Calcitonin gene-related peptide inhibits lipopolysaccharide-induced interleukin-12 release from mouse peritoneal macrophages, mediated by the cAMP pathway

Previously we showed that calcitonin gene-related peptide (CGRP), a neuropeptide, inhibited lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) production and increased interleukin (IL)-6 release at low concentrations via activation of the cAMP pathway in mouse peritoneal macrophages (M phi). In this study we examined whether CGRP could modulate IL-12 release from mouse peritoneal M phi, and if so, what signal transduction pathway was involved. M phi were obtained from the peritoneal exudate of male BALB/c mice. The cells were plated on culture dishes at a density of 5 x 10(5) cells per well and allowed to adhere for 2 hr. After incubation for 24 hr, the M phi were cultured with 0.1 mu g/ml of LPS, alone or together with CGRP (1-1000 nm) for 24 hr. The amount of IL-12 in the cell medium was measured by enzyme-linked immunosorbent assay (ELISA). The results showed that CGRP attenuated LPS-induced IL-12 release in a concentration-dependent manner. Production of IL-12 was decreased from 95.9 +/- 4.6 to 73.4 +/- 5.7 pg/ml by 100 nm CGRP. The two cAMP phosphodiesterase (PDE) inhibitors, 3-isobutyl-1-methyl-xanthine (IBMX) and rolipram, significantly potentiated the CGRP response, and the level of IL-12 was further decreased by 28% and 47%, respectively. However, CGRP had no effect on IL-12 production from unstimulated M phi. The LPS-induced IL-12 release from M phi could also be reduced by forskolin, an activator of adenylate cyclase, and 8-Br-cAMP, an analogue of cAMP. Using the reverse transcription-polymerase chain reaction (RT-PCR), we found that CGRP also decreased the LPS-induced IL-12 p40 mRNA levels. Furthermore, pretreatment with H89 (0.1 mu m or 1 mu m), an inhibitor of cAMP-dependent protein kinase, diminished CGRP effects, IL-12 production and gene expression. These data suggest that LPS-induced IL-12 release and gene expression were attenuated by CGRP via an activated cAMP-protein kinase A (PKA) pathway in mouse peritoneal M phi.