Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit tumor necrosis factor- transcriptional activation by regulating nuclear factor-B and cAMP response element-binding protein/c-Jun. the same concentration. TNF- protein levels were reduced 90% by VIP or PACAP at 10?7m. An antagonist of VPAC1 receptors blocked the action of VIP and PACAP, and a PAC1 antagonist blocked the action of PACAP. A direct demonstration of VIP binding on microglia and the existence Tetrodotoxin of mRNAs for VPAC1 and PAC1 (but not C13orf15 VPAC2) receptors argue for a Tetrodotoxin receptor-mediated effect. The action of VIP is cAMP-mediated because (1) activation of cAMP by forskolin mimics the action; (2) PKA inhibition by H89 reverses the neuropeptide-induced inhibition; and (3) the lipophilic neuropeptide mimic, stearyl-norleucine17 VIP (SNV), which does not use a cAMP-mediated pathway, fails to duplicate the inhibition. We conclude that VIP and PACAP inhibit the production of TNF- from activated microglia by a cAMP-dependent pathway. 055:B5 (Sigma, St. Louis, MO) was resuspended in sterile PBS and stored at ?20C. Forskolin (Sigma) and(C. Wang et al., 1996, 1997; Bartholdi and Schwab, 1997; Hayashi et al., 1997; Streit et al., 1998; Hart et al., 1999), and this temporal profile of cytokine production is mimicked in spinal cord slices (Hart et al., 1999). Total RNA from the spinal cord slices was analyzed with a multiprobe RPA for cytokine mRNAs (see Materials and Methods). Consistent with the data of others (Bartholdi and Schwab, 1997; Streit et al., 1998; Hart et al., 1999), TNF- mRNA was undetected in uninjured cords, but spinal cord transection produced an elevation of mRNAs for TNF- as well as IL-1, IL-1, and IL-6 within 2 hr (Fig. ?(Fig.1).1). Inclusion of the synthetic VPAC1 receptor agonist (10?8m) inhibited TNF- mRNA expression by as much as 50%. Inhibitory effects also were seen on IL-1, IL-6, and IL-10. Open in a separate window Fig. 1. Spinal cord transection induces inflammatory cytokine mRNA expression. Freshly isolated spinal cord slices were incubated with or without 10?8m or 10?9m of a synthetic VPAC1agonist [denoted as or is a probe set untreated with RNase. The lanes on the are protected fragments resulting from RNase treatment. In (Lee et al., 1993; Laskin and Pendino, 1995). A low but detectable level of TNF- mRNA was present in untreated microglia, but LPS treatment (100 ng/ml) raised levels of TNF- mRNA substantially as early as 1 hr after exposure (Fig.?(Fig.2).2). Simultaneous treatment with VIP (10?7m) inhibited LPS-induced TNF- mRNA levels 45% (Fig.?(Fig.3).3). PACAP (10?7m) completely abolished the LPS-induced increase (Fig. ?(Fig.3).3). Trypan blue exclusion confirmed that VIP did not affect the viability of microglial cells (data not shown). These data indicate that the LPS-inducible increase in TNF- gene expression in cultured microglia is inhibited by the authentic neuropeptides VIP and PACAP. Open in a separate window Fig. 2. LPS induces TNF- mRNA in microglia.represents the average of TNF determinations (pg/ml) in duplicate cultures. Cells cultured without LPS did not produce detectable amounts of TNF ( 15.6 pg/ml). Open in a separate window Fig. 5. VIP and PACAP inhibit TNF- protein production via specific receptors. represents the mean SEM of TNF- protein (pg/ml) in three separate cultures. This experiment was repeated with similar results. Data were compared by using an ANOVA with aFisher’s test for comparisons at the 95% confidence level. *Different when compared with cultures treated with LPS and VIP or PACAP. Microglia express VPAC1 and PAC1? receptors Both VIP and PACAP use G-protein-linked seven-transmembrane-domain receptors. VIP has highest efficacy at the VPAC1(or VIP1) and VPAC2(VIP2) receptors, and PACAP has almost equal efficacy at these two receptors as well as a third, the so-called PAC1 receptor (for review, see Dickinson and Fleetwood-Walker, 1999). To determine which of these receptor subtypes is responsible for the neuropeptide effect on microglia, we examined mRNA for the three receptors by using RT-PCR analysis. mRNAs for VPAC1 and PAC1 were expressed in microglia. VPAC2 receptor mRNA was not present even in LPS-treated microglia (Fig.?