Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0406237 on October 5, 2006

Published online before print October 5, 2006
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(Journal of Leukocyte Biology. 2007;81:344-354.)
© 2007 by Society for Leukocyte Biology

Differential involvement of RelB in morphine-induced modulation of chemotaxis, NO, and cytokine production in murine macrophages and lymphocytes

Cataldo Martucci, Silvia Franchi, Donatella Lattuada, Alberto E. Panerai and Paola Sacerdote1

Department of Pharmacology, University of Milan, Milan, Italy

1Correspondence: Dept. of Pharmacology, University of Milano, Via Vanvitelli 32, 20129 Milano, Italy. E-mail: paola.sacerdote{at}unimi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute morphine impairs innate and acquired immunity. The mechanisms involved in immunosuppression have not been well defined yet. The transcription factor NF-{kappa}B is a central regulator of immunity, and of the NF-{kappa}B family, RelB is particularly involved in the expression of genes important in immune responses. We investigated the involvement of RelB in morphine-induced immunosuppression in mice deficient for the RelB factor. RelB–/– mice and wild-type (WT) controls were injected s.c. with morphine 20 mg/Kg, and 1 h later, immune parameters were evaluated. Morphine significantly reduced macrophage production of the proinflammatory cytokines IL-1ß, TNF-{alpha}, and IL-12 in WT animals, and the drug failed to diminish the production of these cytokines in the RelB–/– mice. In contrast, the anti-inflammatory cytokine IL-10 was similarly affected in the two strains. Macrophage NO production was modulated by morphine in WT animals only, and morphine similarly decreased macrophage chemotaxis in the presence or in the absence of RelB. When Th1 and Th2 cytokines were evaluated, we observed a clear morphine-induced reduction of IL-2 and IFN-{gamma} production by WT splenocytes, whereas no effect of the drug was observed in RelB–/– mice. On the contrary, the production of the Th2 cytokines IL-4 and IL-10 was lessened to the same degree by morphine in WT and RelB–/– mice. In conclusion, our data suggest that RelB is an important target for morphine modulation of proinflammatory and Th1 cytokines. They also indicate that morphine uses multiple intracellular pathways to exert its generalized immunosuppression.

Key Words: monocytes • opioid receptors • Th1/Th2 • transcription factors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Opioid drugs remain the mainstay treatment for acute and chronic pain conditions besides still being common drugs of abuse. In vivo administration of the opioid drug morphine induces a decrease of multiple immune parameters, affecting almost all cells of innate and acquired immunity [1 , 2 ]. There is considerable evidence that in vivo morphine modulates most T cell functions. Independently of the stimulus used (polyclonal mitogens, CD3 activation, antigen-specific challenge), T lymphocyte proliferation is decreased by acute and chronic morphine administration [1 2 3 4 5 6 ]. Also, many cytokines such as IL-2 and IFN-{gamma} are greatly affected [1 , 5 , 6 ]. Clear inhibitory effects of in vivo administration of morphine on monocyte/macrophage functions have been described consistently [7 , 8 ]. We previously showed that acute morphine administration significantly reduced IL-12 and IL-10 production by LPS-stimulated macrophages [7 ]. Moreover, morphine also affects precocious steps of macrophage activation by decreasing their chemotactic properties [9 ]. The impairment is evident in circulating, peritoneal, alveolar, or splenic macrophages, indicating a general down-regulation of innate immunity [1 , 7 , 8 ]. Convincing evidence of the morphine immmunosuppressive effect comes from several experiments associating opioids to microbial pathogen infections in the animal as well as in opioid-addicted humans [10 ]. Drugs of abuse are in fact associated with increased susceptibility to infectious diseases, especially opportunistic intracellular microbial infection and HIV infection [11 ]. The effects of morphine on the immune system are mediated throughout central as well as peripheral mechanisms [1 , 12 ]. Morphine interacts with opioid receptors, present on the cells of the immune system, or with receptors within the nervous system. The potential mechanisms by which central opioid receptors modulate peripheral immune responses may involve the hypothalamic pituitary adrenal axis and the autonomic nervous system [12 , 13 ]. It has been demonstrated that lymphocytes and mononuclear phagocytes express classical µ, {kappa}, and {delta} opioid receptors, functionally coupled to signal transduction mechanisms, involving mainly Gi protein activity [1 , 6 , 8 , 13 ]. By binding to these receptors, morphine has been shown to trigger the reported responses in immune cells.

Although morphine immunomodulating properties are clear and increasingly becoming a clinical problem in pain therapy [1 ], its intracellular molecular targets in immune cells are far from being elucidated. Recent evidence has suggested that morphine can impair macrophage function by interfering with transcriptional activation of NF-{kappa}B [14 ]. The transcription factor NF-{kappa}B plays a pivotal role in immune responses, inflammation, apoptosis, and cancer [15 16 17 ]. Five members of this family have been identified in mammals: RelA(p65), c-Rel, RelB, NF-{kappa}B1 (encoding the precursor form p100 and the processed molecule p50), and NF-{kappa}B2 (encoding the precursor p105 and the processed form p52) [15 , 16 ]. The different members form a variety of heterodimers and homodimers. They are associated with inhibitory proteins, the I{kappa}Bs, or with the precursors p100/105, which retain the dimers in the cytoplasm [18 ]. A wide variety of stimuli leads to the degradation of I{kappa}B and of the p100/p105 precursors, resulting in the nuclear translocation of Rel/NF-{kappa}B complexes and the regulation of target genes.

