Complex array of cytokines released by vasoactive intestinal peptide
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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