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From the *Hôpital Bicêtre, AP-HP, Le Kremlin-Bicêtre, cedex, France; and Hôpital Hotel-Dieu, Nantes, France.
Address correspondence and reprint requests to Marie-Pierre Bonnet, MD, Département d’Anesthésie Réanimation Chirurgicale, Groupement Hospitalier Universitaire Sud, Hôpital Bicêtre, 78, rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, cedex, France. Address e-mail to marie-pierre.bonnet{at}abc.aphp.fr.
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood by centrifugation on a density gradient. Monocytes were secondarily separated using a high-gradient magnetic cell sorting kit with specific anti-CD14 antibodies. Monocytes or PBMCs were pretreated with opioid receptors antagonists before being cultured with morphine and peptidoglycan (PGN) from Staphylococcus aureus (specific TLR2 agonist). The amount of TNF, IL-6, and IL-10 was measured in the supernatant enzyme-linked immunosorbent assay.
RESULTS: Proinflammatory cytokines: Morphine significantly inhibited the production of cytokines in a dose and concentration-dependent manner in PGN-stimulated monocytes. µ Opioid receptor activation specifically mediated this morphine-induced TNF and IL-6 inhibition in monocytes. Morphine significantly inhibited the TNF, but not the IL-6 production, in PGN-stimulated PBMCs. The µ opioid receptor was not involved in this morphine-induced TNF inhibition in PBMCs. Antiinflammatory cytokines: IL-10 was not a factor for the inhibition of TNF and IL-6 production after PGN stimulation in either monocytes or PBMCs cultures.
CONCLUSIONS: The µ opioid receptor mediates morphine-induced TNF and IL-6 inhibition in PGN-stimulated monocytes, but not in PBMCs. A direct monocyte-to-lymphocyte contact (PBMCs) alters the inhibitory effects of morphine observed on monocytes alone. IL-10 is not a factor for the inhibition of TNF or for IL-6 production. Interactions between TLR2 and µ opioid intracellular pathways remain to be studied to delineate these morphine immunosuppressive effects.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Morphine’s effects on innate immunity have been mainly studied in different cell populations after exposure of cells to lipopolysaccharide (LPS) from Gram-negative bacteria. However, infections by Gram-positive bacteria have been increasing during the last 20 yr12 and are of poor prognosis.13 Host-defense mechanisms against bacterial infections are under the control of toll-like receptors (TLRs). TLRs play a critical role in innate defense by sensing specific molecular patterns associated with microbial pathogens. Two TLRs are especially involved in specific identification of bacterial components: TLR4 recognizes the LPS from Gram-negative bacteria and TLR2 recognizes cocci Gram-positive components such as peptidoglycan (PGN).14,15 PGN stimulates TLR2, leading to nuclear factor 
-B (NF-B) activation16 and to release of proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-6 (IL-6).17,18 The inhibitory effects of morphine on LPS-induced TNF and IL-6 production have been reported in monocytes.6,7 IL-10 is an antiinflammatory cytokine, which classically inhibits proinflammatory cytokine production, as TNF and IL-6, especially in monocyte cellular models. A potential mechanism of morphine’s inhibitory effects could be through enhancing IL-10 production. Despite the clinical importance of Gram-positive infections, the effects of morphine on the TLR2 signaling pathway in monocytes have not been delineated.
The purpose of the present study was therefore (i) to investigate effects of morphine on TNF and IL-6 production by human monocytes after TLR2 stimulation by PGN, (ii) to examine the implication of the different opioid receptors using specific antagonist receptors, (iii) to test the effects of lymphocyte-to-monocyte contact, and (iv) to study the role of IL-10 in the immunomodulatory effects of morphine.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide [CTOP]); a opioid receptor-specific antagonist (nor-binaltorphimine dihydrochloride [nor-BNI]) and a 
opioid receptor-specific antagonist (naltribene methane sulfonate [NLT]). Lympholyte was purchased from Cederlane (Cederlane®, Tebu-bio Le Perrey-en-Yvelines, France). The gradient magnetic cell sorting kit used to isolate monocytes was purchased from Miltenyi Biotec (Miltenyi Biotec®, Paris) as well as the anti-CD14 antibodies. Mallassez cells for cell count were purchased from Glasstic (Glasstic® Kova® Hycor Biomedical Inc., Garden Grove, CA). All other reagents were purchased from Sigma-Aldrich (Saint Quentin Fallavier) unless specified otherwise.
