitogen concanavalin A (Con A); no immunological changes were found following DPDPE or U69,593 treatment. Calculation of the number of white blood cells per sample showed no differences between rats treated with saline and rats treated with any of the selective agonists. Administration of the opioid antagonist N-methylnaltrexone prior to DAMGO treatment attenuated the DAMGO-induced changes in immune status. Results from the present study indicate that the immunomodulatory effects of opioids can be attributed to interactions with the mu-opioid receptor.

Heroin-induced alterations in leukocyte numbers and apoptosis in the rat spleen.

The present study assessed the effects of acute heroin treatment on the cellularity of the rat spleen and the rate of splenocyte death by necrosis or apoptosis. The results showed that 1 h after a single injection of heroin, the total number of leukocytes in the spleen was decreased in a dose-dependent manner. Prior injection of naltrexone completely blocked heroin's effect, and the heroin-induced decrease in splenic leukocytes was not associated with a heroin-induced increase in circulating leukocytes. A 1-h exposure to heroin did not increase levels of lactate dehydrogenase, a cytosolic enzyme, in supernatants of splenic mononuclear cells cultured for 45 min or 24 h, suggesting that heroin does not increase necrotic death in the spleen. In contrast, a 1-h heroin treatment did increase the percentage of Annexin V(+) cells in 0- and 24-h cultures of splenic mononuclear cells, indicating that heroin increases apoptotic death in the spleen. A 3-h exposure to heroin also produced a significant increase in apoptosis in the spleen. DNA fragmentation, a marker of cells in late stages of apoptosis, could not be detected in fresh splenocytes, but was evident in 24-h cultures of splenic mononuclear cells from saline- and heroin-treated rats. These results demonstrate that a single administration of heroin produces a decrease in the number of splenic leukocytes and an increase in the apoptotic death of splenic mononuclear cells.

Heroin modulates the expression of inducible nitric oxide synthase.

The use of heroin (diacetylmorphine) is associated with a high incidence of infectious disease, and the immunologic alterations responsible for heroin-induced changes in resistance to infection have not been well characterized. The present study tests the hypothesis that expression of inducible nitric oxide synthase (iNOS) is modulated by the administration of heroin. The initial study using rats showed that heroin administration (0, 0.01, 0.1, or 1.0 mg/kg s.c.) results in a pronounced reduction in lipopolysaccharide (LPS)-induced expression of iNOS mRNA in spleen, lung, and liver tissue as measured by RT-PCR. Heroin also produced a reduction in the level of plasma nitrite/nitrate, the more stable end-product of nitric oxide degradation. In a subsequent study, administration of the opioid receptor antagonist, naltrexone (0.1 mg/kg) prior to the injection of heroin (1.0 mg/kg) blocked the heroin-induced reduction of iNOS expression and plasma nitrite/nitrate levels indicating that the effect is mediated via the opioid-receptor. This study provides the first evidence that heroin induces an alteration of iNOS expression, and suggests that a reduction in nitric oxide production may be involved in the increased incidence of infectious diseases amongst heroin users.

Phenotypic and functional assessments of immune status in the rat spleen following acute heroin treatment.

Heroin use is associated with an increased incidence of several types of infections, including HIV. Yet few studies have assessed whether heroin produces pharmacological alterations of immune status that might contribute to the increased rate of infections amongst heroin users. The present study investigated whether a single administration of heroin to rats produces dose-dependent alterations in functional measures of immune status and in the distribution of leukocyte subsets in the spleen. The results showed that heroin produces a dose-dependent, naltrexone-reversible suppression of the concanavalin A-stimulated proliferation of T cells, lipopolysaccharide-stimulated proliferation of B cells, production of interferon-gamma and cytotoxicity of natural killer (NK) cells in the spleen. Heroin's suppressive effect on NK cell activity results in part from a heroin-induced decrease in the relative number of NKR-P1A(hi) CD3- NK cells in the spleen. Heroin also decreases the percent of a splenic granulocyte subset, the CD11b/c+ HIS48(hi) cells, whose function currently is unknown. In contrast, heroin does not alter relative numbers of CD4+ CD3+ T cells, CD8+ CD3+ T cells, CD45+ B cells, NKR-P1A(lo) CD3+ T cells, CD11b/c+ ED1+ (or CD11b/c+ HIS48-) monocytes/macrophages or CD11b/c+ ED1- (or CD11b/c+ HIS48+) total granulocytes in the spleen. Collectively, these findings demonstrate that heroin produces pharmacological effects on functional and phenotypic measures of immune status.

