Neutrophils and monocytes represent the first line of defence against pathogens due to their ability to rapidly migrate into inflamed tissue, to phagocytose micro-organisms and particles and to produce reactive oxygen intermediates. Morphine has been shown to inhibit several of these host defence functions .
The interaction of opioid substances with the immune system has been thoroughly investigated in the past , and inhibitory effects of morphine on leukocyte function have been demonstrated [1,3]. Morphine's effects are mediated by opioid receptors on immunocytes and vascular endothelial cells [2,4,5]. Binding of morphine to the μ opiate receptor sub-type found on endothelial cells, neutrophils and monocytes stimulates nitric oxide (NO) production in these cells .
Recently, the transduction pathways, by which lipopolysaccharide (LPS) activates phagocytes, have been elucidated. CD14 is an innate immunity receptor for LPS found in association with toll-like receptors (TLR) on the surface of cells of the myeloid lineage such as neutrophils, macrophages and monocytes . LPS-induced cell activation is mediated via CD14 as part of a trimolecular receptor cluster consisting of TLR4, MD2 and CD14 : LPS binds to LPS-binding protein (LBP) in plasma and is delivered to the cell surface receptor CD14. Next, LPS is transferred to TLR4 and its accessory protein MD2, an approximately 30 kDa molecule, which physically associates with the extracellular domain of TLR4.
Activator protein 1 (AP-1) is a transcription factor, which is involved in the regulation of proinflammatory mediators at the transcriptional level after stimulation of cells with LPS. The AP-1 complex is composed of either homo- or heterodimers, which consist of protein products derived from the proto-oncogenes c-fos and c-jun . Following activation, AP-1 binds to specific recognition sequences in promotor regions of target genes such as interleukin (IL)-8, IL-1α, IL-2 and inducible nitric oxide synthase (iNOS), thereby modulating the transcription of these genes .
To date, only a few studies have investigated the influence of morphine on intracellular signal transduction processes. In a previous study, we have demonstrated the inhibitory effect of morphine on the activation of nuclear factor (NF)-κB, a transcription factor also involved in proinflammatory gene expression in human blood monocytes and neutrophils .
In this study, we investigated the effects of morphine on CD14 expression and the influence of NO as a second messenger in this process. As a downstream intracellular signalling mechanism, we assessed AP-1 activation in human blood monocytes and neutrophils. To investigate whether morphine effects depend on the expression of μ opioid receptors we used the granulocyte-like HL-60 cell line, which expresses the μ opioid receptor sub-type only after differentiation with retinoic acid .
Materials and methods
The study was approved by the local Ethics Committee of the University Hospital Giessen. Written consent was obtained from all volunteers participating in the study.
Flow cytometric detection of transcription factors was performed as described elsewhere in detail [3,11]. Briefly, 10 mL of heparinized blood was collected from eight healthy volunteers and aliquots of 100 μL blood were incubated with different concentrations of morphine sulphate for 2.5 h or with S-nitroso-N-acetyl-penicillamine (SNAP) as an NO donor. Where indicated, pretreatment with naloxone or the nitric oxide synthase inhibitors Nω-nitro-l-arginine-methylester (l-NAME) or Nω-nitro-l-arginine (NLA) was performed for 10 min at 37°C. Controls were incubated with 0.9% saline. To induce AP-1 activity, samples were stimulated with 100 ng mL−1 LPS for 30 min in a 37°C water bath. Red cell lysis was performed with a commercially available lysing solution (FACS Brand Lysing Solution; BD Biosciences, Heidelberg, Germany). Cells were washed once with phosphate-buffered saline (PBS) before staining of nuclei using a commercially available DNA staining kit (Cycletest Plus DNA Reagent Kit; BD Biosciences). Anti-AP-1 polyclonal rabbit antibody (Santa Cruz Biotechnology, Heidelberg, Germany) was used as primary antibody, a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit monoclonal antibody (Sigma Chemicals, Deisenhofen, Germany) served as secondary antibody. Propidium iodide solution was added for nuclei detection.
Stained nuclei were detected by the fluorescence 2 (FL2) filter. The fluorescence 1 (FL1) detector was used to detect AP-1 activation by measuring emission of green fluorescence at 530 nm corresponding with FITC staining. Fluorescence due to unspecific binding of the secondary antibody was excluded by incubation of whole blood with FITC-labelled IgG alone. Commercially available DNA particles (DNA Quality Control Particles Kit; BD Biosciences) were used for the flow cytometer setup. In total, 20 000 events were recorded for each sample. To evaluate leukocyte sub-populations to which nuclei belonged, we analysed a forward angle light scatter/90° side scatter (FSC/SSC) dot plot. Polymorphonuclear and mononuclear cells were analysed for FITC staining using histogram analysis of the FL1 parameter. The median of channel fluorescence (MCF) was used as an indicator for the intensity of nuclei fluorescence.
