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Anesthetic Pharmacology Preclinical Pharmacology

Section Editor: Marcel E. Durieux

Clinical Pharmacology
Section Editor: Tony Gin

The Opioid Receptor Mediates Morphine-Induced Tumor Necrosis Factor and Interleukin-6 Inhibition in Toll-Like Receptor 2-Stimulated Monocytes
Marie-Pierre Bonnet, MD* He le ` ne Beloeil, MD, PhD* Dan Benhamou, MD* Jean-Xavier Mazoit, MD, PhD* Karim Asehnoune, MD, PhD
BACKGROUND: Morphine possesses immunomodulatory effects but its intrinsic mechanisms, especially in the toll-like receptor 2 (TLR2) signaling pathway, are only partially understood. In this study, we evaluated the effects of morphine on tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-10 (IL-10) production in TLR2-stimulated human monocytes and identified the involvement of the different opioid receptors, and of the lymphocyte-to-monocyte contact. 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. Opioid receptor activation specifically mediated this morphineinduced TNF and IL-6 inhibition in monocytes. Morphine significantly inhibited the TNF, but not the IL-6 production, in PGN-stimulated PBMCs. The 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 opioid receptor mediates morphine-induced TNF and IL-6 inhibition in PGN-stimulated monocytes, but not in PBMCs. A direct monocyteto-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 opioid intracellular pathways remain to be studied to delineate these morphine immunosuppressive effects.
(Anesth Analg 2008;106:11429)

orphine and other opioids are potent immunomodulators.1 It has been suggested that chronic opioid users, for either therapeutic reasons or because of addiction, are more susceptible to bacterial and viral infections.2,3 However, clinical evidence demonstrating enhanced infectious susceptibility in opioid-using chronic pain patients is still missing. Opioid administration affects both innate and adaptative immunity,
From the *Ho pital Bice tre, AP-HP, Le Kremlin-Bice tre, cedex, France; and Ho pital Hotel-Dieu, Nantes, France. Accepted for publication on November 28, 2007. Supported by the association Mises Au Point en Anesthe sie et Re animation, Le Kremlin-Bice tre, France. Presented, in part, during the 47th Congress of the Socie te Franc aise dAnesthe sie-Re animation, Paris, France, September 22, 2005. Address correspondence and reprint requests to Marie-Pierre Bonnet, MD, De partement dAnesthe sie Re animation Chirurgicale, Groupement Hospitalier Universitaire Sud, Ho pital Bice tre, 78, rue du Ge ne ral Leclerc, 94275 Le Kremlin-Bice tre, cedex, France. Address e-mail to marie-pierre.bonnet@abc.aphp.fr. Copyright 2008 International Anesthesia Research Society
DOI: 10.1213/ane.0b013e318165de89

such as antibodies production,4 natural killer activity,5 cytotoxicity, cytokine production,6,7 chimiotaxism,8 and phagocytosis.9 Monocytes play a central role in innate immunity. The presence of opioid receptors on monocytes has been demonstrated,10 strengthening the link between immunity and opioid drugs, although this link has been questioned by others.11 Morphines effects on innate immunity have been mainly studied in different cell populations after exposure of cells to lipopolysaccharide (LPS) from Gramnegative 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 Gramnegative bacteria and TLR2 recognizes cocci Grampositive components such as peptidoglycan (PGN).14,15
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PGN stimulates TLR2, leading to nuclear factor -B (NFB) 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 morphines 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-tomonocyte contact, and (iv) to study the role of IL-10 in the immunomodulatory effects of morphine.

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 20C. 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 4C. 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.

METHODS
Reagents
RPMI 1640 with 1% ultraglutamine was provided by Biowittaker (Cambrex Bioscience, Verniers, Belgique). Phosphate buffered saline was obtained from Gibco, life technologies (Cergy-Pontoise, France). Sterile tubes and 24-well plates were purchased from ATGC biotechnology (Marne-La-Valle e, France). Morphine chlorhydrate (AP-HP) without preservative was obtained from the pharmacy department of Bice tre Hospital. Staphylococcus aureus ultrapure PGN and opioid receptor antagonists were purchased from Fluka BioChemica laboratories (Sigma-Aldrich; Saint Quentin Fallavier, France). Opioid receptor antagonists used were a nonspecific antagonist (naloxone methiodide [NLX]); a opioid receptor-specific antagonist (Phe-Cys-Tyr-D-TrpOrn-Thr-Pen-Thr amide [CTOP]); a opioid receptorspecific 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.