Blood Samples
The study protocol was approved by the IRB. Peripheral blood mononuclear cells (PBMCs) and monocytes were isolated from healthy volunteers after informed consent was obtained. For each donor, 40 mL of blood was used. Exclusion criteria were subjects who were younger than 18-yr-of-age, were pregnant, had used steroids or any medication, or had any focus of infection within the previous 30 days.
Cell Preparation
PBMCs
PBMCs were isolated from blood freshly collected in sodium citrate by use of lympholyte, a density gradient solution. In brief, 4 mL of blood was layered on 4 mL of lympholyte in a sterile 15-mL tube (Falcon; Becton Dickinson) and was centrifuged for 35 min at 800g and at 20°C. The ring of PBMCs (milky interface) was recovered and washed twice with RPMI 1640 medium containing 1% ultraglutamine. Cells were resuspended in RPMI 1640 medium with 0.2% of human serum. PBMCs obtained were directly plated and cultured or used for isolation of monocytes.
Monocytes
A fraction of PBMCs, obtained as described above, was immediately used for monocyte isolation, which was performed by using a high-gradient magnetic cell sorting kit19 in accordance with the manufacturer recommendations (Miltenyi Biotec®, Paris). In brief, PBMCs were suspended in phosphate buffered saline with ethylene diamine tetra acetic acid 2 mm and 5% human serum. Monocytes were magnetically labeled with magnetic microbeads coupled to a specific anti-CD14 antibody added to the cell suspension (i.e., PBMCs) and incubated for 20 min at 4°C. The cell suspension was then passed through the separation column that had been placed in a magnetic field. The magnetically labeled cells (i.e., monocytes) were retained on the column and other cells were eluted from the column. Monocytes were recovered by flushing through the column. Cell purity with this technique is >94% of CD14 cells.
Cultures
PGN, morphine, and opioid receptor antagonists were made fresh for each experiment. For each experimental condition, at the end of the cell culture, we checked the viability of cells by a tryptan blue exclusion test. The viability test was consistently >98% ± 1.15%. In each experiment, the cells cultures were stimulated with PGN (10 µg/mL) for 120 min.
Monocytes Cultures
Monocytes were counted on Mallassez cells before being plated and cultured. Monocytes were plated in 24-well plates (final concentration 1.106 cells/0.5 mL/well) and were cultured in RPMI 1640 with 0.2% of human serum in a 5% CO2 incubator at 37°C. In a pilot study, time (60, 120, 180, 350 min) and concentrations-(10–11, 10–9, 10–7, 10–6, 10–5, 10–4 M) dependent effects of morphine on TNF and IL-6 production were investigated. In the main study, the role of the opioid receptors in morphine-induced inhibition on TNF and IL-6 production was investigated by using opioid receptor antagonists. In brief, monocytes were incubated with control media or pretreated with the indicated concentrations of opioid receptor antagonists for 30 min before morphine (10–5 M) incubation for 180 min. Cultures were stimulated with PGN for 120 min before the culture fluid was harvested and centrifuged at 4°C for 10 min. The supernatant was kept at –70°C before the TNF, IL-6, and IL-10 measurements.
