Mu receptor 4.pdf

7/28/2019 Mu receptor 4.pdf

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Anesthetic Pharmacology

Preclinical PharmacologySection Editor: Marcel E. Durieux

Clinical PharmacologySection Editor: Tony Gin

The Opioid Receptor Mediates Morphine-InducedTumor Necrosis Factor and Interleukin-6 Inhibition inToll-Like Receptor 2-Stimulated Monocytes

Marie-Pierre Bonnet, MD*

Helene Beloeil, MD, PhD*

Dan Benhamou, MD*

Jean-Xavier Mazoit, MD, PhD*

Karim Asehnoune, MD, PhD

BACKGROUND: Morphine possesses immunomodulatory effects but its intrinsicmechanisms, especially in the toll-like receptor 2 (TLR2) signaling pathway, areonly partially understood. In this study, we evaluated the effects of morphine ontumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-10 (IL-10) pro-duction in TLR2-stimulated human monocytes and identified the involvement ofthe different opioid receptors, and of the lymphocyte-to-monocyte contact.METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from freshblood by centrifugation on a density gradient. Monocytes were secondarilyseparated using a high-gradient magnetic cell sorting kit with specific anti-CD14antibodies. Monocytes or PBMCs were pretreated with opioid receptors antago-nists before being cultured with morphine and peptidoglycan (PGN) from Staphy-lococcus 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 productionof cytokines in a dose and concentration-dependent manner in PGN-stimulatedmonocytes. Opioid receptor activation specifically mediated this morphine-induced TNF and IL-6 inhibition in monocytes. Morphine significantly inhibitedthe TNF, but not the IL-6 production, in PGN-stimulated PBMCs. The opioidreceptor 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-6production after PGN stimulation in either monocytes or PBMCs cultures.CONCLUSIONS: The opioid receptor mediates morphine-induced TNF and IL-6inhibition in PGN-stimulated monocytes, but not in PBMCs. A direct monocyte-to-lymphocyte contact (PBMCs) alters the inhibitory effects of morphine observedon monocytes alone. IL-10 is not a factor for the inhibition of TNF or for IL-6production. Interactions between TLR2 and opioid intracellular pathways remainto be studied to delineate these morphine immunosuppressive effects.(Anesth Analg 2008;106:11429)

Morphine and other opioids are potent immuno-modulators.1 It has been suggested that chronic opioidusers, for either therapeutic reasons or because ofaddiction, are more susceptible to bacterial and viralinfections.2,3 However, clinical evidence demonstrat-ing enhanced infectious susceptibility in opioid-usingchronic pain patients is still missing. Opioid adminis-tration affects both innate and adaptative immunity,

such as antibodies production,4 natural killer activity,5

cytotoxicity, cytokine production,6,7 chimiotaxism,8

and phagocytosis.9 Monocytes play a central role ininnate immunity. The presence of opioid receptors onmonocytes has been demonstrated,10 strengtheningthe link between immunity and opioid drugs, al-though this link has been questioned by others.11

Morphines effects on innate immunity have been

mainly studied in different cell populations after expo-sure of cells to lipopolysaccharide (LPS) from Gram-negative bacteria. However, infections by Gram-positive

bacteria have been increasing during the last 20 yr12 andare of poor prognosis.13 Host-defense mechanismsagainst bacterial infections are under the control oftoll-like receptors (TLRs). TLRs play a critical role ininnate defense by sensing specific molecular patternsassociated with microbial pathogens. Two TLRs areespecially involved in specific identification of bacterialcomponents: TLR4 recognizes the LPS from Gram-

negative bacteria and TLR2 recognizes cocci Gram-positive components such as peptidoglycan (PGN).14,15

From the *Hopital Bicetre, AP-HP, Le Kremlin-Bicetre, cedex,France; and Hopital Hotel-Dieu, Nantes, France.

Accepted for publication on November 28, 2007.

Supported by the association Mises Au Point en Anesthesie etReanimation, Le Kremlin-Bicetre, France.

Presented, in part, during the 47th Congress of the Societe FrancaisedAnesthesie-Reanimation, Paris, France, September 22, 2005.