Immunomodulatory effects of morphine withdrawal in the rat are time dependent and reversible by clonidine.

RATIONALE: It is well established that opioids can modulate the immune status following both acute and chronic administration and that tolerance develops to some of these immunomodulatory effects. Few studies, however, have investigated opioid withdrawal-induced immunomodulation and the mechanism by which that process may be mediated. OBJECTIVES: The present study examines the immunomodulatory properties of morphine withdrawal alone and in the presence of the alpha(2)-adrenergic agonist, clonidine. METHODS: Rats drank a morphine solution for 20 days; withdrawal was induced on day 21 by replacing the morphine solution with plain tap water. Measurements of withdrawal-induced weight change and immunomodulation were obtained at several time points after withdrawal induction. Immune status was assessed by determining concanavalin A (Con-A), toxic shock syndrome toxin (TSST-1), and lipopolysaccharide (LPS)-stimulated splenocyte proliferation, splenic ConA-stimulated interferon (IFN)-gamma production, and splenic natural-killer (NK) cell activity. In a separate series of experiments, systemic injections of clonidine (0.001-0.01 mg/kg) were administered during a 12-h withdrawal episode and all measures of immune status were reassessed. RESULTS: Weight change was time dependent, with peak decreases in weight occurring 24 h following withdrawal induction. Rats also exhibited a time-dependent suppression of immune status in all assays except LPS-stimulated proliferation; immunomodulation was most evident 12 h following withdrawal induction. Clonidine dose dependently prevented withdrawal-induced suppression of Con-A and TSST-1-stimulated splenocyte proliferation, Con-A-stimulated splenocyte IFN-gamma production, and splenic NK cell activity. CONCLUSIONS: These findings demonstrate that opioid withdrawal significantly suppresses a subset of immune parameters and that these effects can be prevented by clonidine.

Enhancement of the contact hypersensitivity reaction by acute morphine administration at the elicitation phase.

The present study investigated the effects of morphine on the irritant contact sensitivity (ICS) and contact hypersensitivity (CHS) reaction. ICS was induced by croton oil application on the pinnae of naïve rats. Morphine injected prior to croton oil application did not affect the ICS response when assessed by measurements of pinnae thickness. CHS was induced by applying the antigen 2,4-dinitro-1-fluorobenzene (DNFB) to the pinnae of rats sensitized to DNFB. Rats received an injection of morphine prior to either initial antigen exposure (sensitization) or antigen reexposure (challenge). Morphine prior to challenge, but not sensitization, resulted in a pronounced enhancement of the CHS response as measured by pinna thickness. Quantitative PCR also showed increased IFN-gamma mRNA levels in the inflamed tissue of morphine-treated rats. Naltrexone blocked the morphine-induced enhancement of the CHS response. The differential effects of morphine suggest that opioids have a more pronounced effect on in vivo immune responses that involve immunological memory.

Phenotypic analysis of splenocyte subsets following acute morphine treatment in the rat.

Prior studies by our laboratory demonstrated that a single injection of morphine produces dose-dependent, naltrexone-reversible, suppressive effects in assays of mitogen-stimulated lymphocyte proliferation and natural killer (NK) cell cytotoxicity in the spleen. The present study used flow cytometry to assess directly whether acute morphine treatment produces these immune alterations by altering the leukocyte composition of the spleen. In agreement with our previous findings, morphine suppressed the concanavalin A-stimulated proliferation of T cells, lipopolysaccharide-stimulated proliferation of B cells, and NK cell cytotoxicity in the spleen. However, the same morphine treatment protocol did not alter the total number of splenic leukocytes, the percentage of live splenic leukocytes (as assessed by forward-scatter versus side-scatter histograms), or the relative number of CD4(+)CD3(+) T cells, CD8(+)CD3(+) T cells, CD45RA/B(+) B cells, NKR-P1A(hi)CD3(-) NK cells, NKR-P1A(lo)CD3(+) T cells, CD11b/c(+)HIS48(-) monocytes/macrophages, or CD11b/c(+)HIS48(+) granulocytes in the spleen. These findings indicate that the effects of a single sc dose of morphine on functional measures of immune status in the spleen do not result from a redistribution of splenic leukocytes; instead, morphine's effects likely result from direct alterations in leukocyte activities.

