Morphine compromises bronchial epithelial TLR2/IL17R signaling crosstalk, necessary for lung IL17 homeostasis.

Opportunistic lung infection and inflammation is a hallmark of chronic recreational/clinical use of morphine. We show that early induction of IL17 from the bronchial epithelium, following pathogenic encounter is a protective response, which contributes to pathogenic clearance and currently attributed to TLR2 activation in immune cells. Concurrent activation of TLR2 and IL17R in bronchial epithelium results in the sequestration of MyD88 (TLR2 adapter) by Act1/CIKS (IL17R adapter), thereby turning off TLR2 signaling to restore homeostasis. Morphine inhibits the early IL17 release and interaction between Act1 and MyD88, leading to decreased pathogenic clearance and sustained inflammation. Hence, we propose that therapeutically targeting either TLR2 or IL17 in bronchial epithelia, in the context of morphine, can restore inflammatory homeostasis.

Morphine inhibits migration of tumor-infiltrating leukocytes and suppresses angiogenesis associated with tumor growth in mice.

Tumor cells secrete factors that stimulate the migration of peripheral blood leukocytes and enhance tumor progression by affecting angiogenesis. In these studies, we investigated the effect of morphine, a known immunosuppressant, on leukocyte migration and recruitment to conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells. Our results indicate that morphine treatment reduced the migration and recruitment of tumor-infiltrating leukocytes into Matrigel plugs and polyvinyl alcohol sponges containing conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells when compared with placebo. A reciprocal increase in peripheral blood leukocytes was observed at the time of plug or sponge removal in morphine-treated mice. Decreased angiogenesis was observed in conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells Matrigel plugs taken from morphine-treated wild-type mice when compared with placebo but was abolished in morphine-treated µ-opioid receptor knockout mice. In addition, in vitro studies using trans-well and electric cell substrate impedance sensing system studies reveal for the first time morphine's inhibitory effects on leukocyte migration and their ability to transmigrate across an activated endothelial monolayer. Taken together, these studies indicate that morphine treatment can potentially decrease leukocyte transendothelial migration and reduce angiogenesis associated with tumor growth. The use of morphine for cancer pain management may be beneficial through its effects on angiogenesis.

Morphine induced exacerbation of sepsis is mediated by tempering endotoxin tolerance through modulation of miR-146a.

Development of tolerance to endotoxin prevents sustained hyper inflammation during systemic infections. Here we report for the first time that chronic morphine treatment tempers endotoxin tolerance resulting in persistent inflammation, septicemia and septic shock. Morphine was found to down-regulate endotoxin/LPS induced miR-146a and 155 in macrophages. However, only miR-146a over expression, but not miR-155 abrogates morphine mediated hyper-inflammation. Conversely, antagonizing miR-146a (but not miR-155) heightened the severity of morphine-mediated hyper-inflammation. These results suggest that miR-146a acts as a molecular switch controlling hyper-inflammation in clinical and/or recreational use of morphine.

Morphine modulation of toll-like receptors in microglial cells potentiates neuropathogenesis in a HIV-1 model of coinfection with pneumococcal pneumoniae.

Chronic drug users account for a third of all cases of AIDS in the United States and the progression to AIDS dementia is accelerated in opiate drug abusers. Clinically, microglial activation better correlates with HIV-associated neurocognitive disorders (HAND) than productive HIV-1 infection in the CNS. Moreover, pneumococcal pneumonia is the most common opportunistic infection in individuals with HAND. We show that coinfection with Streptococcus pneumoniae may be a contributing factor in the increased prevalence of HAND in the opioid-dependent population. To date, there have been no studies published implicating the Toll-like receptors (TLR) in the neurocognitive disorders associated with NeuroAIDS in the context of opportunistic infection. Our studies show for the first time, in a morphine-dependent model, synergistic increase and activation of TLR expression in the presence of HIV-1 protein TAT and S. pneumoniae with a significant increase in proinflammatory cytokines (IL-6, TNF-α) levels. Furthermore, concurrent increases in reactive oxygen species and nitric oxide production leading to increased caspase 3 activation are also observed in both murine and human microglial cells. These effects are recapitulated with TLR 2, 4, and 9 cognate ligands (Pam3CSK4, LPS, and CpG) and significantly attenuated in TLR 2 and 4 knock-out mice and TLR2/4 double knock-out mice. Therefor, our findings clearly suggest for the first time that activation of TLRs on microglia cells by morphine and TAT in the context of S. pneumoniae infection may be a potential mechanism for the increased prevalence of HAND in HIV-infected opioid-dependent patients.