Address correspondence and reprint requests to Marie-PierreBonnet, MD, Departement dAnesthesie Reanimation Chirurgicale,Groupement Hospitalier Universitaire Sud, Hopital Bicetre, 78, ruedu General Leclerc, 94275 Le Kremlin-Bicetre, cedex, France. Ad-dress e-mail to [email protected].

Copyright 2008 International Anesthesia Research Society

DOI: 10.1213/ane.0b013e318165de89

Vol. 106, No. 4, April 20081142


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PGN stimulates TLR2, leading to nuclear factor -B(NFB) activation16 and to release of proinflammatorycytokines, such as tumor necrosis factor (TNF) andinterleukin-6 (IL-6).17,18 The inhibitory effects of mor-phine on LPS-induced TNF and IL-6 production have

been reported in monocytes.6,7 IL-10 is an antiinflamma-tory cytokine, which classically inhibits proinflamma-tory cytokine production, as TNF and IL-6, especially inmonocyte cellular models. A potential mechanism of

morphines inhibitory effects could be through enhanc-ing IL-10 production. Despite the clinical importance ofGram-positive infections, the effects of morphine on theTLR2 signaling pathway in monocytes have not beendelineated.

The purpose of the present study was therefore (i)to investigate effects of morphine on TNF and IL-6production by human monocytes after TLR2 stimula-tion by PGN, (ii) to examine the implication of thedifferent opioid receptors using specific antagonistreceptors, (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.

METHODS

Reagents

RPMI 1640 with 1% ultraglutamine was provided byBiowittaker (Cambrex Bioscience, Verniers, Belgique).Phosphate buffered saline was obtained from Gibco,life technologies (Cergy-Pontoise, France). Sterile tubesand 24-well plates were purchased from ATGC bio-technology (Marne-La-Vallee, France). Morphine chlo-rhydrate (AP-HP) without preservative was obtained

from the pharmacy department of Bicetre Hospital.Staphylococcus aureus ultrapure PGN and opioid recep-tor antagonists were purchased from Fluka BioChemicalaboratories (Sigma-Aldrich; Saint Quentin Fallavier,France). Opioid receptor antagonists used were a non-specific antagonist (naloxone methiodide [NLX]); a opioid receptor-specific antagonist (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]). Lympholytewas purchased from Cederlane (Cederlane, Tebu-bio

Le Perrey-en-Yvelines, France). The gradient magneticcell sorting kit used to isolate monocytes was pur-chased from Miltenyi Biotec (Miltenyi Biotec, Paris)as well as the anti-CD14 antibodies. Mallassez cells forcell count were purchased from Glasstic (Glasstic

Kova Hycor Biomedical Inc., Garden Grove, CA). Allother 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 afterinformed consent was obtained. For each donor, 40

mL of blood was used. Exclusion criteria were subjectswho were younger than 18-yr-of-age, were pregnant,had used steroids or any medication, or had any focusof infection within the previous 30 days.

Cell Preparation

PBMCsPBMCs were isolated from blood freshly collected

in sodium citrate by use of lympholyte, a densitygradient solution. In brief, 4 mL of blood was layeredon 4 mL of lympholyte in a sterile 15-mL tube (Falcon;Becton Dickinson) and was centrifuged for 35 min at800g and at 20C. The ring of PBMCs (milky interface)was recovered and washed twice with RPMI 1640medium containing 1% ultraglutamine. Cells wereresuspended in RPMI 1640 medium with 0.2% ofhuman serum. PBMCs obtained were directly platedand cultured or used for isolation of monocytes.

MonocytesA fraction of PBMCs, obtained as described above,

was immediately used for monocyte isolation, whichwas performed by using a high-gradient magnetic cellsorting kit19 in accordance with the manufacturer recom-mendations (Miltenyi Biotec, Paris). In brief, PBMCswere suspended in phosphate buffered saline with eth-ylene diamine tetra acetic acid 2 mm and 5% humanserum. Monocytes were magnetically labeled with mag-netic microbeads coupled to a specific anti-CD14 anti-

body added to the cell suspension (i.e., PBMCs) andincubated for 20 min at 4C. The cell suspension wasthen 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 andother cells were eluted from the column. Monocyteswere recovered by flushing through the column. Cellpurity with this technique is 94% of CD14 cells.

