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 (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.
Liu
J
J
Institute of Vascular Medicine, The Third Hospital, Beijing Medical University, Beijing, China.
Chen
M
M
Wang
X
X
D000818:Animals; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology*; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D015870:Gene Expression; D018664:Interleukin-12 / Q000235:genetics / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology*; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000276:immunology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000276:immunology
D008070:Lipopolysaccharides; D010446:Peptide Fragments; D012333:RNA, Messenger; D018664:Interleukin-12; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015740:Calcitonin Gene-Related Peptide