Hypoxia differentially regulates arterial and venous smooth muscle cell proliferation via PDGFR-ß and VEGFR-2 expression.

Despite intensive research studies, theories have yet to focus on the contribution of hypoxia to patency differences observed clinically between arterial vs. venous grafts. This study investigates the differential hypoxic response of smooth muscle cells (SMC) to hypoxia-derived endothelial cell (EC) growth factors. Initiation of SMC proliferation under hypoxia (<5% O(2)) occurred only after incubation with hypoxic endothelial cell-conditioned media (H-ECM). After the investigation of several possible growth factors in the H-ECM that may be responsible for SMC proliferation, the greatest difference was observed in vascular endothelial growth factor (VEGF-A) and platelet-derived growth factor homodimer B (PDGF-BB) expression. VEGF-A increased (2-fold) significantly (P < 0.05) in arterial-derived smooth muscle cells (ASMC) under hypoxia compared with venous-derived smooth muscle cells (VSMC), which showed no significant change. VSMC showed significant (P < 0.05) increase in VEGFR-2 expression under hypoxia compared with ASMC. Incubation with VEGFR-2-neutralizing antibody/PDGFR antagonist in VSMC before addition of H-ECM resulted in decreased proliferation. ASMC proliferation under hypoxia did not decrease during incubation with VEGFR-2-neutralizing antibody but did decrease upon PDGFR antagonist incubation. Current therapies focusing on treating intimal hyperplasia have negated the fact that combinational therapy might be required to combat induction of SMC proliferation. Clinically, therapy with PDGFR antagonists plus anti-VEGFR-2 may prove to be efficacious in managing SMC proliferation in venous-derived grafts.

Morphine withdrawal stress modulates lipopolysaccharide-induced interleukin 12 p40 (IL-12p40) expression by activating extracellular signal-regulated kinase 1/2, which is further potentiated by glucocorticoids.

Withdrawal stress is a common occurrence in opioid users, yet very few studies have examined the effects of morphine withdrawal (MW) on immune functioning or the role of glucocorticoids in MW-induced immunomodulation. This study investigated for the first time the role of glucocorticoids in MW modulation of LPS-induced IL-12p40, a key cytokine playing a pivotal role in immunoprotection. Using WT and µ-opioid receptor knock-out mice, we show that MW in vivo significantly attenuated LPS-induced IL-12p40 mRNA and protein expression. The role of glucocorticoids in MW modulation of IL-12p40 was investigated using a murine macrophage cell line, CRL2019, in an in vitro MW model. Interestingly, MW alone in the absence of glucocorticoids resulted in a significant reduction in IL-12p40 promoter activity and mRNA and protein expression. EMSA revealed a concurrent decrease in consensus binding to transcription factors NFκB, Activator Protein-1, and CCAAT/enhancer-binding protein and Western blot analysis demonstrated a significant activation of LPS-induced ERK1/2 phosphorylation. Interestingly, although glucocorticoid treatment alone also modulated these transcription factors and ERK1/2 activation, the addition of glucocorticoids to MW samples resulted in a greater than additive reduction in the transcription factors and significant hyperactivation of LPS-induced ERK1/2 phosphorylation. ERK inhibitors reversed MW and MW plus corticosterone inhibition of LPS-induced IL-12p40. The potentiating effects of glucocorticoids were non-genomic because nuclear translocation of glucocorticoid receptor was not significantly different between MW and corticosterone treatment. This study demonstrates for the first time that MW and glucocorticoids independently modulate IL-12p40 production through a mechanism involving ERK1/2 hyperactivation and that glucocorticoids can significantly augment MW-induced inhibition of IL-12p40.

Opioid drug abuse and modulation of immune function: consequences in the susceptibility to opportunistic infections.