Detection of surface CD14 expression
Blood samples were incubated with monoclonal FITC-conjugated anti-CD14 (Camon/Serotec, Germany). Nonfluorescent antibodies against CD14 were used to reach optimal setups at the flow cytometer (FacsCalibur®; BD Biosciences). FITC-labelled antibodies against human IgG (Camon/Serotec) as well as isotype-matched control antibodies were used for instrument settings and to exclude significant unspecific binding. Red cell lysis was performed as described above. Samples were washed, cell nuclei were stained with propidium iodide and flow cytometric analysis was performed. Neutrophils were identified and gated by their special characteristics in FSC and SSC and the median of channel fluorescence intensity was determined.
Cell culture, reagents and induction
The promyelocytic cell line HL-60 was obtained from ATCC (Rockville, MD, USA). Cells were grown in RPMI 1640 media (Gibco BRL, Great Plain, NY, USA) supplemented with 10% heat-inactivated fetal calf serum, HEPES (2.38 g L−1), NaHCO3− (2 g L−1), l-glutamine (0.3 g L−1), pyruvic acid (0.088 g L−1), glucose (4.5 g L−1), penicillin (100 U mL−1) and streptomycin (100 μg mL−1) at 37°C in a humidified 5% CO2/95% air atmosphere. To differentiate HL-60 cells into the granulocytic phenotype, cells were treated for 5 days with 1 μmol retinoic acid, with differentiation being confirmed by nitroblue tetrazolium reducing ability and response to N-formyl-methionyl-leucyl-phenylalanine (FMLP). Together, 107 cells were incubated for 2.5 h with different concentrations of morphine sulphate (Sigma Chemicals) as indicated, followed by a 30-min induction of transcription factors by lipopolysacharide (LPS, Escherichia coli serotype 026B6, Sigma Chemicals).
Preparation of nuclear extracts
Nuclear extracts of HL-60 cell pellets were obtained by a modification of the method described by Schreiber and colleagues . Briefly, cell pellets were washed twice in ice cold PBS and resuspended in 150 μL buffer A (10 mmol HEPES-KOH pH 7.9, 1.5 mmol KCl, 0.5 mmol dithiothreitol, 0.2 mmol PMSF). After a 10-minute incubation on ice, cell lysis was checked microscopically. After centrifugation (12 000 r.p.m., 30 s, 4°C), the supernatant containing the cytoplasmatic protein fraction was discarded. The remaining pellet was washed in PBS, resuspended in 50 μL Puffer C (20 mmol HEPES-KOH pH 7.9, 25% glycerol, 420 mmol NaCl, 1.5 mmol MgCl2, 0.2 mmol EDTA, 0.5 mmol dithiothreitol, 0.2 mmol PMSF) and incubated on ice for 20 min. After centrifugation (12 000 r.p.m., 2 min, 4°C), the supernatant containing the nuclear protein fraction was collected. Protein concentrations of nuclear extracts were determined by the BioRad Protein Assay (BioRad Laboratories, Hercules, CA, USA). The samples were stored at −70°C until further analysis.
Electric mobility shift assay (EMSA)
Five micrograms of nuclear protein was preincubated for 20 min at 22°C with radiolabelled oligonucleotide, poly[dI-dC], and poly-l-lysine in reaction-buffer (100 mmol HEPES, pH 7.6, 5 mmol EDTA, 50 mmol (NH4)2SO4 5 mmol DTT, 1% Tween-20, 150 mmol KCl). The double-stranded oligonucleotides (Santa Cruz Biotechnology) contained the DNA binding site for AP-1: 5′-CGC TTG ATG ACT CAG CCG GAA-3′. After incubation, DNA-protein complexes were resolved by electrophoresis on 7.5% polyacrylamide gels under nondenaturing conditions. Gels were dried for 1 h before autoradiography with intensifying screens.
Isolation of total RNA and reverse transcription polymerase chain reaction (RT-PCR)
HL-60 cells were collected, centrifuged and homogenized in 1 mL Tri Reagent (Molecular Research Center, Cincinnati, OH, USA) for 5 min. After addition of 0.1 mL 1-bromo-3-chloropropane, samples were vigorously mixed and centrifuged for 15 min at 12 000g. The aqueous phase was transferred into a fresh tube. RNA was precipitated with isopropanol, washed with 75% ethanol and air-dried for 10 min. After resuspension in RNAse-free water, RNA was analysed spectrophotometrically and electrophoresed on a 1% agarose gel to ensure purity.
