orphaninFQ secretion by immune cells and functional modulation of interleukin-2

peptides 28 (2007) 2243–2252

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Regulation of nociceptin/orphaninFQ secretion by immune cells and functional modulation of interleukin-2 Thomas R. Miller, Allison J. Fulford * Department of Anatomy, University of Bristol, Southwell Street, Bristol BS2 8EJ, UK

article info

abstract

Article history:

Previous research has demonstrated that numerous populations of immune cell, including

Received 2 August 2007

lymphocytes, synthesize nociceptin (N/OFQ) precursor mRNA although little is known

Received in revised form

regarding the immunological role of N/OFQ. In the present study we have demonstrated

6 September 2007

significant effects of mitogens, pro-inflammatory cytokines, cyclic AMP analogues, gluco-

Accepted 7 September 2007

corticoids and CRF on N/OFQ secretion by rat splenocytes in vitro. N/OFQ (1014 to 1010 M)

Published on line 15 September 2007

was also shown to inhibit proliferation of Con A-activated splenocytes and production of IL2 in vitro. In summary we have shown how a variety of stimuli relevant to inflammation can

Keywords:

regulate endogenous N/OFQ secretion by splenocytes in vitro. We also suggest that N/OFQ

Nociceptin

may promote anti-inflammatory actions via suppression of IL-2 in vivo. # 2007 Elsevier Inc. All rights reserved.

Mitogens IL-2 Inflammation Glucocorticoids Splenocytes

1.

Introduction

Accumulating evidence supports the existence of a bidirectional communication network between the nervous and immune systems involving reciprocal feedback coordinated by a common set of signaling molecules including glucocorticoid hormones, peptides and neurotransmitters. The neuropeptide, nociceptin/orphaninFQ (N/OFQ) has recently been shown to modulate several parameters of the immune system. N/OFQ is a heptadecapeptide that is evolutionarily related to the opioid peptide family [29,38] and exhibits a high degree of homology with the dynorphin A opioid system. Despite the close structural similarity, there is no functional cross talk between opioid and N/OFQ systems at the receptor level, in fact N/OFQ activates its own G-proteincoupled receptor, NOP. N/OFQ has been shown to modulate a wide range of CNS functions in rodents including monoamine

neurotransmitter release [28,41], the hypothalamo–pituitary– adrenal (HPA) axis [6,23,24] and other endocrine functions [16,33], in addition to locomotor [9,21] and anxiety-related behaviors [7,14]. Outside of the CNS, a major location of N/OFQ-NOP synthesis is the peripheral immune system. Various neuropeptides have been localized to lymphoid cells and therefore may contribute to pro- and anti-inflammatory responses. Calcitonin-gene related peptide, substance P, somatostatin and neuromedin-U can directly stimulate T cells to produce cytokines [15,25]. RT-PCR analyses indicate the capacity of various immunocyte populations to synthesize mRNA for the precursor of N/OFQ, preproN/OFQ, and many immune cell types express the full-length NOP receptor mRNA including normal circulating lymphocytes, polymorphonuclear cells and monocytes in addition to T, B and monocytic cell lines [36,42], however relatively little is known regarding the functional

* Corresponding author. Tel.: +44 117 9288692; fax: +44 117 9254794. E-mail address: [email protected] (A.J. Fulford). 0196-9781/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.09.004