PBMCs Cultures
PBMCs were counted on Mallassez cells before being plated and cultured. PBMCs were plated in 24-well plates (final concentration 4.106 cells/0.5 mL/well) and cultured in RPMI 1640 with 0.2% of human serum in a 5% CO2 incubator at 37°C. PBMCs were incubated with control media or pretreated with CTOP (10–5 M) for 30 min before morphine (10–5 M) incubation for 180 min. Cultures were stimulated with PGN for 120 min before the culture fluid was harvested and centrifuged at 4°C for 10 min. The supernatant was kept at –70°C before the TNF, IL-6, and IL-10 measurements.
Cytokine Measurement
The amount of TNF, IL-6, and IL-10 was measured with a commercial enzyme-linked immunosorbent assay kit (DuosetTM, R&D systems, Abingdon, United Kingdom), according to the manufacturer’s instructions. TNF and IL-6 concentrations in the absence of stimulation (control group: C) were always very low in all experiments, indicating that the culture’s plates did not stimulate monocytes or PBMCs.
Statistical Analysis
The distribution of cytokine concentrations in each unit was checked for normality using the Shapiro-Wilk test. The results were evaluated by a one-way analysis of variance followed by a Newman-Keul’s test for intergroup comparison. We used Bonferroni correction for multiple comparisons, and adjusted P for each comparison. Results are expressed as mean ± sem.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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µ Opioid Receptor Mediates Morphine-Induced TNF and IL-6 Inhibition in PGN-Stimulated Monocytes
Nonspecific antagonist NLX, and receptor antagonists (NLT and nor-BNI, respectively) at the concentration of 10–5 M did not prevent the decrease in TNF and IL-6 production induced by morphine. Conversely, the morphine-inhibiting effect was reversed when monocytes were treated with the specific µ opioid receptor antagonist, CTOP, at the concentration of 10–5 M (Fig. 2).
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Monocytes cell cultures were then pretreated with varying CTOP and NLX concentrations (Fig. 3). Opioid receptor antagonists alone did not activate cell cultures. Treatment of monocytes with the nonspecific antagonist NLX reversed morphine-induced TNF and IL-6 inhibition at the concentration of 10–4 M and 10–3 M. CTOP also prevented the decrease in TNF and IL-6 production induced by morphine at the concentration of 10–5 M (Fig. 3).
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A Monocyte-to-Lymphocyte Contact Modifies the Immunosuppressive Effect of Morphine Observed on Monocytes Cultured Alone
It has been shown that TLR2, a receptor for PGN, is expressed mainly on the surface of monocytes/macrophages but also on the surface of T cells.20 It is therefore possible that a monocyte/lymphocyte interaction modifies morphine’s suppressive effects observed on monocytes cultured alone. To test this hypothesis, we therefore examined whether morphine exerted a suppressive effect on the production of TNF and IL-6 in PBMCs (i.e., lymphocytes and monocytes) cultures stimulated with PGN. As shown in Figure 4, the release of TNF but not IL-6 from PGN-stimulated PBMCs was inhibited by morphine. Moreover, CTOP did not prevent the decrease in TNF production induced by morphine. The monocyte/lymphocyte interactions alter the immunosuppressive effect of morphine observed on monocytes alone.
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Antiinflammatory Cytokines Production
IL-10 Is Not Involved in Morphine-Induced TNF and IL-6 Inhibition in PGN-Stimulated Monocytes
IL-10 is an immunosuppressive cytokine produced by a variety of cell types including monocytes and T lymphocytes. Thus, IL-10 seemed to be a potential candidate for the morphine-induced TNF and IL-6 inhibition in PGN-stimulated monocytes.
No IL-10 production was detected in the monocytes cell cultures after PGN stimulation with or without morphine pretreatment (Fig. 5). These results indicate that IL-10 is not a factor for morphine-induced suppression of the production of TNF and IL-6 in cultured monocytes.