After denaturation at 95°C, 1 μg of neutrophil RNA and 3 μg of RNA from HL-60 cells were reverse transcribed at 42°C using Superscript II RNAse H-RT and random hexamer primers (Both Gibco BRL, Gaithesburg, MD, USA). RT products (10 μL) were added to the PCR mix containing primers (sense: 5′-GGT ACT GGG AAA ACC TGC TGA AGA TCT GTG-3′, antisense: 5′-GGT CTC TAG TGT TCT GAC GAA TTC GAG TGG-3′ ) to amplify a 441 bp fragment of the μ opioid receptor gene. Taq DNA polymerase (Gibco BRL) was used to catalyse the reaction. All PCR reactions were followed by an extension cycle at 72° for 10 min. PCR conditions were: 30 cycles (95°C 1 min, 53°C 1 min, 72°C 1 min). Total RNA from SH-SY5Y neuroblastoma cells known to express μ opioid receptors served as a positive control for μ receptor expression. PCR products were analysed on a 2% agarose gel (Sigma-Aldrich, St. Louis, USA) stained with ethidium bromide. β-Actin reference gene primers were used to amplify a 302-bp fragment (primers: sense 5′-GCG AGA AGA TGA CCC AGA TCA TGT T-3′, antisense 5′-GCT TCT CCT TAA TGT CAC GCA CGA T-3′; 20 cycles: 95°C 1 min, 60°C 1 min and 72°C 1 min).
All flow cytometric results are expressed as mean values with standard deviations. Statistical analysis was performed using Friedman's test followed by Wilcoxon-Wilcox procedure, allowing multiple comparisons against a control group. P-values <0.05 were considered significant.
In a first set of experiments, we used HL-60 cells known to express μ opioid receptor transcripts  to investigate the influence of morphine on AP-1 binding. RT-PCR analysis confirmed expression of μ opioid receptor transcripts in differentiated granulocyte-like HL-60 cells but not in undifferentiated HL-60 cells (Fig. 1). To evaluate whether morphine exerts inhibitory effects on AP-1 binding activity in HL-60 cells, we performed a gel shift assay. Compared to LPS stimulation alone, pretreatment with morphine decreased LPS-induced AP-1 DNA binding activity in differentiated HL-60 cells (Fig. 2). This effect was more pronounced after treatment with the higher morphine concentration (50 μmol).
In order to exclude cell-line-specific effects, we determined AP-1 nuclear content in human neutrophils. A whole blood flow cytometric technique was used to avoid neutrophil activation during cell separation. Determination of nuclear AP-1 in LPS-stimulated whole blood revealed a reduction in nuclear AP-1 content in human monocytes and neutrophils after pretreatment with morphine (Fig. 3).
As determined by flow cytometry, incubation of whole blood with morphine inhibited CD14 expression on the surface of human neutrophils in a concentration-dependent manner (Fig. 4).
Since naltrindole was found to have little affinity for the opioid receptors present on neutrophils , while naloxone was very effective in reversing morphine-induced changes, we chose naloxone as an opioid receptor antagonist to investigate whether morphine acts via a classical μ opioid receptor. Exposure to naloxone before morphine treatment abolished morphine-induced inhibition of CD14 expression and AP-1 activity in human blood monocytes and neutrophils (Figs 4 and 5).
In order to determine whether morphine attenuates CD14 expression and AP-1 activation via NO-dependent pathways, the NOS inhibitor NLA (500 μmol) was administered to whole blood 10 min before incubation with morphine. NLA completely antagonized morphine-induced reduction in CD14 expression (Fig. 4). Similarly, the NOS inhibitor l-NAME reversed morphine-induced attenuation of AP-1 nuclear content in human neutrophils and monocytes (Fig. 5). These data demonstrate that morphine exerts its effects on AP-1 activation via NO as a second messenger in both cell types.
In the present report, we demonstrate the following effects of morphine on phagocyte function:
- Morphine attenuates LPS-induced activation of AP-1 in human neutrophils, monocytes and in differentiated granulocyte-like HL-60 cells.
- Morphine inhibits CD14 expression on human neutrophils.
- Inhibition of AP-1 activation by morphine depends on the presence of a μ opioid receptor and can be antagonized by naloxone.
- NO is involved in morphine-stimulated inhibition of AP-1 activity, since NO donors mimic the inhibitory effects of morphine on AP-1 activation, and NO antagonists abrogate morphine's effects.
In the immune system, morphine signalling is thought to be mainly inhibitory , although the exact mechanisms leading to morphine-induced immunosuppression are still under investigation. In previous studies, we have demonstrated that morphine reduces the expression of surface receptors linked to leukocyte functions such as phagocytosis and oxidative burst [1,14]. In accordance with these results, we now report a decrease in CD14 expression in human blood monocytes and neutrophils caused by morphine. This effect appears to be coupled to NO as a second messenger and depends on the expression of μ opioid receptors on the surface of monocytes and polymorphonuclear cells. CD14, a glycosylphosphatidyinositol-anchored protein, is crucial for LPS recognition  and acts in concert with CD11b/CD18 and TLR4 to elicit full gene transcription in response to LPS . Thus, a reduction in CD14 expression, as observed after morphine treatment, may result in reduced transcriptional activity.