RelB alone does not bind to the DNA but must dimerize with p50 or p52 to form transcriptional activators [18 , 19 ]. In the mouse, high levels of the RelB/p50 or p52 heterodimers are mainly restricted to different immune cell populations. In spleen, thymus, and dendritic cells, the RelB/p50 or p52 heterodimers have been shown to have a role in the constitutive expression of {kappa}B-regulated genes [19 , 20 ]. Consistently, the RelB-deficient mice display several different immune abnormalities [21 22 23 ]. The RelB factor appears therefore a good candidate as the target for the immunopharmacological effects of morphine.

In the present study, we have examined whether the effects of acute morphine administered in vivo to mice on several macrophage and splenocyte functions are also maintained in mice deficient for the RelB factor. In these animals, we evaluate the impact of morphine on macrophage chemotaxis, NO production, pro- and anti-inflammatory, and Th1/Th2 cytokines. Moreover, to be reassured of the specificity of RelB to the immune system, we also checked the analgesic response to morphine in RelB wild-type (WT) and knockout (KO) animals. Although morphine analgesia is conserved in RelB-deficient mice, we demonstrate that RelB might be the target for morphine-induced inhibition of proinflammatory and Th1 cytokines and for NO modulation, and to decrease IL-10, IL-4, and macrophage chemotaxis, morphine does not need a functional RelB system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and drugs
The RelB-deficient mice (gift of Dr. Paola Ricciardi Castagnoli and Dr. D. Lo) used in the study have been characterized extensively [19 20 21 22 23 ]. All the animals used were males, their age ranged from 4 to 6 weeks, and they were healthy at the time they were used in these studies. As controls, C57BL/6 (Charles River, Calco Italy) WT, age-matched animals were used. All the animals were kept within the animal facilities of the Department of Pharmacology (University of Milano, Italy) with a light:dark cycle of 12:12 h and had water and food ad libitum. The Institutional Review Board of the Department of Pharmacology of the University of Milano approved all experiments performed. Morphine hydrochloride (Salars, Como, Italy) was dissolved in a solution of 0.9% NaCl and administered s.c.

Evaluation of nociceptive thresholds
The hot-plate test [24 ] was used to assess nociceptive thresholds in untreated mice and to evaluate the analgesic effect induced by acute administration of morphine. In our experimental conditions, the hot plate was maintained at 54°C. Each animal was placed on the heated surface, and the time interval (seconds) between placement and the simultaneous licking of both fore paws was recorded. The cut-off time, chosen to avoid tissue damage, was 30 s. The doses of morphine tested were 1.25 and 2.5 mg/Kg, 30 min after s.c. injection. Thresholds are expressed as latencies in seconds.

Treatment protocols for immunological studies
WT and RelB–/– animals were administered s.c., 1h 20 mg/Kg morphine or saline and were killed by cervical dislocation 1 h after treatment. Dose and timing were chosen on the basis of previous experiments from our laboratory [2 , 4 , 5 , 7 ].

Harvest of resident peritoneal macrophages
Resident peritoneal cells (PC) from saline- and morphine-treated WT and RelB–/– animals were harvested in cold RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO) plus 10% FCS (Gibco-BRL, Life Technology, Italy). Viability of cells was checked by the trypan blue exclusion test. PC from three mice were pooled and resuspended in 10% FCS RPMI 1640 at 1 x 106/ml, and 1 ml/well aliquots were dispensed into 24-well culture plates. Isolation and purification of macrophages were carried out by adherence. After a period of 2 h, nonadherent cells were removed, and adherent cells were washed twice with warm RPMI plus 10% FCS [7 ]. Differential staining with Diff-Quick (Dade, Biomap, Italy) and nonspecific esterase staining with {alpha}-naphthyl acetate (Sigma Chemical Co.) were used to assess the percentage of macrophages in the PC. Moreover, a FACS analysis with the typical macrophage surface marker F4/80 antibody was performed on cell preparation as described below. The percentage of macrophages in the adherent cells was ≥88%.

Macrophage cytokine production
Macrophages from saline- and morphine-treated WT and RelB–/– animals were incubated with 1 µg/ml LPS (serotype Escherichia coli 0111:B4, Sigma Chemical Co.) for IL-10, IL-1ß, and TNF-{alpha} or with 1 µg/ml LPS and 50 U/ml IFN-{gamma} for IL-12 stimulation. The different stimuli were added to the macrophage cultures in a final volume of 1 ml/well RPMI plus 10% FCS, 1% glutamine (Sigma Chemical Co.), 2% penicillin/streptomycin solution (Sigma Chemical Co.), and 0.1% 2-ME (complete medium, Sigma Chemical Co.). The plates were incubated at 37°C and 5% CO2, and supernatants were collected after 24 h in culture and stored frozen at –80°C for cytokine analysis [7 , 25 ].