CulturesPGN, morphine, and opioid receptor antagonists

were made fresh for each experiment. For each experi-mental condition, at the end of the cell culture, wechecked the viability of cells by a tryptan blue exclu-sion 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 CulturesMonocytes were counted on Mallassez cells before

being plated and cultured. Monocytes were plated in24-well plates (final concentration 1.106 cells/0.5 mL/well)and were cultured in RPMI 1640 with 0.2% of humanserum 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 ofmorphine on TNF and IL-6 production were investi-gated. In the main study, the role of the opioid receptors in

morphine-induced inhibition on TNF and IL-6 pro-duction was investigated by using opioid receptor

Vol. 106, No. 4, April 2008 2008 International Anesthesia Research Society 1143


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antagonists. In brief, monocytes were incubated withcontrol media or pretreated with the indicated concen-trations of opioid receptor antagonists for 30 min

before morphine (105 M) incubation for 180 min. Cultureswere stimulated with PGN for 120 min before theculture fluid was harvested and centrifuged at 4C for10 min. The supernatant was kept at 70C before theTNF, IL-6, and IL-10 measurements.

PBMCs CulturesPBMCs were counted on Mallassez cells before being

plated and cultured. PBMCs were plated in 24-wellplates (final concentration 4.106 cells/0.5 mL/well) andcultured in RPMI 1640 with 0.2% of human serum in a5% CO2 incubator at 37C. PBMCs were incubated withcontrol media or pretreated with CTOP (105 M) for 30min before morphine (105 M) incubation for 180 min.Cultures were stimulated with PGN for 120 min beforethe culture fluid was harvested and centrifuged at 4Cfor 10 min. The supernatant was kept at 70C beforethe TNF, IL-6, and IL-10 measurements.

Cytokine MeasurementThe amount of TNF, IL-6, and IL-10 was measured

with a commercial enzyme-linked immunosorbentassay kit (Duoset, R&D systems, Abingdon, UnitedKingdom), according to the manufacturers instruc-tions. TNF and IL-6 concentrations in the absence ofstimulation (control group: C) were always very lowin all experiments, indicating that the cultures platesdid not stimulate monocytes or PBMCs.

Statistical Analysis

The distribution of cytokine concentrations in eachunit was checked for normality using the Shapiro-Wilktest. The results were evaluated by a one-way analysis ofvariance followed by a NewmanKeuls test for inter-group comparison. We used Bonferroni correction formultiple comparisons, and adjusted P for each compari-son. Results are expressed as mean sem.

RESULTS

Proinflammatory Cytokines Production

Suppressive Effects of Morphine on PGN-Induced

TNF and IL-6 Production in MonocytesMorphine alone did not activate monocytes cell cul-tures (Fig. 1A). Concentrations of TNF and IL-6 wereincreased in supernatants of monocytes stimulated withPGN. The release of TNF and IL-6 from PGN-stimulatedmonocytes was inhibited in a dose and time-dependentmanner by morphine (Figs. 1A and B).

Opioid Receptor Mediates Morphine-Induced TNFand IL-6 Inhibition in PGN-Stimulated MonocytesNonspecific antagonist NLX, and receptor antago-

nists (NLT and nor-BNI, respectively) at the concentra-

tion of 10

5

M did not prevent the decrease in TNF andIL-6 production induced by morphine. Conversely, the

morphine-inhibiting effect was reversed when mono-cytes were treated with the specific opioid receptorantagonist, CTOP, at the concentration of 105 M (Fig. 2).

Monocytes cell cultures were then pretreated withvarying CTOP and NLX concentrations (Fig. 3). Opi-oid receptor antagonists alone did not activate cellcultures. Treatment of monocytes with the nonspecificantagonist 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

Figure 1. (A) Morphine inhibits tumor necrosis factor (TNF)and interleukin (IL)-6 production in peptidoglycan (PGN)stimulated monocytes in a dose-dependent manner. Mono-cytes were incubated with control media or with the indicatedconcentrations of morphine for 180 min, and then culturedwith PGN. The supernatant was collected and assayed forconcentration of TNF and IL-6 120 min after PGN stimulation.Data are the mean sem from 12 different volunteers repre-senting 12 different experiments (monocytes from each healthy

volunteer were cultured separately). We used Bonferroni cor-rection for multiple comparisons and adjusted P. *P 0.01versus 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 mor-phine (105 M) at the indicated incubation times and thencultured with PGN. The supernatant was collected and as-sayed for concentration of TNF and IL-6 120 min after PGNstimulation. Data are the mean sem from 12 differentvolunteers representing 12 different experiments (monocytesfrom each healthy volunteer were cultured separately). Weused Bonferroni correction for multiple comparisons and ad-justed P. *P 0.01 versus PGN alone. (C control media;

PGN peptidoglycan; M morphine).