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IL-10 Is Released Through a Contact of T Cells with Monocytes but This Release Is Not Involved in the Antiinflammatory Effects of Morphine
We hypothesized that a cellular interaction between monocytes and T lymphocytes could be involved in IL-10 production after PGN stimulation. IL-10 production was therefore measured in human PBMCs cultures. A basal production of IL-10 (control) was detected in PBMCs cultures, and this production was significantly enhanced after PGN stimulation (Fig. 5). However, pretreatment with morphine did not further enhance the production of IL-10 observed with PGN. These results indicate that IL-10 is not involved in the suppressive effects of morphine on PGN-induced TNF production in PBMCs cultures (Fig. 5).
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence indicate that morphine severely impairs host defense against bacterial invaders. Indeed, it was demonstrated that PBMCs from patients treated with methadone had a significantly impaired capacity to generate reactive oxygen species involved in host immune response.21 In a mouse model of cocci Gram-positive pneumonia, animals treated with morphine had an increased mortality rate, an increased rate of bacterial growth, a decrease in TNF and IL-6 production in bronchoalveolar lavage and a decreased NF-B activation compared with control mice.22 In monocytes or PBMCs, several studies have shown that morphine has an immunosuppressive effect on different cell types and through different signaling pathways. First, Peterson et al. demonstrated in PBMCs that concanavalin A-induced interferon 
production was inhibited by morphine.23 The same authors subsequently showed that morphine decreased TNF production after TLR4 stimulation with LPS.6 Other studies have revealed that morphine decreases phagocytosis activity,24 chimiotaxism,25 and NF-B activity after TLR4 stimulation with LPS.26 Regarding TLRs signaling, morphine’s immunomodulatory effects were evaluated only in the TLR4 pathway. However, TLR2-induced monocytes responses are likely to have important clinical consequences, as Gram-positive organisms are an increasingly growing cause of severe infections associated with organ dysfunction, including septic shock.27 This study shows for the first time that morphine pretreatment induces a time and concentration-dependent inhibition of TNF and IL-6 production in TLR2-stimulated monocytes. Recent evidence has suggested that signals others than those from TLRs could contribute to PGN recognition. Indeed, a family of intracellular proteins, named NOD1 and NOD2, senses degradation products of PGN. However, both systems, i.e., TLR2 and NOD, activate NF-B, leading to the production of proinflammatory cytokines.
The opioid receptor affinity for morphine is 10–9 M,28 and the present results show a significant inhibitory effect of morphine only at a concentration of 10–5 M. However, the concentration of 10–5 M could be clinically relevant, since morphine consumption by patients or drug addicts can be very high and can reach plasma concentrations of 2-5 µM.29 Moreover, morphine’s effects were studied in isolated monocyte cultures. Cell interactions and plasma protein interventions are not considered in such a cell culture model. Another explanation for the inhibitory effect observed in these experiments could be related to the direct toxicity of morphine. However, morphine at a concentration of 10–5 M does not affect cell viability as assessed by tryptan blue exclusion criteria, and the reversibility of morphine’s inhibitory effect by CTOP confirms the absence of toxicity.
In the present study, the inhibitory effects of morphine on TNF and IL-6 production were reversed with naloxone and with a specific µ opioid receptor antagonist (CTOP), but not with a specific or opioid receptor antagonist. The opioid receptor antagonists concentration used was 10–5 M, with an incubation time of 30 min, as previously described.9,25 In monocytes, morphine’s effects on the TLR2 pathway are therefore specifically mediated by µ opioid receptors. In mice with a genetic disruption of the µ opioid receptor (MOR) gene (MORKO),30 morphine’s immunosuppressor effects disappeared, highlighting the mediation of morphine’s immune effects via the µ opioid receptors. The current results show that morphine’s inhibitory effects are reversed by CTOP 10–5 M and reversed by naloxone 10–4 M. CTOP is 2000-fold more specific to the µ opioid receptor than naloxone.31 This could explain why no effect was observed when naloxone was used at the dose of 10–5 M.