Chemotaxis
Peritoneal macrophages from saline- and morphine-treated WT and RelB–/– animals were collected as described above and pooled. Cells and chemoattractant substances were suspended in RPMI 1640 plus BSA 1%. Chemotaxis was measured using a Boyden-modified, 48-well microchemotaxis chamber, in which a polycarbonate filter (Biomap, Agrate Brianza, Italy) with a pore diameter of 5 µm separated the upper and lower compartments. Cells (2x106 cells/ml, 10x104 macrophages/well) were placed in the upper chamber, and aliquots of medium (to evaluate spontaneous mobility) or of the chemoattractant fMLP (10–8M) were added in the lower chamber (to evaluate chemotaxis). The chambers were incubated for 90 min at 37°C in 5% CO2 atmosphere, and then, the migrated cells that adhered to the distal part of the filters were fixed and stained. Migrated cells were quantitated by microscopically counting random fields by a scorer, which was blind to experimental conditions [25 ].

NO determination
Macrophages were collected from saline- and morphine-treated WT and RelB–/– animals and purified as described above. The adherent cells were cultured in triplicate in 24 culture plates at 1 x 106 cells/ml, with or without LPS at the final concentration of 1 µg/ml. Plates were incubated at 37°C 5% CO2 and at 5, 8, 18, and 20 h of incubation. Supernatant (100 µl) was pipetted into a 96-well plate, and an equal volume of modified Griess reagent (Sigma Chemical Co.) was added. Plates were read at wavelength 550 nm. A standard curve was obtained with sodium nitrite, ranging from 0.2 µg/ml to 8 µg/ml. When indicated, N{omega}-nitro-L-arginine (LNNA; Sigma Chemical Co.), an irreversible inhibitor of constitutive NO synthase (cNOS) and a reversible inhibitor of inducible NOS (iNOS), was added to macrophage culture at the concentration of 100 µM, and NO determination was performed as described after 18 h of incubation.

RNA extraction and real-time RT-PCR
Adherent peritoneal macrophages, obtained from saline- and morphine-treated WT and RelB–/– animals, were cultured in the presence or absence of LPS (1 µg/ml) for 6 h. Total RNA was extracted using Trizol reagent (Life Technologies, Carlsbad, CA), according to the manufacturer’s instructions. After purification, RNA was treated with DNase (Ambion, Austin, TX) to avoid false-positive results as a result of amplification of contaminating genomic DNA. Concentrations were determined by the absorbance value of the samples at 260 nm. First-strand cDNA was synthesized from 1 µg total RNA in a 20-µl final volume using a Moloney murine leukemia virus RT (Invitrogen, Carlsbad, CA).

Real-time PCR of iNOS and IL-1ß genes
cDNA (2 µl) was subjected to real-time quantitative PCR using ABI PRISM 7000 (Applied Biosystems, Foster City, CA). TaqMan PCR was performed in 25 µl vol using TaqMan® Universal PCR Master Mix (Applied Biosystems). TaqMan probe/primers specific for GAPDH (Cod. Number Hs99999915_ g1), murine IL-1ß (Mm00434228-m1), and murine iNOS (Cod. Number Mm00440485_m19) were purchased from Taqman® Assays-on-DemandTM gene expression products (Applied Biosystems). All PCR assays were done in triplicate, and before starting, we performed a validation experiment to demonstrate that the efficiencies of target and reference are approximately equal. As controls, we used the reaction mixtures without the cDNA. The reaction conditions were as follows: 50°C for 2 min; 95°C for 10 min, followed by 40 cycles at 95°C for 15 s (denaturation); and 60°C for 1 min (annealing and elongation).

Threshold cycle numbers (CT) were determined with the ABI PRISM 7000 sequence detection system (Version 1.1 software). Gene-specific expression values were normalized to expression values of GAPDH (endogenous control) within each sample. IL-1ß and iNOS mRNA levels were expressed as relative values of cells obtained from morphine-treated mice reported to the calibrator value of cells obtained from saline-treated mice. Relative quantitation was performed using the comparative method. The amount of target, normalized to an endogenous reference and relative to a calibrator, is given by 2–{Delta}{Delta}CT. Briefly, the {Delta}CT value is determined by subtracting the average GAPDH rRNA CT value from the average iNOS CT value in the same sample. The calculation of {Delta}{Delta}CT involves subtraction of the {Delta}CT calibrator value.

Statistical analysis was performed with Prism GraphPad software, and Student’s t-test on {Delta}CT of saline- and morphine-treated animals was used for comparing data.

Collection of splenocytes and cytokine production
Spleens were removed aseptically from saline- and morphine-treated WT and RelB–/– animals, and cells were teased from the spleens by using 20-gauge sterile needles through an incision made in the spleen cuticle [26 ]. Spleen cells were adjusted to 4 x 106 cells/ml medium and incubated for 24 h, with or without 10 µg/ml Con A for IL-2 and IFN-{gamma} and 48 h for IL-10, IL-4, and IL-1ß.

Cytokine ELISA
The levels of TNF-{alpha} and IL-1ß protein (mature murine IL-1ß, MW 17.4 KD) were determined by ELISA kits (OptEIATM, PharMingen, San Diego, CA; CytoSetsTM, Biosource, Camarillo, CA), according to the manufacturers’ instructions. The levels of IL-12 p70 protein were determined by ELISA protocol, as standardized by PharMingen. The anti-IL-12 capture mAb (9 µg/ml) was absorbed on a polystyrene 96-well plate, and IL-12 present in the sample was bound to the antibody-coated wells. The biotinylated anti-IL-12-detecting mAb (0.25 µg/ml) was added to bind the IL-12 captured by the first antibody. After washing, avidin-peroxidase (Sigma Chemical Co.) was added to the wells to detect the biotinylated, detecting antibody, and finally, 2,2'azino-bis(3-ethylbenzthiazoline6-sulfonic acid) (Sigma Chemical Co.) substrate was added. A colored product was formed in proportion to that measured at OD 405 nm. The standards were recombinant cytokines in a range from 30 pg/ml to 4000 pg/ml.