(Fig.66). Open in a separate window Fig. 6. Microglia express mRNAs for VPAC1 and PAC1 but not VPAC2, receptors.andwere collected by using identical iris, gain, and background settings. Biotinylated VIP was visualized with Texas Red-conjugated avidin D. OX-42 was visualized with FITC-conjugated anti-mouse Ig (see Materials and Methods). Scale bar, 12 m. The VIP-induced downregulation of TNF, the presence of VPAC1 and PAC1 mRNAs, the direct demonstration of VIP binding, and the inhibition by receptor-specific antagonists (see Fig. ?Fig.55Fisher’s test for 95% confidence levels. *Different when compared with cultures.Biotinylated VIP was visualized with Texas Red-conjugated avidin D. receptors blocked the action of VIP and PACAP, and a PAC1 antagonist blocked the action of PACAP. A direct demonstration of VIP binding on microglia and the existence of mRNAs for VPAC1 and PAC1 (but not VPAC2) receptors argue for a receptor-mediated effect. The action of VIP is cAMP-mediated because (1) Tetrodotoxin activation of cAMP by forskolin mimics the action; (2) PKA inhibition by H89 reverses the neuropeptide-induced inhibition; and (3) the lipophilic neuropeptide mimic, stearyl-norleucine17 VIP (SNV), which does not use a cAMP-mediated pathway, fails to duplicate the inhibition. We conclude that VIP and PACAP inhibit the production of TNF- from activated microglia by a cAMP-dependent pathway. 055:B5 (Sigma, St. Louis, MO) was resuspended in sterile PBS and stored at ?20C. Forskolin (Sigma) and(C. Wang et al., 1996, 1997; Bartholdi and Schwab, 1997; Hayashi et al., 1997; Streit et al., 1998; Hart et al., 1999), and this temporal profile of cytokine production is mimicked in spinal cord slices (Hart et al., 1999). Total RNA from the spinal cord slices was analyzed with a multiprobe RPA for cytokine mRNAs (see Materials and Methods). Consistent with the data of others (Bartholdi and Schwab, 1997; Streit Tetrodotoxin et al., 1998; Hart et al., 1999), TNF- mRNA was undetected in uninjured cords, but spinal cord transection produced an elevation of mRNAs for TNF- as well as IL-1, IL-1, and IL-6 within 2 hr (Fig. ?(Fig.1).1). Inclusion of the synthetic VPAC1 receptor agonist (10?8m) inhibited TNF- mRNA expression by as much seeing that 50%. Inhibitory results also were noticed on IL-1, IL-6, and IL-10. Open up in another screen Fig. 1. Spinal-cord transection induces inflammatory cytokine mRNA appearance. Freshly isolated spinal-cord slices had been incubated with or without 10?8m or 10?9m of the man made VPAC1agonist [denoted seeing that or is a probe place Tetrodotoxin untreated with RNase. The lanes over the are covered fragments caused by RNase treatment. In (Lee et al., 1993; Laskin and Pendino, 1995). A minimal but detectable degree of TNF- mRNA was within neglected microglia, but LPS treatment (100 ng/ml) elevated degrees of TNF- mRNA significantly as soon as 1 hr after publicity (Fig.?(Fig.2).2). Simultaneous treatment with VIP (10?7m) inhibited LPS-induced TNF- mRNA amounts 45% (Fig.?(Fig.3).3). PACAP (10?7m) completely abolished the LPS-induced boost (Fig. ?(Fig.3).3). Trypan blue exclusion verified that VIP didn’t have an effect on the viability of microglial cells (data not really proven). These data suggest which the LPS-inducible upsurge in TNF- gene appearance in cultured microglia is normally inhibited with the genuine neuropeptides VIP and PACAP. Open up in another screen Fig. 2. LPS induces TNF- mRNA in microglia.represents the common of TNF determinations (pg/ml) in duplicate civilizations. Cells cultured without LPS didn’t produce detectable levels of TNF ( 15.6 pg/ml). Open up in another screen Fig. 5. VIP and PACAP inhibit TNF- proteins production via particular receptors. represents the mean SEM of TNF- proteins (pg/ml) in three split cultures. This test was repeated with very similar results. Data had been compared through the use of an ANOVA with aFisher’s check for comparisons on the 95% self-confidence level. *Different in comparison to civilizations treated with LPS and VIP or PACAP. Microglia exhibit VPAC1 and PAC1?receptors Both VIP and PACAP make use of G-protein-linked seven-transmembrane-domain receptors. VIP provides highest efficacy on the VPAC1(or VIP1) and VPAC2(VIP2) receptors, and PACAP provides almost equal efficiency at both of these receptors and a third, the so-called PAC1 receptor (for review, find Dickinson and Fleetwood-Walker, 1999). To determine which of the receptor subtypes is in charge of the neuropeptide influence on microglia, we analyzed mRNA for the three receptors through the use of RT-PCR evaluation. mRNAs.