1144 Morphine Immunomodulation in Monocytes ANESTHESIA & ANALGESIA


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production induced by morphine at the concentrationof 105 M (Fig. 3).

A Monocyte-to-Lymphocyte Contact Modifies theImmunosuppressive Effect of Morphine Observed onMonocytes Cultured AloneIt 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 istherefore possible that a monocyte/lymphocyte inter-action modifies morphines suppressive effects ob-served on monocytes cultured alone. To test thishypothesis, we therefore examined whether morphineexerted a suppressive effect on the production of TNFand 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-stimulatedPBMCs was inhibited by morphine. Moreover, CTOPdid not prevent the decrease in TNF productioninduced by morphine. The monocyte/lymphocyte in-teractions alter the immunosuppressive effect of mor-phine observed on monocytes alone.

Antiinflammatory Cytokines Production

IL-10 Is Not Involved in Morphine-Induced TNF andIL-6 Inhibition in PGN-Stimulated MonocytesIL-10 is an immunosuppressive cytokine produced

by a variety of cell types including monocytes and Tlymphocytes. Thus, IL-10 seemed to be a potentialcandidate for the morphine-induced TNF and IL-6inhibition in PGN-stimulated monocytes.

Figure 2. Opioid receptor antagonist effects on tumor necro-sis factor (TNF) and interleukin (IL)-6 production in pepti-doglycan (PGN) stimulated monocytes. Monocytes wereincubated with control media or pretreated with opioidreceptor antagonists (105 M) for 30 min before morphine (105

M) incubation for 180 min, and then cultured with PGN. Thesupernatant was collected and assayed for concentration ofTNF and IL-6 120 min after PGN stimulation. Data are themean sem from 11 different volunteers representing 11different experiments (monocytes from each healthy volun-teer were cultured separately). We used Bonferroni correc-tion for multiple comparisons and adjusted P. *P 0.01versus PGN M; : P 0.01 versus PGN. (C control;PGN peptidoglycan; M morphine; ORA opioidreceptor antagonists including NLX, naloxone; NLT, nal-tribene; nor-BNI, nor-binaltorphimine, CTOP).

Figure 3. Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP) and naloxone methiodide (NLX) prevent morphine effects ontumor necrosis factor (TNF) production in peptidoglycan (PGN) stimulated monocytes. Monocytes were incubated withcontrol 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 TNFand IL-6 120 min after PGN stimulation. Data are the mean sem from 12 different volunteers representing 12 differentexperiments (monocytes from each healthy volunteer were cultured separately). We used Bonferroni correction for multiplecomparisons 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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No IL-10 production was detected in the monocytescell cultures after PGN stimulation with or withoutmorphine pretreatment (Fig. 5). These results indicatethat IL-10 is not a factor for morphine-induced suppres-

sion of the production of TNF and IL-6 in culturedmonocytes.

IL-10 Is Released Through a Contact of T Cellswith Monocytes but This Release Is Not Involvedin the Antiinflammatory Effects of MorphineWe hypothesized that a cellular interaction between

monocytes and T lymphocytes could be involved inIL-10 production after PGN stimulation. IL-10 produc-tion was therefore measured in human PBMCs cultures.A basal production of IL-10 (control) was detected inPBMCs cultures, and this production was significantly

enhanced after PGN stimulation (Fig. 5). However, pre-treatment with morphine did not further enhance the

production of IL-10 observed with PGN. These resultsindicate that IL-10 is not involved in the suppressiveeffects of morphine on PGN-induced TNF production inPBMCs cultures (Fig. 5).