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significance of immune-derived N/OFQ and NOP. Emerging evidence shows that the immune NOP receptor modulates proliferation of human lymphocytes in vitro [37,48], regulates antibody production [12] and neutrophil chemotaxis [42]. In addition, N/OFQ precursor knockout mice show attenuated lymphoid organ expression of cytokine transcripts (TNFa and IL-1b) following challenge with staphylococcal enterotoxin A (SEA) and N/OFQ peptide (100 mg) given by peripheral injection in normal mice also stimulates levels of circulating TNFa in response to SEA [11]. These recent findings suggest that N/OFQ precursor gene products, including N/OFQ itself, are able to modulate immune functions in vivo. Interestingly, recent in vitro studies show that exogenously applied N/OFQ peptide in culture reduces TNFa secretion by staphylococcal enterotoxin B (SEB)-activated T cells [48] denoting divergence between in vitro and in vivo experimental findings. Interaction between N/OFQ and glucocorticoids in vivo may therefore be an important consideration given that N/OFQ precursor knockout mice display elevated basal and post-stress plasma glucocorticoid levels compared to wild-type controls [20]. N/OFQ has the potential to modulate immune function via regulation of glucocorticoid secretion [23,24] and via direct actions on immune cells. One possibility is that immune-derived N/OFQ may exert paracrine or autocrine regulation over local inflammatory responses or N/OFQ may interact with peripheral nerves to modulate pain transmission. Like opioids, N/OFQ may serve to regulate both innate and adaptive immunity. Cytokines are the products of innate responses, and these can help to determine the particular type of adaptive response. One mechanism by which N/OFQ could modulate the immune system is by regulation of cytokines, an aspect that warrants further study. The adaptive immune response involves the activation of T cells, of which there are two subsystems, the Th1 and Th2. The classification of these two polarized forms of CD4+ T cells is based on cytokine production patterns. Th1 cells mainly produce TNFb, IL-2, IL12, IL-18 and thereby promote cell-mediated immunity, while Th2 cells principally secrete IL-4, IL-5, IL-6 and IL-10 and promote humoral responses. TNFa is derived from both Th1 and Th2 cells. Th1 and Th2 cells are counterregulatory, hence the system is balanced between cell-mediated and humoral immunity. Maintenance of the appropriate Th1/Th2 balance is critical for protective immunity and disruption of this equilibrium has been implicated in a variety of disorders including chronic stress and autoimmunity. To address the role of N/OFQ in cytokine regulation, the effects of N/OFQ on production of IL-2 by splenic lymphocytes was investigated. In this study we targeted IL-2 as it is known to have broad effects on diverse types of cells of the immune system. IL-2 has both autocrine and paracrine functions. It amplifies T cell responses, supports growth, enhances activation-induced apoptosis, stimulates antibody production and regulates T cell tolerance [26]. Cytokines and corticotrophin-releasing factor (CRF) release opioids from immune cells [40]. Potent peripheral analgesia due to direct injection of CRF can be blocked by both antibodies directed against opioid peptides, including endomorphin-2 and b-endorphin [22] and opioid receptor antagonists, including naloxone [31]. The release of opioid peptides from lymphocytes is calcium-dependent and opioid receptor

specific [3]. Despite observations of elevations in plasma N/OFQ in chronic pain conditions [19], little is known regarding the origin of plasma N/OFQ and indeed whether N/OFQ in immune cells is released and if so, under what conditions. A further aim of this study was therefore to investigate the factors and mechanisms governing secretion of N/OFQ in lymphocyte cultures. This study addressed the regulatory potential of glucocorticoids, cytokines and other inflammatory mediators on N/OFQ secretion by rat lymphocyte cultures.

2.

Materials and methods

2.1.

Animals

Male Wistar rats (200–250 g) were obtained from Harlan (Harlan, Blackthorn, Oxfordshire) and after arrival in the housing facility were allowed to acclimatize to the colony conditions for a week prior to the onset of the experiment. Rats were group-reared (four rats per cage) in standard sawdustlined cages in a holding room subject to a fixed photoperiod (lights on 07:00–19:00 h). Standard rat chow and water were available ad libitum. Ambient temperature of the housing room was 22–23 8C.

2.2.

Splenocyte culture

Rats were decapitated and their spleens removed. Using sterile forceps, cells were dispersed in Hanks balanced salt solution and erythrocyte lysis achieved by suspension in a hypoosmotic solution of ammonium chloride and potassium carbonate for 90 s. Splenocytes were suspended in RPM-1640 medium (Invitrogen) supplemented with streptomycin/penicillin (1%), sodium pyruvate (0.1%), 2-mercaptoethanol (0.1%) and foetal calf serum (10%). Viability of cells was confirmed by trypan blue exclusion and was routinely >95%. Cells were then seeded in flat-bottomed 24-well plates (Corning Glass, Corning, NY) at a density of 5  106 cells/ml prior to the addition of test agents.

2.2.1.

Nociceptin secretion studies

The mitogens, concanavalin A (Con A, 5 mg/ml) or bacterial endotoxin, lipopolysaccharide (LPS, 25 mg/ml, from Escherichia coli 055:B5) were added to cells immediately prior to incubation. Con A is a plant lectin that preferentially stimulates T cells by binding to carbohydrate residues in glycoproteins specifically involved in T-cell activation whereas LPS preferentially activates B cells. Cells were incubated at 37 8C in 95% O2/5% CO2 for 0, 6, 18, 24 or 48 h and then cell-free supernatants were collected for N/OFQ ELISA. In other experiments cells were exposed to CRF in the absence or presence of its antagonist, a-helical CRF for 24 h. In experiments determining the effects of various stimuli on normal splenocytes, cells were exposed to IL-1b (0.5–20 ng/ml) in the absence or presence of IL-1 receptor antagonist (10–500 ng/ml), TNFa (1–10 ng/ml), dexamethasone (1011 to 108 M) or 8-bromo-cAMP (0.2–1.0 mM) for 24 h. Cell-free supernatants were collected and stored at 20 8C prior to measurement of N/OFQ by ELISA.

peptides 28 (2007) 2243–2252

2.2.2.