IL-2, IL-4, IL-10, and IFN-{gamma} production was measured with the same ELISA protocol, except for use of anti-IL-2 and -IL-4 capture mAb at 1 µg/ml and IL-10, IFN-{gamma} at 2 µg/ml; biotinylated anti-IL-10-, -IL-4-, and -IL-2-detecting mAb at 0.5 µg/ml and IFN-{gamma} were used at the concentrations of 1 µg/ml, and the standard curves were ranging for IL-2, IL-4, and IL-10 from 15 pg/ml to 2000 pg/ml and from 32 pg/ml to 4000 pg/ml for IFN-{gamma}.

FACS analysis
Spleen cells or peritoneal macrophages were isolated from saline- and morphine-treated WT and RelB–/– animals as described above. After lysis of erythrocytes (lysing buffer, NH4Cl 0.155 M, KHCO3 0.01 M, Na2EDTA 10–4 M), cells were washed with PBS (NaCl 0.15 M, Na2HPO4 0.015 M, NaH2PO4 0.0014 M), and FcRs were blocked by incubating with FCS for 30 min at room temperature. After incubation, 5 x 105 cells for the sample were centrifuged at 1200 rpm for 5 min. After washing, each sample was resuspended with 100 µl PBS-FCS (unstained control) or primary-specificity antibodies for CD11b (macrophages, 0.5 µg/5x105 cells), F4/80 (macrophages, 0.125 µg/5x105 cells), CD19 (B cells, 0.12 µg/5x105 cells), CD3{epsilon} (all T cells, 1.5 µg/5x105 cells), and IgG2a (isotype control, 0.5 µg/5x105 cells). All the primary antibodies were conjugated directly to the fluorescent tag FITC, except for F4/80, which was conjugated with R-PE, and its matching isotype control was rat IgG2a R-PE . All antibodies were purchased from Caltag (Walter Occhiena, Torino, Italy). After 30 min of dark incubation at 4°C, the cells were washed three times with PBS-FCS. After a final wash, the cells were resuspended in 0.6 ml PBS-FCS.

For each cell surface marker, 20,000-gated cells were analyzed using the FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). The data are reported as percent of positive cells labeled with the indicated antibody. Results were analyzed using Cell Quest software (Becton Dickinson).

Statistical analysis
Data were analyzed by means of one-way ANOVA, followed by Bonferroni’s t-test for multiple comparisons.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Antinociceptive response to morphine is not altered in RelB-deficient mice
The classical CNS response to morphine is to increase nociceptive thresholds. We therefore tested whether antinociception to acute morphine was maintained in the RelB–/– mice. The hot-plate test, a classical method for evaluating nociceptive thresholds to opioids drugs, is used. As reported in Figure 1 , the antinociceptive response, measured 30 min after a suboptimal (1.25 mg/Kg) as well as an optimal (2.5 mg/Kg) analgesic dose of morphine, is similar in the WT and RelB–/– animals. The drug in fact increased nociceptive thresholds significantly over basal values in both strains of animals.


Figure 1
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  		Figure 1. Analgesic responses to acute morphine administration in WT and RelB–/– mice. Nociceptive thresholds were measured using the hot-plate test immediately before (basal) and 30 min after treatment with 1.25 or 2.5 mg/kg morphine injected s.c. The results are expressed in seconds (cut-off time, 30 s). Each bar represents the means ± SD of eight animals. *, P< 0.05, versus basal values.

 
RelB is necessary for morphine-induced inhibition of macrophage IL-1ß, TNF-{alpha}, and IL-12 but not for IL-10 production
The ability of an acute administration of morphine to affect spontaneous and LPS-stimulated cytokine production was assessed in WT and RelB-deficient mice. One hour after morphine injection, peritoneal macrophages were collected from each mouse, pooled, purified as described, and cultured for 24 h in the presence or absence of LPS. In Figure 2 , the levels of the proinflammatory cytokines IL-1ß and TNF-{alpha} are reported. The levels of IL-1ß were slightly lower in RelB–/– mice compared with WT animals. However, the stimulation with LPS induced a significant increase of IL-1ß production by macrophages in both mice strains. The effect exerted by the administration of morphine is different in the WT and KO animals. Although, in fact, morphine significantly decreases the spontaneous and the LPS-stimulated IL-1ß production in normal animals, the inhibitory effect of the drug is totally absent in the RelB–/– animals. The spontaneous and LPS-stimulated IL-1ß levels are in fact not different in macrophages obtained from saline- or morphine-treated animals. Figure 2 also shows that morphine is a potent inhibitor of spontaneous and LPS-stimulated TNF-{alpha} production by macrophages obtained from normal animals. Macrophages from RelB–/– mice stimulated with LPS were deficient in their ability to produce TNF-{alpha} in comparison with WT mice, and the inhibition induced by morphine was lost completely.


