Regulation of VIP production and secretion by murine lymphocytes

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Abstract

Vasoactive intestinal peptide (VIP) is a neuropeptide present in the lymphoid microenvironment with a multiplicity of actions. Two sources for VIP have been described in the immune system, the terminals present in central and peripheral lymphoid organs and the immune cells. Although VIP is synthesized by lymphocytes, there is no evidence demonstrating that VIP is released, and which stimuli are able to induce VIP production and secretion. In this study, we demonstrated for the first time, that agents that mediate important immune functions, such as proliferation and antigenic stimulation (Con A, LPS, and anti-TCR antibody), inflammation (LPS, TNFα, IL-6 and IL-1β) or apoptosis (dexamethasone) induce the production and release of VIP to the lymphoid microenvironment. We conclude that VIP is produced and secreted by lymphocytes and propose that during an immune response, the timely release of VIP within the lymphoid organs and peritoneum should influence the differentiation and/or downregulation of the ongoing response.

Introduction

The immune and neuroendocrine systems represent an integrated network sharing the same molecular signals and their receptors. In 1980 Blalock and Smith (Blalock and Smith, 1980) demonstrated that, in this network, neurons and endocrine cells were not the exclusive sources of hormones and regulatory peptides, describing for the first time that macrophages and lymphocytes were also sources for ACTH and endorphins. Actually, there are at least 20 different neuroendocrine peptides that can be synthesized by cells of the immune system (Blalock, 1992; Dardenne and Savino, 1994). Thus, the presence of hormones and neuropeptides including prolactin (O'Neal et al., 1992) growth hormone (GH) (Weigent et al., 1988), GHRH (Weigent and Blalock, 1990), LHRH (Emanuele et al., 1990) arginine–vasopressin (Geenen et al., 1987), substance P (Weinstock et al., 1989), somatostatin (Fuller and Verity, 1989), proenkephalin A (Rosen et al., 1989), nerve growth factor (Santambrogio et al., 1994) and atrial natriuretic peptide (Vollmar et al., 1990) has been described in different cells of the immune system.

Vasoactive intestinal peptide (VIP) is a 28 aminoacid peptide isolated in 1970 from pig intestinal extracts (Said and Mutt, 1970). It is known today that VIP is an ubiquitous peptide, with a multiplicity of actions and highly conserved sequence in vertebrates (Dimaline, 1989). VIP is a signal molecule of the neuroendocrine-immune network produced by neurons in different areas of the central and peripheral nervous system and by endocrine cells as the pituitary lactotropes and cells of the endocrine pancreas (Davidson et al., 1996). In the immune system, two sources for VIP have been described, the nerve terminals present in central and peripheral lymphoid organs (Bellinger et al., 1997), and the inflammatory and immune cells (Cutz et al., 1978; Goetzl et al., 1988; Gomariz et al., 1990; Weinstock and Blum, 1990). The first identification of VIP immunoreactivity in cells other than neurons was in peritoneal rat mast cells (Cutz et al., 1978), followed by eosinophils (Weinstock and Blum, 1990). The first evidence that lymphocytes produced VIP was the presence of VIP immunoreactivity in thymus, spleen and lymph nodes cells using immunohistochemical methods with a polyclonal antibody (Gomariz et al., 1990). These results were later confirmed by the biochemical characterization of VIP by reverse-phase high performance liquid chromatography (HPLC) and radioimmunoassay (RIA) in lymphoid cell suspensions (Gomariz et al., 1992), and finally VIP synthesis was demonstrated by in situ hybridization and RT–PCR (Gomariz et al., 1993, Gomariz et al., 1994a). Different studies have indicated an important role for VIP in natural and acquired immunity (De la Fuente et al., 1996) such as natural killer cell activity, cytokine production, lymphocyte adhesion and traffic, T and B proliferative responses, and apoptosis. These actions are exerted through a family of VIP receptors that have been recently cloned and sequenced. The type 1 VIP receptor (VIP1/PACAP receptor), coupled to the adenylate cyclase system (Calvo et al., 1996) is expressed in lymphocytes and macrophages (Calvo et al., 1994; Gomariz et al., 1994b; Delgado et al., 1996a; Johnson et al., 1996). The type 2 VIP receptor (VIP2/PACAP receptor), coupled to the adenylate cyclase system and to a calcium chloride channel (Calvo et al., 1996) is expressed in lymphocytes (Delgado et al., 1996b). Finally, a third class of VIP binding site is the PACAP1 receptor that recognizes both forms of the pituitary adenylate cyclase-activating polypeptide (PACAP27 and PACAP38), two peptides structurally related with VIP, and shows a 300- to 1000-fold lower VIP affinity (Rawlings, 1994). This receptor is expressed in macrophages but not in lymphocytes (Pozo et al., 1997) and is coupled with both the adenylate cyclase and the PLC systems (Rawlings, 1994).

Although VIP is synthesized by lymphocytes, there is no evidence demonstrating that VIP is released, and which stimuli are able to induce VIP production and secretion. Therefore, the present study was designed to determine whether agents that mediate important immune functions, such as proliferation, inflammation or apoptosis, also induce the production and release of VIP to the lymphoid microenvironment. Our results demonstrate, for the first time, that VIP secretion from lymphocytes of lymphoid organs and peritoneum is promoted by proliferative stimuli, proinflammatory cytokines and glucocorticoids.

Section snippets

Animals

Adult male Wistar rats (Iffa Credo), weighing 200–250 g, were maintained at constant temperature (23±2°C) on a 12 h light/dark cycle and food and water ad libitum.

Antibodies and reagents

Monoclonal anti-VIP antibody (clone CURE.V55) was a generous gift from Dr. Wong (UCLA School of Medicine, Los Angeles, CA). FITC-labeled F(ab′)2 fragments of sheep anti-mouse IgG was from Sigma (St. Louis, MO). Synthetic VIP, PACAP27, PACAP38, VIP1–12 and VIP10–28 were purchased from Novabiochem (Läufelfingen, Switzerland), and VIP6–28

VIP expression in lymphocytes of central and peripheral lymphoid organs and peritoneum after mitogenic stimulation

In the present study we have used a recently synthesized monoclonal anti-VIP antibody (Wong et al., 1996) to improve previous results obtained in our laboratory with a polyclonal antibody, as well as to develop a competitive ELISA in order to measure the VIP released to the lymphoid microenvironment. Flow cytometry analysis in freshly isolated cells with the monoclonal anti-VIP antibody demonstrates that 37% of thymocytes, 27% of splenocytes, 47% of lymph node lymphocytes, and 15% of peritoneal

Discussion

Several different groups have reported an increase in neuropeptide production by immune cells after mitogenic stimulation in culture (Zurawski et al., 1986; Clarke et al., 1993), and we provided evidence that lymphocytes can produce VIP (Gomariz et al., 1994a). In the present report we also demonstrate their ability to synthesize VIP in response to different stimuli and to secrete this neuropeptide to the culture medium `in vitro'. We have developed an ELISA that was specific and highly

Acknowledgements

This work was supported by grant PB94-0310 from the Spanish Department of Education and Science. We are grateful to Dr. H.C. Wong for the generous gift of the anti-VIP monoclonal antibody (UCLA School of Medicine, Los Angeles, CA).

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