Nociceptin regulation of IL-2 production

Cells were exposed to N/OFQ (1014 to 1010 M) in the absence or presence of peptidic NOP antagonists, [Nphe1, Arg14, Lys15]Nociceptin(1-13)-NH2 (UFP-101, 106 M) for 15 min prior to the activation with Con A (5 mg/ml) for 18 or 48 h. Plates were incubated at 37 8C in 95% O2/5% CO2. Splenocytes were tested for their ability to produce IL-2, after Con A stimulation. Supernatants were stored at 20 8C prior to determination of cytokine content using a commercially available kit.

2.3.

Measurement of nociceptin in splenocyte supernatants

N/OFQ was measured in cell-free supernatants from splenocyte cultures using a commercially available competitive ELISA kit (Phoenix Pharmaceuticals, Belmont, CA). ELISA plates were coated with the IgG fraction of N/OFQspecific monoclonal antibody. Lyophilized culture supernatants reconstituted in PBS containing 10% FCS and biotinylated-N/OFQ were added to each well and incubated for 2 h at room temperature. Bound biotinylated N/OFQ was detected with streptavidin-horseradish peroxidase for 1 h followed by addition of substrate solution, 3,30 ,5,50 -tetramethylbenzidine (TMB) and hydrogen peroxide with the reaction terminated using 2N HCl. Absorbances were read at 450 nm and a standard curve generated using serial dilutions of N/OFQ (1000 ng/ml) in PBS containing 10% FCS. N/OFQ concentration of sample supernatants was determined by extrapolation from the standard curve. The limit of detection of the assay was 0.1 ng/ml and the concentration of N/OFQ in RPMI 1640 medium was routinely < 0.1 ng/ml. The N/OFQ ELISA kit has demonstrable specificity for quantification of N/OFQ in cell, tissue and inflammatory exudate samples and has been used to detect N/OFQ in cultured astrocytes [46], human polymorphonuclear cells and synovial fluid [8].

2.4.

Quantification of secreted cytokines

IL-2 was measured after appropriate dilution using a sandwich ELISA kit (R&D Systems, Minneapolis, MN). These kits provide a reliable and reproducible method to detect bioactive cytokines. Supernatants were harvested at 18 and 48 h for IL-2. For assay, appropriate anti-cytokine ‘capture antibody’ was added to a 96-well flat-bottomed plate and incubated overnight at room temperature. The capture antibody was goat anti-rat IL-2 (1.6 mg/ml) in PBS. Following a wash step (PBS containing 0.05% Tween 20) plates were blocked with 300 ml of 1% bovine serum albumin (BSA, Sigma) in PBS for 1 h at room temperature. One hundred microliters aliquots of serially diluted standards of the recombinant rat IL-2 was added to control wells. One hundred microliters aliquots of test supernatants were added to the experimental wells. The plates were subsequently washed, as above, followed by addition of 100 ml of the biotinylated detection antibody for 2 h at room temperature. Following an additional wash step, wells were incubated in the presence of streptavidin-conjugated horseradish peroxidase (1:200) for 20 min at room temperature. Following this step substrate solution containing H2O2 and chromogen, tetramethylbenzidine in a 1:1 ratio was added to

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the wells. A color developed within 20 min and was determined as the average of duplicate wells at 450 nm using a Labsystems Multiskan Bichromatic microplate reader. Cytokine values were derived from standard curves of the appropriate recombinant cytokine generated using Deltasoft III software. In all assays there was less than 15% experimental error within an individual experiment.

2.5.

MTT assay for splenocyte proliferation

The MTT colorimetric assay (R&D Systems, UK), based on the ability of viable cells to reduce the yellow MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) salt to the blue MTT formazan product, was used to assess rat splenocyte proliferation in vitro. Preliminary experiments determined that splenocyte number and absorbance (570 nm) formed a linear relationship thereby providing a reliable assay for measurement of cell proliferation. Splenocytes (5  105 per well) were dispensed into flat-bottomed 96well plates and exposed to varying doses of N/OFQ (1014 to 1010 M) in the absence or presence of the NOP antagonist, UFP-101 (106 M). Thirty minutes following drug addition splenocyte cultures were activated by Con A (5 mg/ml). Plates were then incubated at 37 8C in 95% O2/5% CO2 for 18 or 48 h. Following incubation, 10 ml MTT reagent was added to each well for 4 h at 37 8C. This step was followed by the addition of detergent reagent (100 ml per well) for 2 h at r.t. to enable complete solubilization of formazan product (plates kept in the dark). The absorbance of the samples was then measured using a microplate reader at 570 nm. Each treatment/assay was based on n = 6 replicate samples.

2.6.