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 g/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 37C. In a pilot study, time (60, 120, 180, 350 min) and concentrations-(1011, 109, 107, 106, 105, 104 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
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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
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antagonists. In brief, monocytes were incubated with control media or pretreated with the indicated concentrations of opioid receptor antagonists for 30 min before morphine (105 M) incubation for 180 min. Cultures were stimulated with PGN for 120 min before the culture fluid was harvested and centrifuged at 4C for 10 min. The supernatant was kept at 70C 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 37C. PBMCs were incubated with control media or pretreated with CTOP (105 M) for 30 min before morphine (105 M) incubation for 180 min. Cultures were stimulated with PGN for 120 min before the culture fluid was harvested and centrifuged at 4C for 10 min. The supernatant was kept at 70C 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 (Duoset, R&D systems, Abingdon, United Kingdom), according to the manufacturers instructions. TNF and IL-6 concentrations in the absence of stimulation (control group: C) were always very low in all experiments, indicating that the cultures 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 NewmanKeuls test for intergroup comparison. We used Bonferroni correction for multiple comparisons, and adjusted P for each comparison. Results are expressed as mean sem.

RESULTS
Proinammatory Cytokines Production
Suppressive Effects of Morphine on PGN-Induced TNF and IL-6 Production in Monocytes Morphine alone did not activate monocytes cell cultures (Fig. 1A). Concentrations of TNF and IL-6 were increased in supernatants of monocytes stimulated with PGN. The release of TNF and IL-6 from PGN-stimulated monocytes was inhibited in a dose and time-dependent manner by morphine (Figs. 1A and B).

Figure 1. (A) Morphine inhibits tumor necrosis factor (TNF) and interleukin (IL)-6 production in peptidoglycan (PGN) stimulated monocytes in a dose-dependent manner. Monocytes were incubated with control media or with the indicated concentrations of morphine for 180 min, and then cultured with PGN. The supernatant was collected and assayed for concentration of TNF and IL-6 120 min after PGN stimulation. Data are the mean sem from 12 different volunteers representing 12 different experiments (monocytes from each healthy volunteer were cultured separately). We used Bonferroni correction for multiple comparisons and adjusted P. *P 0.01 versus PGN alone. (C control media; PGN peptidoglycan; M morphine). (B) Morphine inhibits tumor necrosis factor (TNF) and interleukin (IL)-6 production in peptidoglycan (PGN) stimulated monocytes in a time-dependent manner. Monocytes were incubated with control media or with morphine (105 M) at the indicated incubation times and then cultured with PGN. The supernatant was collected and assayed for concentration of TNF and IL-6 120 min after PGN stimulation. Data are the mean sem from 12 different volunteers representing 12 different experiments (monocytes from each healthy volunteer were cultured separately). We used Bonferroni correction for multiple comparisons and adjusted P. *P 0.01 versus PGN alone. (C control media; PGN peptidoglycan; M morphine).

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 105 M did not prevent the decrease in TNF and IL-6 production induced by morphine. Conversely, the
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morphine-inhibiting effect was reversed when monocytes were treated with the specific opioid receptor antagonist, CTOP, at the concentration of 105 M (Fig. 2). 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 104 M and 103 M. CTOP also prevented the decrease in TNF and IL-6
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production induced by morphine at the concentration of 105 M (Fig. 3). A Monocyte-to-Lymphocyte Contact Modies 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 morphines 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.

