VIP and PACAP enhance IL-6 release and mRNA levels in resting peritoneal macrophages: in vitro and in vivo studies¹

Abstract

Vasoactive intestinal peptide (VIP), a neuropeptide produced by lymphocytes has been previously reported to modulate cytokine expression in T lymphocytes. In this study, we investigated the effects of VIP and of the structurally related neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP38) on the production of IL-6 in unstimulated murine peritoneal macrophages. Both neuropeptides stimulate rapidly, specifically, and similarly the production of IL-6, exerting their action through two different receptor/signal transduction systems, i.e., primarily through the binding to VIP₁/PACAP receptor followed by adenylate cyclase activation, and partially through the activation of protein kinase C following binding to PACAP-R. VIP and PACAP38 regulate the production of IL-6 at a transcriptional level, affecting the de novo synthesis of this cytokine. The stimulatory in vitro effect correlates with the stimulation of IL-6 expression and release in vivo. These studies suggest that VIP/PACAP play a role in immune system homeostasis, participating in the intricate cytokine network and controlling local immune responses. In addition, the understanding of the factors that regulate the expression and release of IL-6 by macrophages is important for the elucidation of the role of IL-6 in health and disease.

Introduction

Macrophages are widely known as cells that play an important role in inflammatory processes as well as in the initiation, maintenance and control of primary and secondary immune responses. The execution of these activities is mediated by complex and multifunctional processes involving macrophage secretory cytokines.

IL-6, one of the pleiotrophic cytokines, regulates both innate and acquired immunity, haematopoiesis and acute-phase reactions (Van Snick, 1990; Hirano, 1994). This cytokine is produced by a variety of cell types including fibroblasts (Weissenbach et al., 1980), monocytes/macrophages (Aarden et al., 1985), T-cell lines (Van Snick et al., 1986) and folliculo-stellate cells of the anterior pituitary (Matsumoto et al., 1993). Furthermore, IL-6 could be classified as a mediator that belongs to the growing list of cytokines and neuropeptides and corresponding receptors shared by the nervous and the immune system.

Among neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP38), the most recently discovered member of the VIP/secretin/glucagon family (Miyata et al., 1989), have been identified as important immunomodulatory peptides.

The immunological actions of VIP and PACAP are exerted through a family of VIP/PACAP receptors that have been recently cloned and sequenced. The PACAP receptor (PACAP-R) binds the two amidated PACAP forms, PACAP27 and PACAP38 with similar affinity, but has a 300 to 1000-fold lower affinity for VIP in human neuroblastoma (Christophe, 1993). This receptor is coupled to the activation of both the adenylate cyclase and PLC systems (Christophe, 1993; Spengler et al., 1993). Experiments in our laboratory demonstrated PACAP-R gene expression in rat peritoneal macrophages (Pozo et al., 1997). The VIP₁/PACAP receptor recognizes with comparable high affinity VIP and both PACAP forms, and is coupled to adenylate cyclase (Calvo et al., 1996; Rawlings and Hezareh, 1996). Lymphocytes and macrophages have been shown to possess the VIP₁/PACAP receptor (Guerrero et al., 1981; Segura et al., 1991; Gomariz et al., 1994a; Delgado et al., 1996a). Finally, the VIP₂/PACAP receptor has similar affinities for PACAP and VIP, and is coupled to the adenylate cyclase system (Calvo et al., 1996; Rawlings and Hezareh, 1996) and to a calcium chloride channel in transfected cells (Inagaki et al., 1994). The expression of VIP₂/PACAP-R has been reported in lymphocytes (Delgado et al., 1996b).

Since nineteen years separate the discovery of VIP (Said and Mutt, 1970) and PACAP (Miyata et al., 1989), the immune actions of PACAP are scarcely studied as compared to VIP. Furthermore, in view of the finding that common receptors recognize VIP and PACAP with similar affinities, some of the immunological actions previously ascribed to VIP must be reviewed. Thereby, functional comparative studies with these two neuropeptides could contribute to the understanding of their precise immune action and the receptor type implicated, as well as the second messengers involved in the transduction signal.

Although PACAP/VIP effects on IL-6 production have been studied in several non-immune cells such as pituitary-stellate cells (Spangelo et al., 1990; Tatsuno et al., 1991a), astrocytes (Gottschall et al., 1994), and osteoblasts (Greenfield et al., 1996), data in cells of the immune system are very scarce. Moreover, to our knowledge there are no reports regarding the effects of VIP/PACAP on IL-6 production in vivo in macrophages.

For these reasons, the purpose of the present study was to investigate the comparative effect of VIP and PACAP on the production of IL-6 by murine peritoneal macrophages. Our results indicate that the two neuropeptides stimulate both in vivo and in vitro IL-6 production at a transcriptional level. Furthermore, we suggest that this stimulation is mediated by both PACAP and VIP₁/PACAP receptors.