Infection rate among intravenous drug users (IDU) is higher than the general public, and is the major cause of morbidity and hospitalization in the IDU population. Epidemiologic studies provide data on increased prevalence of opportunistic bacterial infections such as TB and pneumonia, and viral infections such as HIV-1 and hepatitis in the IDU population. An important component in the intravenous drug abuse population and in patients receiving medically indicated chronic opioid treatment is opioid withdrawal. Data on bacterial virulence in the context of opioid withdrawal suggest that mice undergoing withdrawal had shortened survival and increased bacterial load in response to Salmonella infection. As the body of evidence in support of opioid dependency and its immunosuppressive effects is growing, it is imperative to understand the mechanisms by which opioids exert these effects and identify the populations at risk that would benefit the most from the interventions to counteract opioid immunosuppressive effects. Thus, it is important to refine the existing animal model to closely match human conditions and to cross-validate these findings through carefully controlled human studies. Better understanding of the mechanisms will facilitate the search for new therapeutic modalities to counteract adverse effects including increased infection rates. This review will summarize the effects of morphine on innate and adaptive immunity, identify the role of the mu opioid receptor in these functions and the signal transduction activated in the process. The role of opioid withdrawal in immunosuppression and the clinical relevance of these findings will also be discussed.

Chronic morphine treatment inhibits LPS-induced angiogenesis: implications in wound healing.

Delayed wound healing is a chronic problem in opioid drug abusers. We investigated the role chronic morphine plays on later stages of wound healing events using an angiogenesis model. Our results show that morphine treatment resulted in a significant decrease in inflammation induced angiogenesis. To delineate the mechanisms involved we investigate the role of hypoxia inducible factor 1 alpha (HIF-1 alpha), a potent inducer of angiogenic growth factor. Morphine treatment resulted in a significant decrease in the expression and nuclear translocation of HIF-1 alpha with a concurrent suppression in vascular endothelial growth factor (VEGF) synthesis. Cells of the innate immune system play a dominant role in the angiogenic process. Morphine treatment inhibited early recruitment of both neutrophils and monocytes towards an inflammatory signal with a significant decrease in the monocyte chemoattractant MCP-1. Taken together, our studies show that morphine regulates the wound repair process on multiple levels. Morphine acts both directly and indirectly in suppressing angiogenesis.

Chronic morphine administration delays wound healing by inhibiting immune cell recruitment to the wound site.

Patients prescribed morphine for the management of chronic pain, and chronic heroin abusers, often present with complications such as increased susceptibility to opportunistic infections and inadequate healing of wounds. We investigated the effect of morphine on wound-healing events in the presence of an infection in an in vivo murine model that mimics the clinical manifestations seen in opioid user and abuser populations. We show for the first time that in the presence of an inflammatory inducer, lipopolysaccharide, chronic morphine treatment results in a marked decrease in wound closure, compromised wound integrity, and increased bacterial sepsis. Morphine treatment resulted in a significant delay and reduction in both neutrophil and macrophage recruitment to the wound site. The delay and reduction in neutrophil reduction was attributed to altered early expression of keratinocyte derived cytokine and was independent of macrophage inflammatory protein 2 expression, whereas suppression of macrophage infiltration was attributed to suppressed levels of the potent macrophage chemoattractant monocyte chemotactic protein-1. When the effects of chronic morphine on later wound healing events were investigated, a significant suppression in angiogenesis and myofibroblast recruitment were observed in animals that received chronic morphine administration. Taken together, our findings indicate that morphine treatment results in a delay in the recruitment of cellular events following wounding, resulting in a lack of bacterial clearance and delayed wound closure.

Morphine disrupts interleukin-23 (IL-23)/IL-17-mediated pulmonary mucosal host defense against Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a pathogen that causes serious respiratory disease and meningitis in the immunocompromised drug abuse population. However, the precise mechanisms by which drug abuse compromises the host immune defense to pulmonary S. pneumoniae infection is not fully understood. Using a well-established murine model of opiate abuse and S. pneumoniae lung infection, we explored the influence of morphine treatment on the interleukin-23 (IL-23)/IL-17 axis and related innate immunity. Impairment of early IL-23/IL-17 production caused by morphine treatment was associated with delayed neutrophil migration and decreased pneumococcal clearance. Furthermore, morphine treatment impaired MyD88-dependent IL-23 production in alveolar macrophages and dendritic cells in response to in vitro S. pneumoniae cell infection. Moreover, morphine treatment significantly inhibited the S. pneumoniae-induced phosphorylation of interferon response factor 3 (IRF3), ATF2, and NF-kappaBp65. T-cell receptor delta (TCRdelta)-deficient mice showed a decrease in IL-17 production and a severely weakened capacity to clear lung S. pneumoniae infection. Finally, morphine treatment resulted in diminished secretion of antimicrobial proteins S100A9 and S100A8/A9 during early stages of S. pneumoniae infection. In conclusion, morphine treatment causes a dysfunction in IL-23-producing dendritic cells and macrophages and IL-17-producing gammadeltaT lymphocytes in response to S. pneumoniae lung infection. This leads to diminished release of antimicrobial S100A8/A9 proteins, compromised neutrophil recruitment, and more-severe infection.