Figure 2
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  		Figure 2. Effect of the acute administration of morphine on the production of IL-1ß (A) and TNF-{alpha} (B) by resident peritoneal macrophages from WT and RelB–/– mice. Macrophages were obtained from saline- or morphine-treated animals 1 h after drug administration. Cells from three mice were pooled, separated by adherence, and cultured in the presence or absence of LPS, and levels of IL-1ß and TNF-{alpha} were measured after 24 h. Results presented are from typical experiments containing nine mice/group. Similar results were obtained in three additional experiments. Values are means ± SD. *, P < 0.01, versus saline-treated animals of the same strain; #, P < 0.05, versus WT animals.

 
We reported previously that morphine depressed IL-12 and IL-10 macrophage production [7 ]. As shown in Figure 3 , we observe a clear-cut decrease of spontaneous and LPS + IFN-{gamma}-stimulated IL-12 concentrations in normal mice. In RelB–/– mice, IL-12 levels were lower than in WT animals, and morphine was unable to depress IL-12 production further. In fact, macrophages obtained from saline-treated and morphine-treated RelB-deficient mice released the same amount of IL-12.


Figure 3
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  		Figure 3. Effect of the acute administration of morphine on the production of IL-12 (A) and IL-10 (B) by resident peritoneal macrophages from WT and RelB–/– mice. Macrophages were obtained from saline- or morphine-treated animals 1 h after drug administration. Cells from three mice were pooled, separated by adherence, and cultured in the presence or absence of 1 µg/ml LPS + 50 U/ml IFN-{gamma} for IL-12 or with LPS alone for IL-10 stimulation. Levels of cytokines were measured after 24 h. Results presented are from typical experiments containing nine mice/group. Similar results were obtained in three additional experiments. Values are means ± SD. *, P < 0.01, versus saline-treated animals of the same strain; #, P < 0.05, versus WT animals.

 
In the same figure, the effect of morphine on the production of IL-10 is also depicted. Contrary to what was observed previously for the proinflammatory cytokines, the levels of this anti-inflammatory cytokine are comparable in normal and RelB-deficient animals. Moreover, it is interesting that the absence of RelB does not affect the ability of morphine to suppress IL-10 production. Indeed, a significant and similar reduction of spontaneous and LPS-stimulated IL-10 production is induced by morphine in WT and RelB-deficient animals.

To further study the effect of morphine on IL-1ß production, real-time RT-PCR was applied to measure the steady-state mRNA levels of this cytokine. Macrophages collected from saline- or morphine-treated mice, WT and RelB–/–, were cultured for 6 h in the presence or absence of LPS. Steady-state IL-1 mRNA expression determined from the IL-1:GAPDH mRNA ratio in macrophages obtained from saline animals was set as 1 arbitrary unit and compared with the IL-1:GAPDH mRNA ratio after morphine treatment. As shown in Figure 4 , the levels of IL-1 mRNA were decreased significantly by morphine treatment in WT animals only.


Figure 4
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  		Figure 4. IL-1ß mRNA expression in macrophages obtained from morphine-treated animals. Macrophages were obtained 1 h after saline or morphine treatment of WT and RelB–/– animals and cultured with or without LPS for 6 h. Results are shown as IL-1ß mRNA expression in relation to GAPDH and are presented as a fold decrease relative to saline-treated animals. Data are means ± SD of four animals. *, P < 0.05.

 
Morphine similarly affects macrophage chemotaxis in RelB–/– and WT animals
It is well known that macrophage migration is affected significantly by morphine as well as by other opioids. From the experiments described in Figure 5 , it clearly emerges that RelB is not required for morphine-induced inhibition of chemotaxis. Morphine in fact causes a significant and comparable inhibition of spontaneous migration and fMLP-induced chemotaxis in normal and RelB–/– animals.


Figure 5
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  		Figure 5. Morphine-induced inhibition of spontaneous and fMLP-stimulated chemotaxis of macrophages obtained from WT or RelB–/– mice. Macrophages were obtained from saline- or morphine-treated animals 1 h after drug administration. The viability of macrophages was checked, and they were added in the chemotaxis chamber in the presence of medium only or with optimal concentrations of fMLP (10 nM). The values represent the mean ± SD of eight animals/group. *, P < 0.01, versus migration of macrophages obtained from saline-treated animals.

 
RelB is important for morphine stimulation of NO macrophage production
We tested the effect of morphine on macrophage NO production in the two strains of mice. The effect of the acute administration of morphine on the production of NO by macrophages obtained from normal and RelB-deficient animals was checked after 5, 8, 18, and 20 h in cultures stimulated or not with LPS (Fig. 6 ). In the absence (Fig. 6A) and in the presence (Fig. 6B) of LPS, macrophages obtained from WT animals treated with morphine produced a higher amount of NO in comparison with those from saline-treated WT animals. In contrast, unstimulated, (Fig. 6A) and LPS-induced production of NO (Fig. 6B) were not increased after morphine administration in RelB–/– animals.


Figure 6
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  		Figure 6. Concentration of NO in unstimulated (A) and LPS-timulated (B) macrophage cultures from saline- and morphine-treated WT and RelB–/– mice. Macrophages were obtained from saline- or morphine-treated animals 1 h after drug administration and were purified and cultured with or without LPS at the final concentration of 1 µg/ml. At 5, 8, 18, and 20 h of incubation, 100 µl supernatant was pipetted into a 96-well plate, and an equal volume of modified Griess reagent was added. The data are expressed as mean ± SD of six animals/group. *, P < 0.01, versus WT saline-treated animals.