DISCUSSION

The present study demonstrates that morphine inhib-its TNF and IL-6 production in TLR2-stimulated mono-cytes in a time and concentration-dependent manner. Opioid receptors specifically mediate this morphine-induced TNF and IL-6 inhibition. A direct monocyte-to-lymphocyte contact (PBMCs) alters the inhibitory effectsof morphine observed on monocytes alone. IL-10 is not afactor for the inhibition of TNF and IL-6 production.

Several lines of evidence indicate that morphine

severely impairs host defense against bacterial invad-ers. Indeed, it was demonstrated that PBMCs from

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 for180 min, and then cultured with PGN. The supernatant was collected and assayed for concentration of TNF and IL-6 120 minafter PGN stimulation. Data are the mean sem from 10 different volunteers representing 10 different experiments (PBMCsfrom each healthy volunteer were cultured separately). We used Bonferroni correction for multiple comparisons and adjustedP (*P 0.01 versus PGN). (C control; PGN peptidoglycan; M morphine).

Figure 5. Interleukin-10 (IL-10) produc-tion after peptidoglycan (PGN) stimula-tion of peripheral blood mononuclearcells (PBMCs) and monocytes with orwithout morphine pretreatment. PBMCsand monocytes were incubated withcontrol media or with morphine (105

M) for 180 min, and then culturedwith PGN. The supernatant was col-lected and assayed for concentrationof IL-10. Data are the mean semfrom eight different volunteers repre-senting eight different experiments(PBMCs and monocytes from eachhealthy volunteer were culturedseparately). We used Bonferroni cor-rection for multiple comparisons andadjusted P (*P 0.025 versus con-trol). (C control; PGN pepti-doglycan; M morphine).



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patients treated with methadone had a significantlyimpaired capacity to generate reactive oxygen speciesinvolved in host immune response.21 In a mousemodel of cocci Gram-positive pneumonia, animalstreated with morphine had an increased mortalityrate, an increased rate of bacterial growth, a decreasein TNF and IL-6 production in bronchoalveolar lavageand a decreased NF-B activation compared withcontrol mice.22 In monocytes or PBMCs, several stud-

ies have shown that morphine has an immunosup-pressive effect on different cell types and throughdifferent signaling pathways. First, Peterson et al.demonstrated in PBMCs that concanavalin A-inducedinterferon production was inhibited by morphine.23

The same authors subsequently showed that mor-phine decreased TNF production after TLR4 stimula-tion with LPS.6 Other studies have revealed thatmorphine decreases phagocytosis activity,24 chimio-taxism,25 and NF-B activity after TLR4 stimulationwith LPS.26 Regarding TLRs signaling, morphinesimmunomodulatory effects were evaluated only in the

TLR4 pathway. However, TLR2-induced monocytesresponses are likely to have important clinical conse-quences, as Gram-positive organisms are an increas-ingly growing cause of severe infections associatedwith organ dysfunction, including septic shock.27 Thisstudy shows for the first time that morphine pretreat-ment induces a time and concentration-dependentinhibition of TNF and IL-6 production in TLR2-stimulated monocytes. Recent evidence has suggestedthat signals others than those from TLRs could con-tribute to PGN recognition. Indeed, a family of intra-cellular proteins, named NOD1 and NOD2, senses

degradation products of PGN. However, both sys-tems, i.e., TLR2 and NOD, activate NF-B, leading tothe production of proinflammatory cytokines.

The opioid receptor affinity for morphine is 109

M,28 and the present results show a significant inhibi-tory effect of morphine only at a concentration of 105

M. However, the concentration of 105 M could beclinically relevant, since morphine consumption bypatients or drug addicts can be very high and canreach plasma concentrations of 25 M.29 Moreover,morphines effects were studied in isolated monocytecultures. Cell interactions and plasma protein inter-

ventions are not considered in such a cell culturemodel. Another explanation for the inhibitory effectobserved in these experiments could be related to thedirect toxicity of morphine. However, morphine at aconcentration of 105 M does not affect cell viability asassessed by tryptan blue exclusion criteria, and thereversibility of morphines inhibitory effect by CTOPconfirms the absence of toxicity.