Drugs

N/OFQ and UFP-101 were obtained from Tocris UK. Human and rat CRF and CRF antagonist (a-helical CRF), dexamethasone and 8-bromo cAMP were obtained from Sigma–Aldrich, UK. Recombinant rat TNFa, human IL-1 and recombinant IL-1 receptor antagonist (IL-1ra) were obtained from R&D Systems, Minneapolis, MN. The N/OFQ ELISA kit was obtained from Phoenix Pharmaceuticals Europe. All drugs and peptides were dissolved in distilled water except for dexamethasone which was reconstituted in 70% ethanol and subsequently diluted in RPMI1640 medium. All other reagents were obtained from Sigma–Aldrich, UK.

2.7.

Data analysis

ANOVA was used to compare N/OFQ secretion in control versus drug-treated cells. The results of each treatment group are expressed as mean  one standard error of the mean (S.E.M.). N/OFQ secretion data in ng/ml (n = 4–6 independent experiments) were compared using either two- or one-way ANOVA where appropriate followed by post hoc analysis using the Fisher PLSD test. IL-2 production data (n = 4–8 independent experiments) and MTT assay data (n = 3–4 independent experiments) following treatment with N/OFQ or N/OFQ antagonists were analyzed using one-way ANOVA with post hoc analysis by the Fisher PLSD test. In all cases the significance level was set at 5%.

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3.

Results

3.1.

Nociceptin secretion by lymphocytes

3.1.1.

Effect of proliferative stimuli on N/OFQ secretion

To determine whether N/OFQ production could be modulated by cell activation, cell suspensions were incubated in the absence or presence of the T cell mitogen Con A (5 mg/ml) or the B cell mitogen LPS (25 mg/ml) compared to control over time (0, 6, 18, 24 and 48 h). Cell culture supernatants were collected and analyzed for N/OFQ-like immunoreactivity by ELISA. N/OFQ secretion did not change significantly over time in unstimulated culture supernatants (see Fig. 1). Treatment with mitogens resulted in a significant increase in N/OFQ secretion across time (F = 20.49, P = 0.001) corresponding to a 55–65% increase in secretion compared to the respective control (see Fig. 1). In Con A-treated cells N/OFQ levels rose at 18 and 24 h and increased further by 48 h with each value significantly different compared to the respective controls (P < 0.05). In LPS-stimulated cultures N/OFQ levels rose from 18 to 48 h and were significantly different to controls at the 24 and 48 h time points (P < 0.05). There was no significant difference in the profile of mitogen-induced N/OFQ secretion (F = 0.868, P = 0.511 n.s.).

3.1.2. Effect of pro-inflammatory cytokines on N/OFQ secretion To further characterize the mediators involved in the regulation of secretion of N/OFQ from splenocytes the effect of the pro-inflammatory cytokine, IL-1b was investigated. Cell culture supernatants were collected 24 h after incubation with IL-1b in the absence and presence of IL-1 receptor antagonist and analyzed for N/OFQ-like immunoreactivity using ELISA. IL-1b significantly increased basal N/OFQ secretion in rat splenocytes after 24 h in culture compared to control (F = 3.776, P = 0.019, see Fig. 2A). The increase in N/OFQ secretion was most marked at the 5 ng/ml concentration of IL-1b. Two-way ANOVA revealed that co-incubation with IL-1

Fig. 2 – Effect of (A) the pro-inflammatory cytokine IL-1b on N/OFQ secretion from rat splenocytes in vitro in the absence and presence of IL-1 receptor antagonist and (B) IL-1 receptor antagonist alone on rat splenocyte N/OFQ secretion. Cell culture supernatants were collected 24 h after incubation with the agents and analyzed for N/OFQlike immunoreactivity using ELISA. Data represent the mean values W S.E.M. of four independent experiments conducted in duplicate. *P < 0.05 vs. control; #P < 0.05 vs. respective IL-1b treatment, two-way ANOVA with post hoc Fisher PLSD test.

receptor antagonist (200 ng/ml) completely attenuated the IL-1b-induced (5–20 ng/ml) increase in N/OFQ secretion (F = 29.386, P = 0.0056) confirming the specificity of the response. When administered alone IL-1 receptor antagonist (10–500 ng/ml) had no significant effect on N/OFQ secretion (see Fig. 2B). TNFa also significantly increased N/OFQ secretion (F = 19.35, P = 0.0002, see Fig. 3) in an apparently bell-shaped manner with maximum increase seen with the 5 ng/ml TNFa concentration.

Fig. 1 – Effect of proliferative stimuli on N/OFQ secretion. Rat splenocytes (5 T 106/ml) were incubated in the presence or absence of Con A (5 mg/ml) or LPS (25 mg/ml) for different time periods. Cell culture supernatants were collected and analyzed for N/OFQ-like immunoreactivity by ELISA. Data represent the mean W S.E.M. of four independent experiments conducted in duplicate. *P < 0.05 control vs. Con A treatment; #P < 0.05 vs. control, two-way ANOVA with post hoc Fisher PLSD test.