Figure 2. Opioid receptor antagonist effects on tumor necrosis factor (TNF) and interleukin (IL)-6 production in peptidoglycan (PGN) stimulated monocytes. Monocytes were incubated with control media or pretreated with opioid receptor antagonists (105 M) for 30 min before morphine (105 M) incubation for 180 min, and then cultured with PGN. The supernatant was collected and assayed for concentration of TNF and IL-6 120 min after PGN stimulation. Data are the mean sem from 11 different volunteers representing 11 different experiments (monocytes from each healthy volunteer were cultured separately). We used Bonferroni correction for multiple comparisons and adjusted P. *P 0.01 versus PGN M; : P 0.01 versus PGN. (C control; PGN peptidoglycan; M morphine; ORA opioid receptor antagonists including NLX, naloxone; NLT, naltribene; nor-BNI, nor-binaltorphimine, CTOP).

Antiinammatory 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.

Figure 3. Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP) and naloxone methiodide (NLX) prevent morphine effects on tumor necrosis factor (TNF) production in peptidoglycan (PGN) stimulated monocytes. Monocytes were incubated with control media or pretreated with the indicated concentrations of naloxone or CTOP for 30 min before morphine (105 M) incubation for 180 min, and then cultured with PGN. The supernatant was collected and assayed for concentration of TNF and IL-6 120 min after PGN stimulation. Data are the mean sem from 12 different volunteers representing 12 different experiments (monocytes from each healthy volunteer were cultured separately). We used Bonferroni correction for multiple comparisons and adjusted P. *P 0.01 versus PGN M; : P 0.01 versus PGN. (C control; PGN peptidoglycan; M morphine; ORA opioid receptor antagonists including NLX, naloxone; NLT, naltribene; nor-BNI, nor-binaltorphimine, CTOP).
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Figure 4. Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP) does not prevent morphine effects on tumor necrosis factor (TNF) and interleukin (IL)-6 production in peptidoglycan (PGN) stimulated peripheral blood mononuclear cells (PBMCs). PBMCs were incubated with control media or pretreated with CTOP (105 M) for 30 min before morphine (105 M) incubation for 180 min, and then cultured with PGN. The supernatant was collected and assayed for concentration of TNF and IL-6 120 min after PGN stimulation. Data are the mean sem from 10 different volunteers representing 10 different experiments (PBMCs from each healthy volunteer were cultured separately). We used Bonferroni correction for multiple comparisons and adjusted P (*P 0.01 versus PGN). (C control; PGN peptidoglycan; M morphine). Figure 5. Interleukin-10 (IL-10) production after peptidoglycan (PGN) stimulation of peripheral blood mononuclear cells (PBMCs) and monocytes with or without morphine pretreatment. PBMCs and monocytes were incubated with control media or with morphine (105 M) for 180 min, and then cultured with PGN. The supernatant was collected and assayed for concentration of IL-10. Data are the mean sem from eight different volunteers representing eight different experiments (PBMCs and monocytes from each healthy volunteer were cultured separately). We used Bonferroni correction for multiple comparisons and adjusted P (*P 0.025 versus control). (C control; PGN peptidoglycan; M morphine). 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. IL-10 Is Released Through a Contact of T Cells with Monocytes but This Release Is Not Involved in the Antiinammatory 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
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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).