 
As reported in Table 1 , when macrophages were incubated with the relative, selective cNOS inhibitor LNNA (an irreversible inhibitor of cNOS and a reversible inhibitor of iNOS), the morphine-induced NO increase observed in WT mice was prevented in unstimulated and LPS-stimulated macrophages, just showing a preferential involvement of cNOS in the observed effect.


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  		Table 1. LNNA Inhibition of Morphine-Induced Increase of NO Production

 
The levels of iNOS expression were evaluated by means of real-time PCR. The basal level of iNOS expression in WT mice was higher than that in RelB–/– animals (178-fold; P<0.01). In unstimulated macrophages obtained from WT mice, the administration of morphine induces a significant decrease of iNOS expression in comparison with saline-treated animals (4.19-fold less; P<0.05). In contrast, no significant alteration of iNOS mRNA expression was observed after morphine in RelB–/– animals. As expected, in LPS-stimulated macrophages, iNOS expression was induced significantly in WT and RelB–/– animals (data not shown). Morphine reduced it significantly only in WT animals (2.3-fold less; P<0.05).

RelB is required for morphine inhibition of Th1 but not Th2 cytokines
T lymphocyte activity and cytokine production have been reported to be particularly affected by morphine [1 , 4 ]. The Con A-induced splenocyte IL-1ß production was lower in RelB–/– animals than in controls (Fig. 7 ). Moreover, although morphine exerted a significant inhibition on IL-1ß production by unstimulated and Con A-stimulated splenocytes obtained from normal animals, it failed to suppress the cytokine production in RelB-deficient animals (Fig. 7) . Afterwards, we analyzed the Th1 and Th2 cytokine profile in normal and RelB–/– animals after morphine. In particular, we evaluated IL-2 and IFN-{gamma} as prototype Th1 and IL-4 and IL-10 as prototype Th2 cytokines [26 27 28 29 ].


Figure 7
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  		Figure 7. Effect of the acute administration of morphine on the production of IL-1ß by splenocytes obtained from WT and RelB–/– mice. Splenocytes obtained from morphine- and saline-treated WT and RelB–/– mice were stimulated with 10 µg/ml Con A. IL-1ß levels were measured 48 h later. Results presented are from typical experiments containing eight mice/group. Similar results were obtained in three additional experiments. Values are means ± SD. *, P < 0.01, versus saline-treated animals of the same strain; #, P < 0.05, versus WT animals.

 
As reported in Figure 8 , in saline-treated animals, no striking differences were observed in the production of IL-2, IFN-{gamma}, IL-4, and IL-10 between WT and RelB–/– animals, considering spontaneous and Con A-stimulated production. The acute administration of morphine depressed Th1- and Th2-stimulated cytokine production significantly in C57BL/6 animals, whereas morphine did not affect IL-2 and IFN-{gamma} production in RelB–/– animals (Fig. 8A and 8B) . In contrast to what was observed for the Th1 cytokines, morphine retained its ability to diminish IL-4 and IL-10 production after Con A stimulation, also in cells obtained from RelB–/– mice. As reported in Figure 8C and 8D , a similar, highly significant decrease of these cytokines was present in morphine-treated animals, independently on the mouse strain.


Figure 8
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  		Figure 8. Effect of the acute administration of morphine on the production of IL-2 (A), IFN-{gamma} (B), IL-10 (C), and IL-4 (D) by splenocytes obtained from WT and RelB–/– mice. Splenocytes, obtained 1 h after saline or morphine treatment of WT and RelB–/– mice, were stimulated with 10 µg/ml Con A. IL-2 and IFN-{gamma} levels were measured 24 h later and IL-10 and IL-4 levels, 48 h later. Results presented are from typical experiments containing eight mice/group. Similar results were obtained in three additional experiments. Values are means ± SD of eight animals. *, P < 0.01, versus saline-treated animals of the same strain.

 
The lack of RelB or morphine treatment does not affect the composition of spleen and peritoneal macrophage population
To rule out the possibility that RelB deficiency or morphine treatment could alter the number and composition of splenic as well as the peritoneal macrophage population, a FACS analysis was performed on cells obtained from saline- and morphine-treated WT and RelB–/– mice. As reported in Table 2 , no variation was present in the number of T lymphocytes and macrophages in the spleen. The percentage of B lymphocytes in saline RelB–/– mice appears slightly elevated, although not significantly different from saline-WT mice. After morphine, the percentage of B lymphocytes in WT and KO animals is not different, and it appeared a small, although significant, alteration between saline- and morphine-treated RelB–/– mice.


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  		Table 2. FACS Analysis of Spleen and PC Populations in Saline- and Morphine-Treated Normal and RelB–/– Mice

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The acute administration of morphine is associated with a generalized immunosuppression, involving functions of innate and acquired immunity [1 2 3 4 5 6 7 8 ]. The opioid-induced immunomodulation has been linked to an increased frequency and severity of infections and constitutes a clinical challenge in pain therapy. However, little is known about the molecular mechanisms by which morphine modulates the immune cells.