In the present study, the inhibitory effects of mor-phine on TNF and IL-6 production were reversed withnaloxone and with a specific opioid receptor antag-onist (CTOP), but not with a specific or opioid

receptor antagonist. The opioid receptor antagonistsconcentration used was 105 M, with an incubation

time of 30 min, as previously described.9,25 In mono-cytes, morphines effects on the TLR2 pathway aretherefore specifically mediated by opioid receptors.In mice with a genetic disruption of the opioidreceptor (MOR) gene (MORKO),30 morphines immu-nosuppressor effects disappeared, highlighting the me-diation of morphines immune effects via the opioidreceptors. The current results show that morphines

inhibitory effects are reversed by CTOP 10

5

M andreversed by naloxone 104 M. CTOP is 2000-fold morespecific to the opioid receptor than naloxone.31 Thiscould explain why no effect was observed when nalox-one was used at the dose of 105 M.

TLR2 agonists induce the production of proinflam-matory cytokines (TNF, IL-6), especially through theactivation of the NF-B pathway. At least two differ-ent mechanisms mediated by opioid receptors might

be involved in morphine-induced TNF and IL-6 inhi-bition in TLR2 stimulated monocytes. First, chronicexposure to agonists of classical opiate receptor

(1/2) increases cytosolic cAMP through a Gi/-coupled receptor mechanism, and there is strongevidence that this increase of cAMP acts as an inhib-itor of NF-B.32,33 However, the time of exposure thatdefines a chronic exposure to morphine in cell culturesremains controversial. Second, morphine exerts itsimmunomodulatory effects in immunocytes (i.e.,granulocytes, monocytes) through the nonclassical 3opiate receptor.34,35 This receptor causes immunosup-pression, at least in part, via the nitric oxide-stimulated depression of NF-B nuclear binding. Ourresults also show an apparent stimulatory effect of

CTOP on IL-6 production (Figs. 2 and 3). An effect ofCTOP on IL-6 production that is not solely due to itsaction at the opioid receptor cannot be excluded.

It is generally accepted that cell-to-cell interactionsbetween monocytes and T cells are required for aneffective immune response.36 To gain further insight intothe immunosuppressive effects of morphine, we studiedthe role of lymphocyte-to-monocyte contact throughPBMCs cultures (i.e., monocytes and lymphocytes). Atleast two distinct mechanisms by which monocyte-lymphocyte interaction could interfere with the sup-pressive effect of morphine might be involved.36 First,

the expression of cell-surface molecules associatedwith the cell-to-cell contact between monocytes and Tcells (CD28 and/or CTLA-4 on T cells and theirligands CD80 and/or CD86 on monocytes; and CD40on monocytes and CD40 ligand on T cells) may bealtered. Second, the inhibitory mediators released bylymphocytes (IL-10, IL-5, IL-4) could modulate theeffects 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 mor-phine, indicating that a monocyte/lymphocyte inter-

action interferes with morphines suppressive effectsobserved in monocytes cultured alone.



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IL-10 is a major antiinflammatory cytokine, knownto inhibit TNF and IL-6 production in human mono-cytes after LPS stimulation by decreasing NF-B acti-vation. An increased level of IL-10 in the cellularcultures could, therefore, be a likely explanation forthe morphine inhibitory effects observed in the presentresults. However, no IL-10 production was detected inmonocyte cultures, regardless of the experimentalconditions used. We hypothesized that a cell interac-tion between monocytes and lymphocytes could beinvolved in the induction of IL-10 production.36 Tostudy the effect of monocyteslymphocytes interac-tion on IL-10 production, we cultured PBMCs. IL-10production was increased after stimulation with PGNin PBMCs cultures, but this increase was not modifiedin the presence of morphine. Thus, IL-10 does not playa role in morphines inhibition of TNF production inPBMCs cultures after stimulation with PGN.

In conclusion, this study demonstrates that there is aninhibitory effect of morphine on proinflammatory cyto-kine production in human monocytes after TLR2 stimu-lation, and that this inhibition is mediated solely by the opioid receptor. A direct monocyte-to-lymphocytecontact (PBMCs) alters the inhibitory effects of morphineobserved on monocytes alone. IL-10 is not a factor for theinhibition of TNF and IL-6 production. Finally, this workhighlights the interaction between the TLR2 signalingpathway and the opioid receptor signaling pathway.Intracellular mechanisms leading to this inhibitory effectof morphine in the TLR2 signaling pathway remain to bestudied.

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