Opiate abuse, innate immunity, and bacterial infectious diseases.

The first line of defense against invading bacteria is provided by the innate immune system. Morphine and other opiates can immediately disrupt the body's first line of defense against harmful external bacteria. Opiate, for example morphine, abuse degrades physical and physiologic barriers, and modulates phagocytic cells (macrophages, neutrophils) and, nonspecific cytotoxic T cells (gammadelta T), natural killer cells, and dendritic cells, that are functionally important for carrying out a rapid immune reaction to invading pathogens. In vitro studies with innate immune cells from experimental animals and humans and in vivo studies with animal models have shown that opiate abuse impairs innate immunity and is responsible for increased susceptibility to bacterial infection. However, to better understand the complex interactions between opiates, innate immunity, and bacterial infection and develop novel approaches to treat and even prevent bacterial infection in the opiate-abuse population, there is an urgent need to fill the numerous gaps in our understanding of the cellular and molecular mechanisms by which opiate abuse increases susceptibility to bacterial infection.

Morphine withdrawal inhibits IL-12 induction in a macrophage cell line through a mechanism that involves cAMP.

There are very few studies that examine the effects that morphine withdrawal has on immune functioning, and of these even fewer describe the mechanisms by which withdrawal brings about these changes. Our previous work demonstrated that morphine withdrawal contributed to Th cell differentiation by biasing cells toward the Th2 lineage. A major finding from these studies was that IL-12 was decreased following withdrawal, and it was concluded that this decrease may be a mechanism by which morphine withdrawal is mediating Th2 polarization. Therefore, it was the aim of the current studies to develop an in vitro model to examine the process of morphine withdrawal and to understand the signaling mechanisms that withdrawal may use to effect IL-12 production through the use of this model. It was demonstrated and concluded that morphine withdrawal may be effecting IL-12 production by increasing cAMP levels, which activates protein kinase A. Protein kinase A activation then prevents the phosphorylation and subsequent degradation of IkappaB, which in turn prevents translocation of the NF-kappaB p65 subunit to the nucleus to transactivate the IL-12 p40 gene, ultimately resulting in decreased IL-12 production following LPS stimulation.

Morphine induces defects in early response of alveolar macrophages to Streptococcus pneumoniae by modulating TLR9-NF-kappa B signaling.

Resident alveolar macrophages and respiratory epithelium constitutes the first line of defense against invading lung pneumococci. Results from our study showed that increased mortality and bacterial outgrowth and dissemination seen in morphine-treated mice were further exaggerated following depletion of alveolar macrophages with liposomal clodronate. Using an in vitro alveolar macrophages and lung epithelial cells infection model, we show significant release of MIP-2 from alveolar macrophages, but not from lung epithelial cells, following 4 h of exposure of cells to pneumococci infection. Morphine treatment reduced MIP-2 release in pneumococci stimulated alveolar macrophages. Furthermore, morphine treatment inhibited Streptococcus pneumoniae-induced NF-kappaB-dependent gene transcription in alveolar macrophages following 2 h of in vitro infection. S. pneumoniae infection resulted in a significant induction of NF-kappaB activity only in TLR9 stably transfected HEK 293 cells, but not in TLR2 and TLR4 transfected HEK 293 cells, and morphine treatment inhibited S. pneumoniae-induced NF-kappaB activity in these cells. Moreover, morphine treatment also decreased bacterial uptake and killing in alveolar macrophages. Taken together, these results suggest that morphine treatment impairs TLR9-NF-kappaB signaling and diminishes bacterial clearance following S. pneumoniae infection in resident macrophages during the early stages of infection, leading to a compromised innate immune response.

Morphine induces CD4+ T cell IL-4 expression through an adenylyl cyclase mechanism independent of the protein kinase A pathway.