Our work clearly suggests that one target of morphine-induced immunomodulation inside immune cells could be the NF-{kappa}B system and in particular, the RelB factor. RelB readily associates with p50 and p52, forming potent transcriptional activators. The transcription factor RelB is fundamental for immune homeostasis. Moreover, although the other members of this gene family have different patterns of expression in all tissues, the expression of RelB is restricted to immune tissues [20 , 30 ]. In line with this specific RelB localization, we report that the antinociceptive activity of morphine, which is a CNS-mediated activity, is not impaired in RelB-deficient animals. The maintenance of the analgesic effect of morphine in the RelB–/– mice shows that in these animals, a general derangement of the opiate system is not present. In contrast, when we evaluated the effects of morphine on macrophage and T cell functions in RelB–/– animals in comparison with WT animals, many differences emerged.

The macrophages play a central role in innate and adaptive immunity [31 ]. They are fundamental cells of the innate immune response, and their ability to be chemotactically attracted to the site of initial microbial invasion or to an inflammatory focus is crucial for the full activation of the immune/inflammatory response that follows [31 ]. Macrophages are the main source of the proinflammatory cytokines IL-1ß and TNF-{alpha} as well as of the major anti-inflammatory cytokine IL-10 [27 , 31 , 32 ]. Macrophages also synthesize and release IL-12, the critical factor driving the development of Th1 cells [32 ]. Morphine significantly modulates all macrophage functions. In WT C57BL/6 animals, in fact, the administration of the drug led to a decreased production of the proinflammatory cytokines IL-1ß, TNF-{alpha}, and IL-12 as well as of the anti-inflammatory cytokine IL-10, in accordance with what we observed previously in the BALB/c mouse [7 ]. TNF-{alpha} and IL-12 production by macrophages obtained from saline-treated RelB–/– mice was significantly lower than WT control, and that of IL-1ß did not differ much from WT controls. These results are in agreement with what was already reported in RelB–/– mice [20 ]. However, in the absence of the RelB factor, all the effects of morphine on these cytokines disappeared, clearly indicating that the presence of RelB is necessary for morphine to decrease the proinflammatory cytokines. Considering the reduced levels of TNF-{alpha} and IL-12 in the RelB–/– mice, the possibility exists that the system leading to the production of these cytokines may already be suppressed maximally by the lack of RelB. However, our data and those by Caamano et al. [20 ] show that the TNF-{alpha} and IL-12 machineries are still active in the RelB–/– mice, as demonstrated by the fact that LPS or other stimuli are able to increase the production of the cytokines by our peritoneal macrophages as well as by bone marrow macrophages [20 ]. Moreover, we also observed that in a different condition known to depress macrophage function throughout steroid elevation, such as prolonged restrain stress [33 ], TNF-{alpha} and IL-12 production by cells obtained from RelB–/– was suppressed further (C. Martucci, S. Franchi, A. E. Panerai, P. Sacerdote., in preparation).

As the release of IL-1ß is regulated at several post-transcriptional and post-translational levels, we also measured steady-state mRNA for this cytokine. The results obtained clearly indicate that in WT animals, morphine is able to decrease IL-1ß transcription, as indicated by the lower mRNA level. Similarly to what was observed for the released protein, the effect of morphine on IL-1ß mRNA is lost in RelB–/–, suggesting that RelB is important for the transcriptional regulation of the cytokine. We cannot rule out, however, that other mechanisms can also be present downstream of IL-1ß gene expression.

The anti-inflammatory cytokine IL-10 seems to be less dependent on the presence of RelB. Not only are the levels of IL-10 confirmed not to be different in WT and RelB-deficient mice [20 ], but also, this cytokine is still sensible to the suppression induced by morphine, also in KO animals. It appears therefore that not all the effects of morphine on the immune system are mediated by the RelB factor.

The ability of macrophages to be chemotactically attracted by the bacterial-derived fMLP is conserved in the RelB-deficient macrophages, and morphine potently suppresses it [9 ]. The importance of the NF-{kappa}B system in inducing the migration of cells is in fact minor, and the activity of morphine has been shown to be a result of its interference with fMLP and chemokine receptors at the levels of interactions of these receptors with the G proteins [9 ]. The persistence of morphine-induced inhibition of IL-10 and chemotaxis in RelB–/– animals confirms that opioid receptors in the cells obtained from the KO animals are not deranged.

NO, a molecule with diverse, physiological function, is produced in vivo throughout the conversion of L-arginine to L-citrulline by different NOS isoforms: Morphine has been shown to be an effective immunomodulator, acting in part by stimulating NO production in neutrophils, monocytes, and endothelial cells [34 ]. Our data show that morphine treatment stimulates NO production in macrophages of WT mice in the presence or the absence of LPS. On the contrary, morphine never incremented the level of NO in RelB–/– mice.

Other reports have shown that morphine induces cNOS activation, and the NO produced thereafter regulates iNOS expression by inhibiting the binding of NF-{kappa}B to DNA [34 ]. LNNA is an irreversible inhibitor of cNOS and a reversible inhibitor of iNOS, and it is widely used to discriminate between the two isoforms [35 ]. The disappearance of NO increase after pretreatment of macrophages with this inhibitor seems to indicate a major involvement of cNOS in the effect of morphine on NO production in normal mice, similarly to what was reported for other cell types [34 ]. The role of cNOS is confirmed further by the observation that the NO increase appears to be completely independent from the induction of iNOS, as we measured an inhibitory effect of morphine on iNOS expression in WT animals. Qu et al. [36 ] have shown that cNOS inhibition up-regulates NF-{kappa}B activation with induction of iNOS expression. In contrast, the simultaneous inhibition of NF-{kappa}B restored cNOS normal expression and activity. On the basis of these published data and our results, we can hypothesize that morphine, by inhibiting NF-{kappa}B-mediated iNOS expression in WT mice, could lead to a hyperactivation of cNOS and to an increased NO production. However, in defective RelB mice, the lack of RelB prevents the inhibition of iNOS by morphine, as we showed, and the consequent activation of cNOS, blocking the overproduction of NO by morphine.

