Elsevier

Neuropeptides

Volume 37, Issue 2, April 2003, Pages 111-119
Neuropeptides

Complex array of cytokines released by vasoactive intestinal peptide

https://doi.org/10.1016/S0143-4179(03)00022-2Get rights and content

Abstract

A complex mixture of five cytokines has been shown to be released by vasoactive intestinal peptide (VIP). Cytokines were measured in paired samples of culture medium and astroglial cytosol by capillary electrophoresis. This is the first description of VIP-mediated release for TNF-α, IL-3, G-CSF and M-CSF from astrocyte cultures. Kinetic studies after VIP treatment demonstrated a gradual but incomplete depletion of cytosolic cytokine levels, with differences observed among the cytokines. Significant increases in release were apparent within 15–30 min for all cytokines. As the recognized VIP receptors (VPAC1 and VPAC2) are linked to adenylate cyclase and also interact with pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), both this homologous peptide and 8-bromo cAMP were investigated and compared to VIP-mediated release. Treatment with 1 mM 8-bromo cAMP produced cytokine release similar in amount to 0.1 nM PACAP-38, but significantly less (<50%) in comparison to 0.1 nM VIP. PACAP-38 and VIP exhibited similar EC50’s for the release of G-CSF and TNF-α; however, the maximal release was 4–6 times greater for VIP than for PACAP-38. This similarity in potency suggested a VPAC-like receptor; however, the greater efficacy for VIP in comparison to PACAP-38, combined with a lack of cAMP production at subnanomolar concentrations of VIP, suggested a mechanism not currently associated with VPAC receptors. For M-CSF, IL-3 and IL-6, the EC50’s of VIP were 3–30 times more potent than those of PACAP-38 in producing release. These studies suggested that multiple mechanisms mediate cytokine release in astrocytes: (1) a low efficacy release produced by PACAP-38 that is cAMP-mediated and (2) a high efficacy, VIP-preferring mechanism that was not linked to cAMP. In summary, subnanomolar concentrations of VIP released a complex array of cytokines from astrocytes that may contribute to the mitogenic and neurotrophic properties of this neuropeptide in the central nervous system.

Introduction

Vasoactive intestinal peptide (VIP), a 28 amino acid polypeptide widely distributed in the peripheral and central nervous systems (Gozes and Brenneman, 1989), has neurotrophic and growth-promoting actions (Brenneman and Eiden, 1986; Brenneman et al., 1990; Gressens et al., 1993; Shoge et al., 1998). Previous studies have shown that these developmentally important functions are contingent on interactions with astrocytes (Brenneman et al., 1987; Brenneman et al., 1990). Among the glial mediators of VIP are: cytokines interleukin-1 (IL-1) α and β (Brenneman et al., 1992, Brenneman et al., 1995), interleukin-6 (IL-6) (Gottschall et al., 1994), a protease inhibitor (protease nexin-1) (Festoff et al., 1996), chemokines (RANTES and macrophage inflammatory protein-1α) (Brenneman et al., 1999) and growth factors (activity dependent neurotrophic factor) (Brenneman and Gozes, 1996), neurotrophin-3 (Blondel et al., 2000) and activity-dependent neuroprotective protein (ADNP) (Bassan et al., 1999). The association of a neurotrophic peptide and the release of potentially proinflammatory cytokines is enigmatic. For example, previous studies have demonstrated that IL-1α and β are toxic to neurons (Brenneman et al., 1993; Downen et al., 1999) and these cytokines have been associated with neurodegenerative disease (Neuman and Wekerle, 1998; Rothwell and Luheshi, 2000). However, in developing cultures, IL-1α can increase neuronal survival at low concentration and related studies have indicated that antiserum to IL-1α can produce neuronal cell death (Brenneman et al., 1992). Furthermore, in the same cultures, application of IL-1α to more mature cultures resulted in neurotoxicity, suggesting a strong developmental requirement or age-dependent vulnerability to this cytokine (Brenneman et al., 1993). These and many other studies indicate that the actions of cytokines are complex, interactive and highly concentration dependent.

Vasoactive intestinal peptide has recognized functions in both the immune and nervous system. Several recent reviews have emphasized that VIP has important regulatory roles in immunity that involved both the transcriptional control of expression of cytokines and modulation of their release (Ganea and Delgado, 2001). In addition, the expression of VIP and its receptors can be regulated by numerous cytokines. (Dorsam et al., 2000). In the periphery, VIP is known to act as an immunosuppressive agent (DelaFuente et al., 1996) and to have an anti-inflammatory function (Kim et al., 2000). In the central nervous system, VIP has been shown to be among the substances that are neuroprotective and regulate neuronal survival (Brenneman et al., 1988; Said et al., 1995). Among the cytokines previously shown to be released by VIP are IL-1α, IL-1β (Brenneman et al., 1992) and IL-6 (Kanterman et al., 1990). In addition to regulating IL-1α release, previous studies have indicated that VIP treatment produced a transient decrease in IL-1α mRNA in astroglial cultures (Brenneman et al., 1995), strongly suggesting multiple levels of response between this peptide and cytokines. VIP has been shown to inhibit endotoxin-induced expression and release of TNF-α (Delgado et al., 1998).

In the present study, VIP is shown to potently release five cytokines. Three of these (M-CSF, G-CSF and IL-3) are shown for the first time to be associated with the action of VIP. Our studies provide a broader perspective on the complexity of VIP-elicited cytokine release and suggest that many of neurodevelopmental functions associated with this neuropeptides are linked to the actions of this complex mixture of glia-derived substances.

Section snippets

Materials

Antiserum and cytokine standards for all the cytokines were obtained from R&D Systems and Cistron Biotech (Pinebrook, NJ). Filters employed in processing the cytokine samples were obtained from Millipore. The chemiluminescent substrate utilized in ELISA assays was AMPPD (3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy)phenyl-1,2-dioxetane) obtained from Tropix (Bedford, MA). VIP was obtained from Peninsula Laboratories (Belmont, CA) and tetrodotoxin from Sigma Chemical (St. Louis, MO).

Cell culture

Results

VIP-mediated release of TNF-α, IL-3 and IL-6 into the medium is shown in Fig. 1A and the companion analyses of the depletion of these cytokines from the corresponding astrocyte cytosol are shown in Fig. 1B. The patterns of release of these three cytokines, while similar, were not identical with respect to VIP–EC50 or the amount of VIP required to elicit maximal efficacy of release. TNF-α exhibited both the highest concentration in the cellular cytosol and the greatest amount released by VIP.

Discussion

The present study has shown that the neurotrophic peptide VIP can release a complex mixture of cytokines from cultured astrocytes. While previous studies have shown that IL-1α, IL-1β and IL-6 were released by this peptide, the current study demonstrated for the first time that VIP releases IL-3, TNF-α, M-CSF and G-CSF from astrocytes. The pharmacological and kinetic properties of the VIP-mediated release revealed significant differences and similarities among the cytokines. An important finding

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