Impaired host defense mechanisms after major operative procedures and trauma are recognized as important factors in the development of infectious complication. Trauma is associated with impaired cellular immunity and CD4+ T cell Th2 differentiation. We have previously implicated morphine treatment as a possible mechanism for Th2 differentiation after injury. In this investigation we first establish that morphine treatment in vivo results in Th2 differentiation and that this effect is mediated through a naltrexone-sensitive opioid receptor. We investigated the intracellular mechanism by which morphine controls CD4+ T cell differentiation and demonstrate that morphine treatment in vitro 1) increases anti CD3/CD28 Ab-induced CD4+ T cell IL-4 protein synthesis, IL-4 mRNA, and GATA-3 mRNA accumulation through a pertussis toxin-sensitive receptor; 2) results in a dose-dependent increase in anti-CD3/CD28 Ab-induced CD4+ T cell cytoplasmic cAMP concentration; and 3) increases the forskolin-stimulated cytoplasmic cAMP level through a pertussis toxin-sensitive receptor. We also demonstrate that chronic morphine treatment increases anti-CD3/CD28 Ab-induced IL-4 promoter activity and IL-4 immunoprotein expression through a p38 MAPK-dependent, but protein kinase A- and Erk1/Erk2-independent, mechanism.

Morphine impairs host innate immune response and increases susceptibility to Streptococcus pneumoniae lung infection.

Chronic morphine use impairs host innate immune response and increases susceptibility to bacteria and virus. In this study a novel mouse model of chronic morphine treatment, followed by intranasal inoculation with Streptococcus pneumoniae, was used to investigate microbial events and host innate immune response. Our results show that chronic morphine treatment markedly delayed neutrophil recruitment and increased bacterial burden in the lung, spleen, and blood with a subsequent increase in mortality. In morphine-treated animals, before neutrophil recruitment, a significant decrease in TNF-alpha, IL-1, IL-6, MIP-2, and KC was observed both in bronchoalveolar lavage fluids and in lung tissue. In the early phase of infection, we found that accumulation of galectin-3 in the alveolar space of streptococcus-infected lungs was decreased after morphine treatment. The transcription factor NF-kappaB in lung resident cells was also inhibited after morphine treatment. Taken together, these results suggest that chronic morphine treatment in an S. pneumoniae infection model suppresses NF-kappaB gene transcription in lung resident cells, which, in turn, modulates the transcriptional regulation of MIP-2 and inflammatory cytokines. The decreased synthesis of MIP-2 and inflammatory cytokines coupled with the decreased release of galectin-3 result in reduced migration of neutrophils to the site of infection, thereby increasing susceptibility to S. pneumoniae infection after morphine treatment.

Chronic morphine treatment differentiates T helper cells to Th2 effector cells by modulating transcription factors GATA 3 and T-bet.

Chronic morphine treatment in animal models has been shown to alter a number of immune parameters including suppression of cellular immunity. T helper cell differentiation into Th2 effector cell may be a major contributing factor to impaired cellular immunity following chronic drug abuse. We had previously shown that chronic morphine treatment in vivo and in vitro decreases IL-2 and IFNgamma (Th1) protein levels and increases IL-4 and IL-5 (Th2) protein levels in a time-dependent manner. In addition in this paper, we show that chronic morphine treatment resulted in a decrease in IFNgamma and IL-2 mRNA and an increase in IL-4 and IL-5 mRNA accumulation in murine splenocytes. Furthermore, chronic morphine treatment inhibited IFNgamma promoter activity and increased IL-4 promoter activity in respective promoter transfected primary T cells. In addition, we also demonstrate that chronic morphine treatment resulted in an increase in GATA 3 binding to DNA consensus elements in electromobility shift assays and an increase in GATA 3 protein and mRNA levels. In contrast, chronic morphine treatment resulted in a decrease in T-bet mRNA levels. From these data, we conclude that chronic morphine treatment differentiates T helper cell to Th2 effector cells by modulating key master switches that results in committing T helper cell to a Th2 phenotype.

Morphine negatively regulates interferon-gamma promoter activity in activated murine T cells through two distinct cyclic AMP-dependent pathways.