So far, little is known about morphine's ability to regulate gene transcription in immune cells. In a previous investigation, we could demonstrate that morphine inhibits LPS-induced activation of NF-κB, a transcription factor that regulates the production of proinflammatory mediators in human blood monocytes and neutrophils . We now report that morphine is able to decrease activation of LPS-induced AP-1 DNA binding activity in human blood neutrophils, monocytes and in granulocyte-like HL-60 cells. Without LPS stimulation, morphine has no effect on basal AP activity. These results are in accordance with a previous study demonstrating significantly decreased levels of AP-1-protein complex formation in nuclear extracts prepared from morphine-treated rat thymocytes stimulated with PHA or IL-1 . Furthermore, morphine-treated thymocytes showed decreased steady-state levels of bioactive IL-2 and IL-2 mRNA , suggesting that the decrease in AP-1 activity may inhibit the synthesis of inflammatory mediators. Genes for proinflammatory mediators such as cytokines and cytokine receptors contain numerous sites for regulation by NF-κB and AP-1 transcription factors in their promoter regions . Many factors affect the formation and activity of AP-1 dimers through protein-specific interactions or by phosphorylation of pre-existing complexes . Intracellular kinases as well as second messengers such as NO are involved in this process. For example, NO-donor compounds inhibit the DNA-binding activity of transcription factors. This suggests that cellular NO provides another control mechanism for modulating the expression of specific responsive genes . Furthermore, there is evidence that NO mediates its inhibitory effect on AP-1 activity by reacting specifically with the conserved cysteine residues in Jun and Fos . AP-1 complexes can vary markedly in their ability to stimulate gene transcription. Thus, the activity of AP-1 is regulated by stimuli that either modulate the de novo synthesis of AP-1 subunits or influence the activity of previously formed AP-1 dimers. It has been shown that morphine treatment inhibits the PHA- and IL-1-stimulated synthesis of c-fos at the mRNA level . Under identical conditions, c-Jun mRNA levels are not altered .
It has previously been demonstrated that μ opioid receptor expression on circulating leukocytes can be regulated by opioid compounds  as well as by proinflammatory mediators . Furthermore, the expression of a μ opioid receptor splice variant, called μ3 receptor, has been identified at the molecular level in human monocytes and neutrophils . This μ3 receptor mediates NO-dependent functional changes of immunocytes . In addition, it was shown that acute morphine exposure to human leukocytes results in specific and significant alterations in gene expression . Our results support the crucial role of μ opioid receptors for immunoregulation and suggest a regulatory role of AP-1 in morphine-induced immunoinhibition.
Apart from their function in the inflammatory response, AP-1 (Fos/Jun) transcription factors play multiple roles in the functional development of haematopoietic precursor cells into mature blood cells . Furthermore, AP-1 (Fos/Jun) transcription factor complexes can promote or inhibit distinct apoptotic pathways in many cell types, including cells of haematopoietic origin. Recently, morphine's regulatory role in immune cell differentiation and apoptosis has been elucidated in immunocytes [25-27]. The proapoptotic effects of morphine have been found to be NO-dependent, suggesting a possible role of AP-1 inhibition by morphine-induced NO release in regulation of immunocyte apoptosis . Thus, morphine may control blood cell homeostasis by programmed cell death of terminally differentiated cells such as neutrophils and by regulation of growth/survival factors associated with apoptosis, thereby terminating or at least attenuating inflammatory processes.
The decrease in CD14 expression may provide evidence for one of the signalling pathways involved in morphine-induced changes. However, the intracellular pathways affected by morphine are complex and interact with functional activity. In this context, morphine's effects on the other components of the receptor cluster that mediates LPS-induced cell activation, in particular MD 2 and TLR 4, remain to be elucidated. Our results do not discriminate between AP-1 inhibition as an indirect effect of reduced CD14 expression on one hand and direct interaction of morphine with transcriptional factors and their DNA binding capacity on the other hand.
In summary, our data demonstrate for the first time that morphine inhibits LPS-induced AP-1 DNA-binding activity in differentiated HL-60 cells as well as in human blood monocytes and neutrophils by an NO-dependent process. The inhibition of transcription factors induced by morphine correlates with a decrease in CD14 receptor expression, which is crucial for LPS signalling. Our results may contribute to explain the inhibitory effects of morphine on cells of the innate immune system.
This work was in part supported by the following grants: Deutsche Forschungsgemeinschaft (We 2440-1/1), Bonn, Germany (I.D.W.).
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