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

Abstract

Previous research has demonstrated that numerous populations of immune cell, including lymphocytes, synthesize nociceptin (N/OFQ) precursor mRNA although little is known regarding the immunological role of N/OFQ. In the present study we have demonstrated significant effects of mitogens, pro-inflammatory cytokines, cyclic AMP analogues, glucocorticoids and CRF on N/OFQ secretion by rat splenocytes in vitro. N/OFQ (10⁻¹⁴ to 10⁻¹⁰ M) was also shown to inhibit proliferation of Con A-activated splenocytes and production of IL-2 in vitro. In summary we have shown how a variety of stimuli relevant to inflammation can regulate endogenous N/OFQ secretion by splenocytes in vitro. We also suggest that N/OFQ may promote anti-inflammatory actions via suppression of IL-2 in vivo.

Introduction

Accumulating evidence supports the existence of a bi-directional 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-protein-coupled 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 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 (TNFα and IL-1β) following challenge with staphylococcal enterotoxin A (SEA) and N/OFQ peptide (100 μg) given by peripheral injection in normal mice also stimulates levels of circulating TNFα 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 TNFα 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 TNFβ, IL-2, IL-12, 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. TNFα 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 β-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.