3.1.3. Effect of glucocorticoids on N/OFQ release by rat splenocytes Synthetic glucocorticoids have previously been shown to regulate N/OFQ levels in neuroblastoma cell lines [43]. We were interested in the possibility that splenocyte-derived N/OFQ is also subject to regulation by glucocorticoids. To investigate this aim, rat splenocytes were incubated with different concentrations of the synthetic glucocorticoid receptor agonist, dexamethasone (1011 to 108 M) and culture supernatants collected 24 h later and analyzed for N/OFQ-like immunoreactivity using

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Fig. 3 – Effect of the pro-inflammatory cytokine TNFa on N/ OFQ secretion from rat splenocytes in vitro. Cell culture supernatants were collected 24 h after incubation with the agent and analyzed for N/OFQ-like immunoreactivity using ELISA. Data represent the mean values W S.E.M. of four independent experiments conducted in duplicate. ***P < 0.001 vs. control; #P < 0.05 vs. either TNFa treatment, one-way ANOVA with post hoc Fisher PLSD test.

ELISA. ANOVA revealed a dose-related increase in N/OFQ secretion compared to controls in splenocytes following 24 h in culture (F = 5.614, P = 0.006, see Fig. 4). In splenocytes exposed to high dose Dex (108 M) N/OFQ levels were increased to a level 70% above the respective control.

3.1.4.

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Fig. 5 – Effect of the neuropeptide, corticotrophin releasing factor (CRF) on N/OFQ secretion by Con A-activated (5 mg/ ml) rat splenocytes in vitro. Splenocytes (5 T 106/ml) were exposed to CRF for 24 h in culture. N/OFQ production was significantly increased by CRF (10S14 to 10S10 M). The CRF induced increase in N/OFQ production was attenuated by the CRF antagonist, a-helical CRF (10S8 M). Data represent the mean values W S.E.M. of four independent experiments conducted in duplicate. *P < 0.05, **P < 0.01 compared to untreated control, #P < 0.05 vs. respective CRF treatment, two-way ANOVA with post hoc Fisher PLSD test.

Effect of CRF on N/OFQ secretion by rat splenocytes

In order to determine whether CRF is involved in the regulation of N/OFQ secretion, rat splenocytes were challenged with CRF in the absence or presence of the CRF

antagonist, a-helical CRF and N/OFQ production in culture supernatants determined by ELISA. N/OFQ production by unstimulated splenocytes at 24 h exposed to CRF challenge (1014 to 1010 M) resulted in a significant increase in N/OFQ production (F = 10.43, P = 0.032, Fig. 5) with the highest dose of CRF (1010 M) being the most effective. The CRF-induced increase in N/OFQ production by splenocytes was completely attenuated by co-administration of the CRF antagonist, ahelical CRF (108 M) confirming mediation by CRF receptors (F = 4.04, P = 0.043). On its own the treatment with a-helical CRF (108 M) for 24 h of culture had no significant effect on production of N/OFQ (F = 1.839, P = 0.194).

3.1.5. Effect of the cyclic AMP analogue, 8-bromo-cAMP on N/ OFQ secretion by rat splenocytes

Fig. 4 – Effect of glucocorticoids on N/OFQ release by rat splenocytes in vitro. Splenocytes were incubated with different concentrations of the synthetic glucocorticoid receptor agonist, dexamethasone (10S11 to 10S8 M). Culture supernatants were collected following 24 h incubation and analyzed for N/OFQ-like immunoreactivity using ELISA. Data represent the mean values W S.E.M. of four independent experiments conducted in duplicate. *P < 0.05, **P < 0.01 compared to vehicle control, one-way ANOVA with post hoc Fisher PLSD test.

A high dose of the cyclic AMP analogue, 8-bromo-cAMP (1.0 mM) significantly increased basal levels of N/OFQ in splenocytes after 24 h in culture (F = 4.977, P = 0.032, see Fig. 6). A low dose of 8-bromo-cAMP (0.2 mM) was without effect and was not different to the N/OFQ level of secretion from unstimulated cells. However, in splenocytes treated with high dose 8-bromo cAMP N/OFQ secretion was increased 80% above the unstimulated control.

3.2.