DISCUSSION
The present study demonstrates that morphine inhibits TNF and IL-6 production in TLR2-stimulated monocytes in a time and concentration-dependent manner. Opioid receptors specifically mediate this morphineinduced TNF and IL-6 inhibition. A direct monocyte-tolymphocyte 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. Several lines of evidence indicate that morphine severely impairs host defense against bacterial invaders. Indeed, it was demonstrated that PBMCs from
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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, morphines 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 TLR2stimulated 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 109 28 M, and the present results show a significant inhibitory effect of morphine only at a concentration of 105 M. However, the concentration of 105 M could be clinically relevant, since morphine consumption by patients or drug addicts can be very high and can reach plasma concentrations of 25 M.29 Moreover, morphines 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 105 M does not affect cell viability as assessed by tryptan blue exclusion criteria, and the reversibility of morphines 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 opioid receptor antagonist (CTOP), but not with a specific or opioid receptor antagonist. The opioid receptor antagonists concentration used was 105 M, with an incubation
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time of 30 min, as previously described.9,25 In monocytes, morphines effects on the TLR2 pathway are therefore specifically mediated by opioid receptors. In mice with a genetic disruption of the opioid receptor (MOR) gene (MORKO),30 morphines immunosuppressor effects disappeared, highlighting the mediation of morphines immune effects via the opioid receptors. The current results show that morphines inhibitory effects are reversed by CTOP 105 M and reversed by naloxone 104 M. CTOP is 2000-fold more specific to the opioid receptor than naloxone.31 This could explain why no effect was observed when naloxone was used at the dose of 105 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 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 (1/2) increases cytosolic cAMP through a Gi/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 3 opiate receptor.34,35 This receptor causes immunosuppression, at least in part, via the nitric oxidestimulated 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 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 monocytelymphocyte 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 morphines suppressive effects observed in monocytes cultured alone.
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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 monocyteslymphocytes 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 morphines 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 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 opioid receptor signaling pathway. Intracellular mechanisms leading to this inhibitory effect of morphine in the TLR2 signaling pathway remain to be studied. REFERENCES
1. Risdahl JM, Khanna KV, Peterson PK, Molitor TW. Opiates and infection. J Neuroimmunol 1998;83:4 18 2. Peterson PK, Sharp BM, Gekker G, Portoghese PS, Sannerud K, Balfour HH Jr. Morphine promotes the growth of HIV-1 in human peripheral blood mononuclear cell cocultures. AIDS 1990;4:869 73 3. Starec M, Rouveix B, Sinet M, Chau F, Desforges B, Pocidalo JJ, Lechat P. Immune status and survival of opiate- and cocainetreated mice infected with Friend virus. J Pharmacol Exp Ther 1991;259:74550 4. Morgan EL. Regulation of human B lymphocyte activation by opioid peptide hormones. Inhibition of IgG production by opioid receptor class (mu-, kappa-, and delta-) selective agonists. J Neuroimmunol 1996;65:2130 5. Lysle DT, Coussons ME, Watts VJ, Bennett EH, Dykstra LA. Morphine-induced alterations of immune status: dose dependency, compartment specificity and antagonism by naltrexone. J Pharmacol Exp Ther 1993;265:1071 8 6. Chao CC, Molitor TW, Close K, Hu S, Peterson PK. Morphine inhibits the release of tumor necrosis factor in human peripheral blood mononuclear cell cultures. Int J Immunopharmacol 1993;15:44753 7. Roy S, Cain KJ, Chapin RB, Charboneau RG, Barke RA. Morphine modulates NF kappa B activation in macrophages. Biochem Biophys Res Commun 1998;245:392 6 8. Makman MH, Bilfinger TV, Stefano GB. Human granulocytes contain an opiate alkaloid-selective receptor mediating inhibition of cytokine-induced activation and chemotaxis. J Immunol 1995;154:132330 9. Tomassini N, Renaud F, Roy S, Loh HH. Morphine inhibits Fc-mediated phagocytosis through mu and delta opioid receptors. J Neuroimmunol 2004;147:1313