Differences in NK cell function in mice bred for high and low aggression: genetic linkage between complex behavioral and immunological traits?

In previous studies, we found differences in cellular immune responsiveness in Institute for Cancer Research (ICR) mice selectively bred for high and low levels of aggression. Compared to the high aggressive line, the low aggressive line had low levels of natural killer (NK) and T cell activity and increased susceptibility to tumor development. To dissect further this novel association, experiments were designed to test two competing hypotheses. The first hypothesis was that the phenotypic expression of the line differences in NK cell activity are dependent on and regulated by the expression of high and low levels aggressive behavior in the lines. The alternative hypothesis was that the differences in immune status are independent of the expression of aggression by the lines, suggesting linkage between a subset of genes involved in determining these complex behavioral and immunological traits or pleiotropic gene effects on both traits. In Experiment 1, three conditions of postweaning social experience (mice singly housed, group housed within line, or group housed between lines) were tested in males to determine whether experiential conditions which modify the expression of aggression would in turn modify the line differences in NK cell activity. This experiment revealed that the difference in NK cell activity between high aggressive and low aggressive male mice was attributable to line only. The different postweaning social conditions examined had no effect on modifying the differences in NK activity, and social dominance hierarchy did not correlate with levels of NK cell activity. Whereas males of the high and low lines exhibit differences in aggressive behaviors across most contexts, females do not exhibit such differences except in response to an intruder during the postpartum period. Therefore, in Experiment 2 we compared the NK cell activity of nulliparous females of the high and low aggressive lines. Under these conditions, females of the low aggressive line had low levels of NK activity compared to high aggressive females (differences comparable to those seen between males of the high and low lines). Taken together, these experiments lend support to the hypothesis that this association may be due to a genetic linkage between subsets of genes involved in determining these complex behavioral and immunological traits, or may possibly represent a fortuitous association which occurred during the selective breeding.

Endogenous opioids regulate the expression of inducible nitric oxide synthase by splenocytes.

In the present study, we tested the hypothesis that lipopolysaccharide (LPS)-induced expression of nitric oxide synthase (iNOS) by splenocytes is modulated through the activation of endogenous opioids in the central nervous system. The initial studies determined the parameters of LPS-induced expression of iNOS by splenocytes. Rats were injected with LPS at doses of 0, 1, 10, 100, and 1000 microg/kg, and measures of both iNOS mRNA and protein showed a dose-dependent increase in expression. In a time course study, rats received 100 microg/kg LPS and were killed at 0, 2, 4, 8, and 16 h postinjection. Both iNOS mRNA and protein expression was detectable at the 2-h time point, with peak expression occurring at 8 h. To evaluate the involvement of endogenous opioids, the opioid receptor antagonist naltrexone was administered at 0, 0.1, 1, or 10 mg/kg s.c. in combination with LPS (100 microg/kg), with a second injection of naltrexone at the same dose 4 h after the injection of LPS. Naltrexone induced a pronounced dose-dependent reduction in iNOS mRNA and protein expression by splenocytes. The modulation of iNOS expression occurs via central opioid receptors as intracerebroventricular administration but not peripheral administration of N-methylnaltrexone, the quaternary form of naltrexone that does not readily cross the blood-brain barrier, reduced the expression of iNOS. For all of the manipulations, nitrite/nitrate levels in the plasma showed effects similar to those for iNOS mRNA and protein. Collectively, these findings indicate that central opioid receptors are involved in the in vivo regulation of splenic nitric oxide production.

Role of central mu-opioid receptors in the modulation of nitric oxide production by splenocytes.

Previous studies have shown that administration of morphine results in alterations of splenic macrophage nitric oxide production. The present studies were conducted to determine the subtype of opioid receptor involved in the modulation of macrophage nitric oxide production. Moreover, the present work was directed at determining whether nitric oxide production is regulated through opioid receptors in the central nervous system (CNS) or via opioid receptors found directly on splenocytes. The study shows that intracerebroventricular (i.c.v.) administration of the mu-selective opioid agonist, DAMGO, to rats dose-dependently increases the production of nitric oxide by splenocytes stimulated with toxic shock syndrome toxin (TSST-1). The effect of DAMGO is blocked by prior i.c.v. administration of N-methylnaltrexone. In contrast, i.c.v. administration of the kappa-selective agonist, U69,593, and the delta-selective agonist, DPDPE, have no significant effect on the production of nitric oxide. Furthermore, the in vitro administration of DAMGO, DPDPE, or U69,593 to splenocytes cultures does not significantly alter the production of nitric oxide by splenocytes. In addition, the present work shows that elevation of nitric oxide production by i.c.v. administration of DAMGO produces functional changes in splenic lymphocytes. Collectively, these results indicate that mu-opioid receptors within the CNS are involved in the regulation of splenic nitric oxide production.