N/OFQ and regulation of cytokine production

The effect of N/OFQ on IL-2 production was evaluated by exposing splenocytes to N/OFQ in vitro for 18–48 h. Stimulation of rat splenocytes with Con A (5 mg/ml) resulted in prolifera-

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Fig. 6 – Effect of the cyclic AMP analogue, 8-bromo-cAMP (0.2–1.0 mM), on N/OFQ secretion by rat splenocytes in vitro. High dose 8-bromo-cAMP (1.0 mM) significantly increased N/OFQ-like immunoreactivity in culture supernatants after 24 h. Data represent the mean values W S.E.M. of four independent experiments conducted in duplicate. *P < 0.05 vs. untreated control, one-way ANOVA with post hoc Fisher PLSD test.

tion and production of detectable levels of IL-2. The production of IL-2 was significantly decreased after exposure to N/OFQ for 18 h (F = 3.199, P = 0.021, see Fig. 7A). The greatest reduction occurred at 1014 M N/OFQ relative to control wells lacking peptide. A similar profile for IL-2 production following N/OFQ treatment was seen after 48 h in culture although this effect did not reach statistical significance (F = 0.646, P = 0.635, see Fig. 7B). The significant reduction in IL-2 secretion by N/OFQ (1014 M) at 18 h was attenuated in the presence of the selective peptidic NOP antagonist, UFP-101 (106 M) demonstrating mediation through NOP receptors.

3.3.

N/OFQ regulation of splenocyte proliferation

The proliferation of splenocytes in response to Con A (5 mg/ml) was significantly attenuated by N/OFQ after 18 h in culture (see Fig. 8A). N/OFQ (1014 M) resulted in significant 20% reduction compared to Con A alone-treated control cultures (F = 3.04, P = 0.039). There was a similar profile for N/OFQ’s effect on proliferation of Con A-activated splenocytes after 48 h (see Fig. 8B, P < 0.05). In both cases the significant reduction in cell proliferation seen with N/OFQ (1014 M) was completely attenuated by UFP-101 (106 M) (P < 0.05).

4.

Discussion

Recently the neuropeptide N/OFQ has been demonstrated to modulate several immune parameters including proliferation of human PBMCs [37,48], monocyte [47] and neutrophil chemotaxis [42] and mast cell histamine release [18]. These studies were based on the observed effects of exogenously applied N/OFQ whereas the functional significance of endogenous N/OFQ in monocytic and lymphocytic cells is less well understood. In the present study we addressed the regulation of endogenous N/OFQ production by immune cells in vitro and

Fig. 7 – Effect of N/OFQ (10S14 to 10S8 M) on secretion of IL-2 from Con A (5 mg/ml)-activated splenocytes after (A) 18 h or (B) 48 h in culture. Bioactive IL-2 was quantified using a selective IL-2 ELISA kit (see Section 2). N/OFQ significantly reduced IL-2 secretion at the lowest dose tested (10S14 M). This effect of N/OFQ appeared to be mediated by NOP receptors as the NOP antagonist, UFP-101 (10S6 M) reversed the inhibitory effect of N/OFQ. A similar profile of N/OFQ response was seen in activated rat splenocytes at the 48 h time-point, although this did not reach significance. The NOP antagonist, UFP-101 (10S6 M) again appeared to reverse the inhibitory effect of N/OFQ. Data represent the mean values W S.E.M. of five to eight independent experiments (samples analyzed in duplicate). *P < 0.05 vs. Con A alone, two-way ANOVA with post hoc Fisher PLSD test.

present several novel findings concerning the ability of mitogens, cytokines, glucocorticoids and CRF to modulate production of immunological N/OFQ. We also provide further insight into the immunological actions of peripheral N/OFQ and describe N/OFQ-induced modulation of splenocyte proliferation and of production of a major cytokine protein, IL-2. N/OFQ was secreted by unstimulated and mitogen-activated cultures in vitro at concentrations equivalent to that seen for the opioid peptide, dynorphin, in circulating rat lymphocytes in vitro [4]. This finding is in accord with several studies that have measured neuropeptide secretion in immune cells in vitro, including vasoactive intestinal peptide (VIP) [27],

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Fig. 8 – Effect of N/OFQ (10S14 to 10S10 M) on proliferation of Con A-activated splenocytes was significantly reduced following 18 h (panel A) or 48 h (panel B) in culture. The NOP antagonist, UFP-101 (10S6 M) significantly attenuated inhibitory effects of N/OFQ at both time points. Data represent the mean values W S.E.M. of three to four independent experiments (samples analyzed in sextuplicate). *P < 0.05 vs. Con A alone, two-way ANOVA with post hoc Fisher PLSD test.

b-endorphin [3], dynorphin and met-enkephalin [4], although levels of both N/OFQ and dynorphin in the immune cells appear to be 10–30-fold lower than that for other neuropeptides [4,27]. Despite the relatively low levels of N/OFQ secreted by unstimulated splenocytes, upon cell stimulation N/OFQ secretion is significantly increased 18–24 h after stimulation providing a source of endogenous N/OFQ able to engage in paracrine/autocrine regulation of ongoing immune functions. This timescale in N/OFQ peptide secretion following mitogenic stimulation is in agreement with the kinetic profile reported for other immune-derived peptides, such as VIP [27]. The functional relevance of immunological N/OFQ is dependent on expression of functional N/OFQ receptors. The broad distribution of N/OFQ receptor (NOP) mRNA and presence of N/OFQ binding sites [35,37,50] in the immune system have been identified. NOP transcripts are found in lymphoid organs and immune tissues of human, rat, mouse and porcine origin,