10. Lopker A, Abood LG, Hoss W, Lionetti FJ. Stereoselective muscarinic acetylcholine and opiate receptors in human phagocytic leukocytes. Biochem Pharmacol 1980;29:13615 11. Gaveriaux C, Peluso J, Simonin F, Laforet J, Kieffer B. Identification of kappa- and delta-opioid receptor transcripts in immune cells. FEBS Lett 1995;369:272 6 12. Cohen J, Abraham E. Microbiologic findings and correlations with serum tumor necrosis factor-alpha in patients with severe sepsis and septic shock. J Infect Dis 1999;180:116 21 13. Brun-Buisson C, Doyon F, Carlet J. Bacteremia and severe sepsis in adults: a multicenter prospective survey in ICUs and wards of 24 hospitals. French Bacteremia-Sepsis Study Group. Am J Respir Crit Care Med 1996;154:61724 14. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, Akira S. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 1999;11:44351 15. Lembo A, Kalis C, Kirschning CJ, Mitolo V, Jirillo E, Wagner H, Galanos C, Freudenberg MA. Differential contribution of Tolllike receptors 4 and 2 to the cytokine response to Salmonella enterica serovar Typhimurium and Staphylococcus aureus in mice. Infect Immun 2003;71:6058 62 16. Takeuchi O, Akira S. Toll-like receptors; their physiological role and signal transduction system. Int Immunopharmacol 2001;1: 62535 17. Wang ZM, Liu C, Dziarski R. Chemokines are the main proinflammatory mediators in human monocytes activated by Staphylococcus aureus, peptidoglycan, and endotoxin. J Biol Chem 2000;275:20260 7 18. Wang JE, Jorgensen PF, Almlof M, Thiemermann C, Foster SJ, Aasen AO, Solberg R. Peptidoglycan and lipoteichoic acid from Staphylococcus aureus induce tumor necrosis factor alpha, interleukin 6 (IL-6), and IL-10 production in both T cells and monocytes in a human whole blood model. Infect Immun 2000;68:396570 19. Miltenyi S, Muller W, Weichel W, Radbruch A. High gradient magnetic cell separation with MACS. Cytometry 1990;11:231 8 20. Kabelitz D. Expression and function of Toll-like receptors in T lymphocytes. Curr Opin Immunol 2007;19:39 45 21. Peterson PK, Gekker G, Brummitt C, Pentel P, Bullock M, Simpson M, Hitt J, Sharp B. Suppression of human peripheral blood mononuclear cell function by methadone and morphine. J Infect Dis 1989;159:480 7 22. Wang J, Barke RA, Charboneau R, Roy S. Morphine impairs host innate immune response and increases susceptibility to Streptococcus pneumoniae lung infection. J Immunol 2005;174:426 34 23. Peterson PK, Sharp B, Gekker G, Brummitt C, Keane WF. Opioid-mediated suppression of interferon-gamma production by cultured peripheral blood mononuclear cells. J Clin Invest 1987;80:824 31 24. Tubaro E, Borelli G, Croce C, Cavallo G, Santiangeli C. Effect of morphine on resistance to infection. J Infect Dis 1983;148:656 66 25. Perez-Castrillon JL, Perez-Arellano JL, Garcia-Palomo JD, Jimenez-Lopez A, De Castro S. Opioids depress in vitro human monocyte chemotaxis. Immunopharmacology 1992;23:57 61 26. Welters ID, Menzebach A, Goumon Y, Cadet P, Menges T, Hughes TK, Hempelmann G, Stefano GB. Morphine inhibits NF-B nuclear binding in human neutrophils and monocytes by a nitric oxide-dependent mechanism. Anesthesiology 2000;92: 1677 84 27. Cockerill FR, III, Hughes JG, Vetter EA, Mueller RA, Weaver AL, Ilstrup DM, Rosenblatt JE, Wilson WR. Analysis of 281,797 consecutive blood cultures performed over an eight-year period: trends in microorganisms isolated and the value of anaerobic culture of blood. Clin Infect Dis 1997;24:40318 28. Stahl KD, van Bever W, Janssen P, Simon EJ. Receptor affinity and pharmacological potency of a series of narcotic analgesic, anti-diarrheal and neuroleptic drugs. Eur J Pharmacol 1977;46: 199 205 29. LeVier DG, McCay JA, Stern ML, Harris LS, Page D, Brown RD, Musgrove DL, Butterworth LF, White KL Jr, Munson AE. Immunotoxicological profile of morphine sulfate in B6C3F1 female mice. Fundam Appl Toxicol 1994;22:525 42 30. Gaveriaux-Ruff C, Matthes HW, Peluso J, Kieffer BL. Abolition of morphine-immunosuppression in mice lacking the muopioid receptor gene. Proc Natl Acad Sci USA 1998;95:6326 30

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Morphine Immunomodulation in Monocytes

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Vol. 106, No. 4, April 2008

2008 International Anesthesia Research Society

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