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

Immunology. 2000 Sep;101(1):61-7. doi: 10.1046/j.1365-2567.2000.00082.x.


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 (Mphi). In this study we examined whether CGRP could modulate IL-12 release from mouse peritoneal Mphi, and if so, what signal transduction pathway was involved. Mphi were obtained from the peritoneal exudate of male BALB/c mice. The cells were plated on culture dishes at a density of 5 x 105 cells per well and allowed to adhere for 2 hr. After incubation for 24 hr, the Mphi were cultured with 0.1 microg/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 Mphi. The LPS-induced IL-12 release from Mphi 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 microM or 1 microM), 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 Mphi.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcitonin Gene-Related Peptide / pharmacology*
  • Cyclic AMP / physiology*
  • Cyclic AMP-Dependent Protein Kinases / physiology
  • Gene Expression
  • Interleukin-12 / genetics
  • Interleukin-12 / metabolism*
  • Lipopolysaccharides / immunology*
  • Macrophages, Peritoneal / drug effects
  • Macrophages, Peritoneal / immunology*
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Peptide Fragments / pharmacology
  • RNA, Messenger / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction / immunology


  • Lipopolysaccharides
  • Peptide Fragments
  • RNA, Messenger
  • Interleukin-12
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • Calcitonin Gene-Related Peptide