Severity, time, and beta-adrenergic receptor involvement in surgery-induced immune alterations.

Although investigations of surgical stress in animals have reported immune alterations, surprisingly little is known about the variables or mechanisms contributing to the effect. Thus, we completed a series of experiments investigating the immune-altering effects of surgery severity, time of maximal immune alterations, and recovery, as well as the involvement of beta-adrenergic receptors in surgery-induced immune alterations in Lewis rats. Immune alterations included natural killer (NK) cell cytotoxicity as well as B- and T-cell proliferation. Results showed increased immune suppression with larger incisions (6 cm > 3 cm > anesthesia > saline). In addition, maximal immune alterations induced by surgery occurred after 24 h; anesthesia effects predominated at the earlier time points. Recovery of immune status varied depending on the immunological measure of interest. Although NK cell cytotoxicity returned to control values within 2 days, B-cell proliferation remained suppressed for at least 8 days, and T-cell proliferation did not begin to recover until 4-8 days following the surgical procedure. To assess the mechanisms involved in surgery-induced immune alterations, follow-up assessments evaluated the effect of nadolol, a beta-adrenergic receptor antagonist, on surgery-induced immune alterations. Results show that nadolol blocks the surgery-induced reduction in B- and T-cell proliferation but has no effect on the suppression of NK cell cytotoxicity. These results indicate the need to consider surgical severity and postoperative time of immune assessment when investigating the immune-altering effects of surgery. Importantly, activation of beta-adrenergic receptors appears to play a modulatory role in surgery-induced immune alterations.

Differential tolerance to morphine's immunomodulatory effects following continuous administration.

Rats were continuously infused with either morphine or saline via an osmotic minipump for 20 consecutive days. Effects on immune status were assessed on the twentieth day of the chronic administration period following a bolus injection of morphine administered 1 h prior to sacrifice. The morphine injection suppressed measures of splenic natural killer (NK) cell activity, mitogen-stimulated T-cell proliferation, and gamma-interferon (IFN) production in rats that received saline via the minipump. In rats that received chronic morphine via the minipump, the morphine injection also suppressed mitogen-stimulated splenocyte proliferation and gamma-IFN production but did not suppress NK cell activity. These data indicate that chronic morphine administration via osmotic minipumps leads to differential tolerance to the immunomodulatory effects of morphine. These findings support previous results indicating differential tolerance development within the immune system following chronic morphine administration via the drinking water.

Conditioned immunomodulation: investigations of the role of endogenous activity at mu, kappa, and delta opioid receptor subtypes.

The present investigations were designed to determine the role of activity at mu, kappa, and delta opioid receptor subtypes in conditioned immunomodulation by evaluating the effects of selective opioid receptor antagonists on conditioned stimulus-induced alterations in immune status. Lewis rats were exposed to an aversive conditioned stimulus that was developed through pairings with electric footshock. This aversive conditioned stimulus induces a reduction in splenic natural killer cell activity, splenocyte proliferation in response to mitogens, and diminished levels of interferon-gamma (IFN-gamma) production by splenocytes. Intracerebroventricular (i.c.v.) administration of the opioid antagonist naltrexone or the mu 1-selective antagonist naloxonazine blocked conditioned alterations of immune status, indicating that activity at mu-opioid receptors is involved in conditioned immunomodulation. Further support for the involvement of mu-opioid receptors within the central nervous system is provided by data showing that peripheral administration of naloxonazine, at doses shown to be effective when administered i.c.v., had no effect on conditioned alterations of immune status. Ventricular administration of the kappa receptor antagonist nor-binaltorphimine (nor-BNI) did not antagonize the immunomodulatory effects of the conditioned stimulus. Administration of the delta receptor antagonist naltrindole also did not antagonize the conditioned alterations of immune status. Collectively, the results of this study indicate that the alterations of immune status produced by an aversive conditioned stimulus require activity at mu-opioid receptors, possibly mu 1, within the central nervous system.