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‐α (TNF‐α) production and increased interleukin (IL)‐6 release at low concentrations via activation of the cAMP pathway in mouse peritoneal macrophages (Mφ). In this study we examined whether CGRP could modulate IL‐12 release from mouse peritoneal Mφ, and if so, what signal transduction pathway was involved. Mφ were obtained from the peritoneal exudate of male BALB/c mice. The cells were plated on culture dishes at a density of 5 × 105 cells per well and allowed to adhere for 2 hr. After incubation for 24 hr, the Mφ were cultured with 0·1 µg/ml of LPS, alone or together with CGRP (1–1000 n m) 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 n m 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 Mφ. The LPS‐induced IL‐12 release from Mφ 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 µm or 1 µm), 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 Mφ.