We then characterized the effects of morphine on cytokines produced by splenocytes, obtained from WT animals and RelB-deficient mice. When we considered the percentage of cell population in spleen, only a slight alteration in the number of B lymphocyte appeared to be present in RelB–/– mice. This difference is unlikely to have a role in the differential modulation of cytokines by morphine, as in particular, we analyzed the production of the Th1 cytokines IL- 1ß, IL-2, and IFN-{gamma} and of the Th2 cytokines IL-4 and IL-10. As we reported several time, the acute administration of morphine induced a relevant decrease of all the cytokines evaluated in the WT animals [2 , 3 , 5 ]. The inhibition of IL-4 cytokine, which we observed after morphine treatment, is in contrast with results for other groups, showing that morphine can induce a Th2 switch [37 ]. However, the stimulation of Th2 responses was observed mainly after chronic treatment with the drug or during morphine withdrawal [37 , 38 ]. We also observed that the inhibitory effect of morphine on IL-4 after chronic stimulation is different from acute administration (unpublished results). It is possible that a single administration of a high dose of morphine, such as we use in the present work, exerts a general immunosuppression, and prolonged treatment leads to a fine modulation of immune responses [26 ].

As happened for the macrophage cytokines, Th1 and Th2 cytokines were affected differentially by morphine in the RelB-deficient mice. The opioid in fact was unable to suppress the Th1 cytokine IFN-{gamma}. The production of IFN-{gamma} is decreased in RelB–/– mice; indeed, it has been reported that among the T lymphocyte cytokines, IFN-{gamma} results to be the more affected by the lack of RelB, whose nuclear translocation has been correlated with the ability of T cells to produce this cytokine [39 ]. We must report, however, that as discussed above for TNF-{alpha} and IL-12 macrophage production, in a stress model characterized by elevated corticosterone levels, it is still possible to further decrease the level of IFN-{gamma} in RelB–/– mice (C. Martucci, S. Franchi, A. E. Panerai, P. Sacerdote, in preparation).

In contrast, the levels of IL-2 were similar in RelB KO animals and in WT, and morphine failed to suppress it in the RelB-deficient animals, and splenocyte production of IL-10 and IL-4 was similarly decreased in C57 and RelB–/– mice.

It appears therefore that morphine uses different intracellular pathways to elicit a profound and generalized immunosuppression. RelB is relevant for morphine to suppress proinflammatory and Th1 cytokines. Roy and co-workers [8 , 14 ] showed that the administration of acute and chronic morphine interferes with transcriptional activation of NF-{kappa}B in macrophages, preventing its translocation to the nucleus. Our data support these results and indicate that the member of the NF-{kappa}B family target of morphine is RelB–/– in macrophages and in T cells.

Functional opioids receptors are present on macrophages and splenocytes, and morphine has been shown to elicit its effects mainly binding to the µ opioid receptor present on these cells [13 , 14 , 40 , 41 ]. The activation of the µ opioid receptor coupled to Gi/o proteins leads to an alteration of cAMP intracellular levels as well as of altered Ca++ fluxes inside the cells [1 , 42 ]. The modulation of these important signal molecules can interfere with the phosphorylation pathways activated by LPS and Con A, which converge on NF-{kappa}B/RelB for the transcriptional activation of proinflammatory and Th1 cytokines. However, we can speculate that the binding of morphine to its receptor with the following cAMP and Ca++ modulation should affect other transcriptional factors, such as GATA-3 and T-bet, leading to the inhibition of IL-10 and IL-4 [43 , 44 ].

It has to be noted, however, that RelB is implicated in the regulation of many genes, not all well known, important for the development and regulation of the immune system and that its lack is not functionally compensated by the other member of the NF-{kappa}B/Rel family [18 19 20 21 22 23 ]. The possibility therefore exists that the absence of RelB could alter the expression of cellular components that are downstream of opioid receptor activation and that this factor might not be a direct target of opiate action. The observation that the production of some cytokines, macrophage- and lymphocyte-derived, as well as other functions are normal and regulated by morphine in these animals, seems, however, to indicate the absence of alterations, which could prevent a full cell response. Together with the data showing a direct modulation by morphine of the NF-{kappa}B system [8 , 14 , 34 ], it can be hypothesized that RelB could be a target for the opioid action.

In conclusion, our data widen the understanding of the cellular and molecular targets of morphine action within the immune system and indicate that morphine uses multiple intracellular pathways to exert its generalized immunosuppression. RelB does not seem to be involved in the early steps of macrophage activation, such as chemotaxis, and it has a role in morphine modulation of NO production. RelB is an important target for the modulation by morphine of proinflammatory and Th1 cytokines but not for anti-inflammatory and Th2 cytokines.


   ACKNOWLEDGEMENTS
 
We thank Dr. Paola Ricciardi Castagnoli and Dr. D. Lo for the gift of the RelB–/– mice.

Received April 3, 2006; revised August 23, 2006; accepted September 11, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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