PACAP-cytokine interactions govern adrenal neuropeptide biosynthesis after systemic administration of LPS

Abstract

We have examined induction of neuropeptide expression in adrenal medulla after treatment of mice with lipopolysaccharide (LPS), a model for septic shock, which activates both immune and stress responses in vivo. Messenger RNAs encoding vasoactive intestinal polypeptide (VIP) and galanin, both modulators of steroidogenesis in neighboring adrenal cortex, are up-regulated at 24 h (eight-fold for VIP and two-fold for galanin) after LPS injection, and remain elevated for the following 24 h. Up-regulation of VIP and galanin by LPS is abrogated in pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice, suggesting an interaction between LPS, or LPS-induced cytokines, and PACAP released in adrenal medulla from the splanchnic nerve. Treatment of cultured chromaffin cells with 100 nM PACAP and 10 nM tumor necrosis factor-alpha (TNF-alpha), a cytokine whose production is elevated by LPS, results in long-term synergistic up-regulation of VIP and galanin mRNA. PACAP blocks the earlier induction by TNF-alpha of mRNA encoding inhibitor of NF-kappaB alpha (I kappaB alpha), normally a negative autoregulator of TNF-alpha signaling through nuclear factor-kappaB (NF-kappaB), without affecting the induction of TNF-alpha-induced protein 3 (TNFAIP3), another NF-kappaB-dependent gene induced by TNF-alpha in chromaffin cells. By acting downstream of NF-kappaB to inhibit I kappaB alpha gene induction by TNF-alpha, PACAP may block I kappaB alpha-dependent negative autoregulation of TNF-alpha signaling through NF-kappaB, prolonging TNF-alpha-dependent signaling to neuropeptide-encoding genes in chromaffin cells. This mechanism may also underlie PACAP-dependent neuropeptide gene induction by LPS in vivo.

Fig. 1

VIP and galanin, but not CgA mRNA, are upregulated in adrenal gland after 24 and 48 h LPS challenge. C57BL/6 males were injected with LPS (1 mg/kg) or saline solution. After 24 h and 48 h, adrenal glands were collected and the expression of VIP (upper panel), galanin (middle panel) and CgA (lower panel) was analyzed by Q-RT-PCR. Results are expressed as fold increase over corresponding control values and represent means ± SEM of four or five determinations for each condition. NS, P>0.05; *, P<0.05; **, P<0.01 vs. the corresponding control (Student’s t test).

Fig. 2

LPS significantly increases galanin and VIP mRNA levels, by a PACAP-dependent mechanism. PACAP KO and WT mice were injected with LPS (1 mg/kg) or saline solution and adrenal glands were collected after 24 h. VIP (A) and galanin (B) mRNA levels, determined by Q-RT-PCR, are expressed as fold increase over control wild type mice values and represent means ± SEM of four or five determinations for each condition from the mean of two different experiments. NS, P>0.05; *, P<0.05 vs. the corresponding control (one-way ANOVA test, Tukey posttest).

Fig 3

VIP and galanin mRNA are stimulated by TNF-α but not by LPS in BCCs after 24 h. BCCs were exposed to either TNF- α (10 nM), or LPS (10 μg/ml) for 24 hours. VIP (A) and galanin (B) mRNA levels, determined by Q-RT-PCR, are expressed as fold increase over corresponding control values and represent means ± SEM of three determinations for each condition. NS, P>0.05; *, P<0.05 vs. the corresponding control (one-way ANOVA test, Dunnett post test).

Fig. 4

PACAP and TNF-α synergistically up-regulate neuropeptide gene expression. BCCs were exposed to either TNF-α (10 nM), PACAP (100 nM) or both agents, for 48 hours. VIP (A) and galanin (B) mRNA levels, determined by Q-RT-PCR, are expressed as fold increase over corresponding control values and represent means ± SEM of four determinations for each condition from one experiment representative of three different experiments. **, P<0.01; ***, P<0.001 combined treatment (PACAP plus TNF-α)vs. individual treatment (PACAP or TNF-α) using one-way ANOVA test, with Tukey posttest.

Fig. 5

PACAP attenuates TNF-α induction of IκBα but not TNFAIP3 mRNA. BCCs were exposed to either TNF-α (10 nM), PACAP (100 nM) or both agents, for 24 hours. IκBα (A) and TNFAIP3 (B) mRNA levels, determined by Q-RT-PCR, are expressed as fold increase over corresponding control values and represent means ± SEM of four determinations for each condition from one experiment representative of three different experiments. NS, P>0.05; *, P<0.05; ***, P<0.001 vs. the corresponding control (one-way ANOVA test, Tukey posttest).