TLR2 agonists induce the production of proinflammatory cytokines (TNF, IL-6), especially through the activation of the NF-B pathway. At least two different mechanisms mediated by µ opioid receptors might be involved in morphine-induced TNF and IL-6 inhibition in TLR2 stimulated monocytes. First, chronic exposure to agonists of classical opiate receptor (µ1/µ2) increases cytosolic cAMP through a Gi/o-coupled receptor mechanism, and there is strong evidence that this increase of cAMP acts as an inhibitor of NF-B.32,33 However, the time of exposure that defines a chronic exposure to morphine in cell cultures remains controversial. Second, morphine exerts its immunomodulatory effects in immunocytes (i.e., granulocytes, monocytes) through the nonclassical µ3 opiate receptor.34,35 This receptor causes immunosuppression, at least in part, via the nitric oxide-stimulated depression of NF-B nuclear binding. Our results also show an apparent stimulatory effect of CTOP on IL-6 production (Figs. 2 and 3). An effect of CTOP on IL-6 production that is not solely due to its action at the µ opioid receptor cannot be excluded.
It is generally accepted that cell-to-cell interactions between monocytes and T cells are required for an effective immune response.36 To gain further insight into the immunosuppressive effects of morphine, we studied the role of lymphocyte-to-monocyte contact through PBMCs cultures (i.e., monocytes and lymphocytes). At least two distinct mechanisms by which monocyte-lymphocyte interaction could interfere with the suppressive effect of morphine might be involved.36 First, the expression of cell-surface molecules associated with the cell-to-cell contact between monocytes and T cells (CD28 and/or CTLA-4 on T cells and their ligands CD80 and/or CD86 on monocytes; and CD40 on monocytes and CD40 ligand on T cells) may be altered. Second, the inhibitory mediators released by lymphocytes (IL-10, IL-5, IL-4) could modulate the effects of morphine observed in monocytes cultures. In the current results, the release of TNF, but not IL-6, from PGN-stimulated PBMCs was inhibited by morphine, indicating that a monocyte/lymphocyte interaction interferes with morphine’s suppressive effects observed in monocytes cultured alone.
IL-10 is a major antiinflammatory cytokine, known to inhibit TNF and IL-6 production in human monocytes after LPS stimulation by decreasing NF-B activation. An increased level of IL-10 in the cellular cultures could, therefore, be a likely explanation for the morphine inhibitory effects observed in the present results. However, no IL-10 production was detected in monocyte cultures, regardless of the experimental conditions used. We hypothesized that a cell interaction between monocytes and lymphocytes could be involved in the induction of IL-10 production.36 To study the effect of monocytes-lymphocytes interaction on IL-10 production, we cultured PBMCs. IL-10 production was increased after stimulation with PGN in PBMCs cultures, but this increase was not modified in the presence of morphine. Thus, IL-10 does not play a role in morphine’s inhibition of TNF production in PBMCs cultures after stimulation with PGN.
In conclusion, this study demonstrates that there is an inhibitory effect of morphine on proinflammatory cytokine production in human monocytes after TLR2 stimulation, and that this inhibition is mediated solely by the µ opioid receptor. A direct monocyte-to-lymphocyte contact (PBMCs) alters the inhibitory effects of morphine observed on monocytes alone. IL-10 is not a factor for the inhibition of TNF and IL-6 production. Finally, this work highlights the interaction between the TLR2 signaling pathway and the µ opioid receptor signaling pathway. Intracellular mechanisms leading to this inhibitory effect of morphine in the TLR2 signaling pathway remain to be studied.
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Footnotes |
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Supported by the association Mises Au Point en Anesthésie et Réanimation, Le Kremlin-Bicêtre, France.
Presented, in part, during the 47th Congress of the Société Française d’Anesthésie-Réanimation, Paris, France, September 22, 2005.
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REFERENCES |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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B nuclear binding in human neutrophils and monocytes by a nitric oxide-dependent mechanism. Anesthesiology 2000;92:1677–84[Web of Science][Medline]B, and cyclic AMP response element binding protein. J Immunol 1999;163:954–62This article has been cited by other articles:
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