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including thymus, spleen and lymph nodes, in addition to normal circulating monocytes, lymphocytes and neutrophils [1,35,36,42] and human lymphoid cell lines (U937 monocytic, CEM and MOLT-4 T leukemic and EBV B cell lines) [36]. Splenocyte cultures represent a mixed population of immunocytes, including T, B and NK lymphocytes, monocytes and macrophages therefore further analysis is required to identify the splenocyte subpopulations that actively secrete N/ OFQ upon mitogenic stimulation. N/OFQ precursor mRNA has been localized to the CD19+ B cell subset of unstimulated human peripheral blood lymphocytes and the CD3+ T cell subset following stimulation with phytohemagluttin (PHA) [1]. N/OFQ is secreted by human neutrophils upon degranulation [8] therefore our findings indicate that N/OFQ secretion may be derived from both polymorphonuclear and mononuclear leukocytes. Biochemical characterization of lymphocytederived N/OFQ-like immunoreactivity is also required in order to determine the predominant molecular forms of N/OFQ secreted by these cells in response to inflammatory mediators and mitogenic stimuli. Furthermore, in accord with recent reports that N/OFQ precursor gene knockout mice show altered responsiveness to SEA challenge in vivo [11], it would be of interest to know whether other N/OFQ precursor-encoded peptides, such as nocistatin [34] also serve immunomodulatory roles. In our studies, splenocyte cultures activated with the mitogens, Con A or LPS secrete equivalent amounts of N/OFQ suggesting that both T and B lymphocytes contribute significant amounts of N/OFQ to the inflammatory milieu. N/OFQ secretion was increased by diverse stimuli involved in the regulation of the inflammatory response. The proinflammatory cytokines, IL-1b and TNFa both caused a marked increase in secretion of N/OFQ from unstimulated splenocytes, with the effect of TNFa being particularly potent. The effect of IL-1b was mediated by the IL-1 receptor as coincubation with IL-1 receptor antagonist completely prevented this effect of IL-1b. On its own the anti-inflammatory cytokine IL-1 receptor antagonist had no significant effect on N/OFQ secretion from unstimulated splenocytes. The effects of IL-1b and TNFa reported here are also consistent with the effects of the T cell-independent B cell mitogen and macrophage stimulator, LPS on N/OFQ secretion from splenocyte cultures seen in the present study. In unstimulated splenocytes CRF also increased N/OFQ secretion in a dose-related fashion and by a CRF receptor-mediated mechanism. Previous research has demonstrated that CRF upregulates production of opioid peptides from lymphocytes and lymph node immune cells, including b-endorphin, met-enkephalin and dynorphin [3,4,17]. In the case of b-endorphin this CRF-induced increase was shown to be mediated indirectly via an IL-1 effect [17]. Furthermore, IL-1b has also been shown to augment secretion of b-endorphin and dynorphin from cultured lymphocytes [3,4]. These immune-derived opioids exert local analgesic effects in tests of nociceptive threshold in inflamed rat paws [4] most likely via interaction with opiate receptors expressed by peripheral sensory nerves [44]. The enhanced secretion of N/OFQ by splenic lymphocytes by CRF, IL-1 b and TNF a seen in the present study also suggests that N/OFQ may contribute to peripheral protective antinociceptive responses in vivo. The abundant distribution of NOP receptor mRNA expression

