Vasoactive intestinal peptide and pituitary adenylyl cyclase-activating polypeptide inhibit tumor necrosis factor-alpha production in injured spinal cord and in activated microglia via a cAMP-dependent pathway

Abstract

Tumor necrosis factor-alpha (TNF-alpha) production accompanies CNS insults of all kinds. Because the neuropeptide vasoactive intestinal peptide (VIP) and the structurally related peptide pituitary adenylyl cyclase-activating polypeptide (PACAP) have potent anti-inflammatory effects in the periphery, we investigated whether these effects extend to the CNS. TNF-alpha mRNA was induced within 2 hr after rat spinal cord transection, and its upregulation was suppressed by a synthetic VIP receptor agonist. Cultured rat microglia were used to examine the mechanisms underlying this inhibition because microglia are the likely source of TNF-alpha in injured CNS. In culture, increases in TNF-alpha mRNA resulting from lipopolysaccharide (LPS) stimulation were reduced significantly by 10(-7) m VIP and completely eliminated by PACAP at the same concentration. TNF-alpha protein levels were reduced 90% by VIP or PACAP at 10(-7) m. An antagonist of VPAC(1) receptors blocked the action of VIP and PACAP, and a PAC(1) antagonist blocked the action of PACAP. A direct demonstration of VIP binding on microglia and the existence of mRNAs for VPAC(1) and PAC(1) (but not VPAC(2)) receptors argue for a 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-norleucine(17) 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-alpha from activated microglia by a cAMP-dependent pathway.

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 VPAC(8) or VPAC(9), respectively] for 2 hr. RNA was extracted and assayed by RPA for cytokine mRNAs (A). The lane markedProbe is a probe set untreated with RNase. The lanes on the right are protected fragments resulting from RNase treatment. In B, the levels of TNF-α mRNA were quantified by using a PhosphorImager with IPLab Gel H software and expressed as a ratio of TNF-α mRNA to GAPDH mRNA. These ratios were assigned arbitrary units for comparison. At time 0, TNF-α mRNA is not detectable (N.D.). This experiment was repeated with virtually identical results.

Fig. 2.

LPS induces TNF-α mRNA in microglia.A, Cultured microglia were treated with LPS (100 ng/ml) for various times, and RNA was assayed by RPA as described in Figure 1.B, TNF-α mRNA was expressed as a ratio to GAPDH mRNA and plotted as arbitrary units. TNF-α mRNA was elevated as early as 1 hr after LPS treatment.

Fig. 3.

VIP and PACAP inhibit the LPS-induced increase in TNF-α mRNA in microglia. Cultured microglia were treated for 3 hr with LPS (100 ng/ml) with or without VIP or PACAP at 10⁻⁷m or with the neuropeptides after a 30 min pretreatment with H89 (10⁻⁶m). A, RPA analysis of TNF-α mRNA was performed as described in Figure 1. B,TNF-α mRNA was measured in arbitrary units and expressed as a ratio to GAPDH mRNA for comparisons among treatments.

Fig. 4.

LPS induces TNF-α protein accumulation over time. Cultured microglia were stimulated with 100 ng/ml of LPS. The supernatants were collected at different times and assayed for TNF-α protein accumulation by ELISA. Each point 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).

Fig. 5.

VIP and PACAP inhibit TNF-α protein production via specific receptors. A, Microglia were treated for 6 hr with various concentrations of LPS (0.1–1000 ng/ml) in the absence or presence of 10⁻⁸m VIP or PACAP. TNF-α accumulation was assayed by ELISA. Each pointrepresents the mean ± SEM of TNF-α protein (pg/ml) in three separate cultures. This experiment was repeated with identical results. Data were compared by using an ANOVA with a post hocFisher's test for comparisons at the 95% confidence level. At all concentrations of LPS above 1 ng/ml, the inhibition produced by VIP and PACAP is significantly different from LPS alone. B, Microglia were treated with LPS (100 ng/ml) and various concentrations of VIP or PACAP for 6 hr. Data are expressed as the mean of TNF-α produced from two separate cultures. Microglia were treated with VIP (C) or PACAP (D) and different concentrations of VPAC₁ and PAC₁antagonists in the presence of LPS (100 ng/ml) for 6 hr. Eachpoint represents the mean ± SEM of TNF-α protein (pg/ml) in three separate cultures. This experiment was repeated with similar results. Data were compared by using an ANOVA with apost hoc Fisher's test for comparisons at the 95% confidence level. *Different when compared with cultures treated with LPS and VIP or PACAP.

Fig. 6.

Microglia express mRNAs for VPAC₁ and PAC₁ but not VPAC₂, receptors.A, mRNA from untreated microglia or microglia treated LPS for 3 hr was prepared and examined by RT-PCR (see Materials and Methods). B, Rat brain and astrocytic RNA were used as positive controls for VPAC₂.

Fig. 7.

Microglia bind biotinylated VIP. Shown are confocal images of cultured microglia after a 2 hr incubation with LPS (100 ng/ml) together with 10⁻¹⁰mbiotinylated VIP (A) or after a 1.5 hr incubation with LPS followed by 10⁻¹⁰mbiotinylated VIP for 30 min (B). To determine binding specificity, we treated HAPI cells for 210 min with LPS. Biotinylated VIP (10⁻¹⁰m) was added for the final 30 min (C). Sister cultures were treated the same except for the inclusion of 1 μmunlabeled VIP during the final 150 min of LPS administration (D). Confocal images in C andD were 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 μm.

Fig. 8.

Forskolin mimics the inhibition by neuropeptides of microglial TNF-α production. Microglia were treated with LPS (100 ng/ml) and various concentrations of forskolin (bars) or VIP (curve). Supernatants were collected after 6 hr and assayed for TNF-α by ELISA. Data are expressed as the mean ± SEM of TNF-α protein in three or four separate cultures and were compared by using an ANOVA with a post hoc Fisher's test for 95% confidence levels. *Different when compared with cultures treated with LPS alone.

Fig. 9.

H89 reverses the neuropeptide inhibition of LPS-induced TNF-α production. Microglia were treated with LPS (100 ng/ml) alone (Control) or with VIP (10⁻⁷m) after a 30 min preincubation with various concentrations of H89. Supernatants were assayed for TNF-α production after 6 hr. Data are expressed as the mean ± SEM of TNF-α protein in three or four separate cultures and were compared by using an ANOVA with a post hoc Fisher's test for 95% confidence levels. All concentrations of H89 produced a significant reversal of the VIP-induced inhibition of LPS-induced TNF-α production. Cells treated with H89 alone or H89 plus VIP did not produce detectable amounts of TNF (<15.6 pg/ml).

Fig. 10.

SNV fails to mimic the VIP-induced inhibition of TNF-α production. Cultured microglia were treated with various concentrations of SNV or VIP in the presence of LPS (100 ng/ml). Data are expressed as the mean ± SEM of TNF-α protein in three or four separate cultures and were compared by using an ANOVA with apost hoc Fisher's test for 95% confidence levels. Only those cultures with VIP showed a significant inhibition of the LPS-induced TNF-α production.