To explore the mechanism by which morphine promotes the incidence of HIV infection, we evaluated the regulatory role of morphine on the interferon-gamma (IFN-gamma) promoter in activated T cells from wild type and mu-opioid receptor knockout mice. Our results show that morphine inhibited anti-CD3/CD28-stimulated IFN-gamma promoter activity in a dose-dependent manner. Chronic morphine treatment of T cells increased intracellular cAMP. To evaluate the role of cAMP in morphine's modulatory function, the effects of dibutyryl cyclic AMP and forskolin were investigated. Both dibutyryl cyclic AMP and forskolin treatment inhibited IFN-gamma promoter activity. Treatment with pertussis toxin, but not with a protein kinase A inhibitor, antagonized morphine's inhibitory effects. Morphine inhibited phosphorylation of ERK1/2 and p38 MAPK; in addition, morphine treatment in the presence of either ERK1/2 or p38 MAPK inhibitor (PD98059 or SB203580) resulted in an additive inhibition of IFN-gamma promoter activity. The transcription factor activator protein-1, NF-kappaB, and nuclear factor of activated T cells (NFAT) were negatively regulated by morphine. Overexpression of NF-kappaB p65 rescued the inhibitory effect of morphine on IFN-gamma promoter activity. However, only when NFATc1 was co-overexpressed with c-fos was the inhibitory effect of morphine on IFN-gamma promoter counteracted. The inhibitory effects of morphine were not observed in T cells obtained from mu-opioid receptor knockout mice, suggesting that morphine modulation of IFN-gamma promoter activity is mediated through the mu-opioid receptor. In summary, our data indicate that morphine modulation of IFN-gamma promoter activity is mediated through two distinct cAMP-dependent pathways, the NF-kappaB signaling pathway and the ERK1/2, p38 MAPK, AP-1/NFAT pathway.

Mu-opioid receptor mediates chronic restraint stress-induced lymphocyte apoptosis.

Psychological stress is associated with immunosuppression in both humans and animals. Although it was well established that psychological stressors stimulate the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system, resulting in the release of various hormones and neurotransmitters, the mechanisms underlying these phenomena are poorly understood. In this study, mu-opioid receptor knockout (MORKO) mice were used to investigate whether the mu-opioid receptor mediates the immunosuppression induced by restraint stress. Our results showed that wild-type (WT) mice subjected to chronic 12-h daily restraint stress for 2 days exhibited a significant decrease in splenocyte number with a substantial increase in apoptosis and CD95 (Fas/APO-1) expression of splenocytes. The effects are essentially abolished in MORKO mice. Furthermore, inhibition of splenic lymphocyte proliferation, IL-2, and IFN-gamma production induced by restraint stress in WT mice was also significantly abolished in MORKO mice. Interestingly, both stressed WT and MORKO mice showed a significant elevation in plasma corticosterone and pituitary proopiomelanocortin mRNA expression, although the increase was significantly lower in MORKO mice. Adrenalectomy did not reverse restraint stress-induced immunosuppression in WT mice. These data clearly established that the mu-opioid receptor is involved in restraint stress-induced immune alterations via a mechanism of apoptotic cell death, and that the effect is not mediated exclusively through the glucocorticoid pathway.

The immunosuppressive effects of chronic morphine treatment are partially dependent on corticosterone and mediated by the mu-opioid receptor.

Wild-type and mu-opioid receptor knockout (MORKO) mice were used to investigate the role of corticosterone (CORT) and the mu-opioid receptor (MOR) in chronic morphine-mediated immunosuppression. We found that although plasma CORT concentrations in CORT infusion (10 mg/kg/day) and morphine-pellet implantation (75 mg) mice were similar (400-450 ng/ml), chronic morphine treatment resulted in a significantly higher (two- to threefold) inhibition of thymic, splenic, and lymph node cellularity; inhibition of thymic-lymphocyte proliferation; inhibition of IL-2 synthesis; and activation of macrophage nitric oxide (NO) production when compared with CORT infusion. In addition, results show that the inhibition of IFN-gamma synthesis and splenic- and lymph node-lymphocyte proliferation and activation of macrophage TNF-alpha and IL-1beta synthesis occurred only with chronic morphine treatment but not with CORT infusion. These morphine effects were abolished in MORKO mice. The role of the sympathetic nervous system on morphine-mediated effects was investigated by using the ganglionic blocker chlorisondamine. Our results show that chlorisondamine was able to only partially reverse morphine's inhibitory effects. The results clearly show that morphine-induced immunosuppression is mediated by the MOR and that although some functions are amplified in the presence of CORT or sympathetic activation, the inhibition of IFN-gamma synthesis and activation of macrophage-cytokine synthesis is CORT-independent and only partially dependent on sympathetic activation.