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throughout dorsal and ventral horns of spinal cord, and in most primary afferent neurons [30] is consistent with a role for N/OFQ in pain modulation. Previous research has demonstrated the regulation of the biosynthesis, processing and secretion of the N/OFQ peptide in mouse neuroblastoma cells [39,43] and there are a number of similarities regarding the regulation of N/OFQ secretion by neuroblastoma cells and rat splenocytes. Secretion of N/OFQ peptide was subject to regulation by the synthetic glucocorticoid, dexamethasone (Dex). Dex alone at physiological concentrations dose-relatedly stimulated splenocyte N/OFQ secretion, suggesting that endogenous glucocorticoids may be able to regulate lymphoid N/OFQ secretion in vivo. Studies conducted in adrenalectomized rats are therefore required to determine whether HPA axis hormones are able to regulate N/OFQ secretion by lymphocytes in vivo. We have shown recently that psychological and physical stressors modulate expression of N/OFQ precursor and NOP gene transcripts in the brain [10] therefore stress may also regulate peripheral N/OFQ synthesis and secretion. This is relevant with regard to related endogenous opioid peptides that have been implicated in the mediation of stress-induced effects on lymphocyte functions [52], with the m-opiate receptor being strongly implicated in these actions [49]. N/OFQ has been shown to have negligible effect on forskolin-stimulation of cAMP levels in porcine splenocytes [35], however a more recent study reports that N/OFQ suppresses intracellular cAMP production by stimulated polymorphonuclear neutrophils [8]. In the present study we showed that the cAMP analogue, 8-br-cAMP, stimulated N/OFQ peptide secretion by rat splenocytes in a concentrationrelated manner with the 10 mM dose doubling basal N/OFQ secretion. These results suggest that secretion of N/OFQ can be modulated through intracellular cAMP signaling and that elevated N/OFQ may be a factor involved in cAMP-mediated effects on lymphocytes such as suppression of cell proliferation and production of IL-2. Other groups have demonstrated the ability of cyclic AMP analogues to induce expression of the prepro-nociceptin gene [2,39]. Regulation of N/OFQ production by cAMP analogues in neuronal and lymphoid cells is consistent with the presence of potential cyclic AMP response-element binding protein (CREB) transcription factor binding sites in the promoter region of the pre-proN/OFQ gene [51]. Diterpenes, such as forskolin, have been shown to enhance N/OFQ promoter activity [51] thus the effects of 8br-cAMP in the present study may be explained at the level of gene transcription. Although N/OFQ actions in pain are controversial elevated N/OFQ levels have been detected in cerebrospinal fluid after nerve root compression in pigs [5], inflammatory synovial fluids from patients with arthritis [8] and in the plasma of patients with chronic pain [19]. Peripheral anti-inflammatory actions have been reported for N/OFQ previously including inhibition of the release of substance P, somatostatin and calcitonin gene-related peptide (CGRP) from sensory nerve endings [13,32]. Our present findings suggest that N/OFQ regulates cytokine secretion providing some support for N/ OFQ in inflammation. Further work is required to identify whether N/OFQ exerts generalized non-specific effects on immune cells activated by Con A or cytokine-specific

responses, involving IL-2. IL-2 is an inflammatory cytokine which plays an important role in the mediation of adaptive immunity and is involved in the clonal expansion of T lymphocytes. In accordance with the observation that N/OFQ inhibited IL-2 production by Con A-activated splenocytes, we subsequently showed that the exogenous peptide also significantly suppressed proliferation of Con A-activated splenocytes. Both actions of N/OFQ appeared to involve activation of NOP receptors as the selective NOP antagonist, UFP-101 reversed N/OFQ effects. Exogenous N/OFQ has previously been shown to reduce proliferation of phytohemagglutinin-activated PBMCs [37]. We propose that the N/OFQ-induced suppression of PHA-activated PBMC proliferation is also dependent on a reduction in IL-2 secretion. N/OFQ effects on lymphocyte proliferation appear to be dependent on the mechanism of activation of the T cell population. A recent study has shown that in human PBMCs sub-nanomolar doses of N/OFQ cause upregulation of expression of T cell activation markers, including CD28 and CD25 (the a chain of the IL-2 receptor) and enhanced proliferation of human PBMCs activated by superantigen (SEB) only [48]. This same study showed that in re-stimulated T cells exposed to CD80, subnanomolar doses of N/OFQ actually reduce cell proliferation. Thus, it appears that T cell activation with polyclonal mitogens, PHA or Con A compared with direct T cell receptor engagement has important implications for the modulatory actions of N/OFQ tested in vitro. N/OFQ’s immunomodulatory properties are qualitatively similar to those of VIP, which also suppresses IL-2 production [45]. Considering the findings from the present study of splenocyte cultures we suggest that N/OFQ may serve a potential anti-inflammatory role. In response to pro-inflammatory cytokine release, N/OFQ levels in the inflammatory milieu are increased in a counterregulatory fashion. An antiinflammatory role for N/OFQ is supported on the basis of the stimulatory effects of dexamethasone on N/OFQ secretion and the observed suppressive effects of exogenous N/OFQ on Con A-stimulated splenocyte proliferation and IL-2 secretion. N/OFQ-NOP receptor actions may subserve a protective role in vivo involving deactivation and attenuation of the ongoing immune response. However, N/OFQ and the complex regulation of pro-inflammatory and anti-inflammatory cytokines requires detailed investigation, especially in the light of the SEA responsiveness of N/OFQ precursor knockout mice recently shown in vivo [11]. In conclusion we have demonstrated for the first time how endogenous N/OFQ secretion in lymphocyte cultures is regulated by inflammatory mediators and glucocorticoids. We have characterized the effect of N/OFQ on the proliferation of, and IL-2 secretion by, activated rat splenocytes in order that the mechanisms by which N/OFQ can modulate immune function can be further delineated. The present research suggests that immune cells of the spleen provide a source of N/OFQ with significant immunoregulatory potential.

Acknowledgement This study was supported by funds from the University of Bristol.

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