Objectives Occurring of acute coronary syndromes (ACSs) displayed circadian rhythms as well as the levels of catecholamines and pro-inflammatory cytokines in vivo. Such circadian rhythms were proved to exist also in mast cells which secreted plenty of pro-inflammatory cytokines and accumulated in the shoulder region of human coronary atherosclerosis plaques. Rev-erb α, a circadian rhythm modulating molecular, has been shown to modulate the release of pro-inflammatory cytokines in vitro and in vivo. Interestingly, an enhancing effect of epinephrine on pro-inflammatory cytokine release has been observed recently in human gingival fibroblasts. We hypothesised that there might be an cross-talking between catecholamine and circadian rhythm which could modulate production of pro-inflammatory cytokines in mast cells. We investigated whether epinephrine affects pro-inflammatory cytokines released from mast cells and whether rev-erb α modulates pro-inflammatory cytokines production in mast cells.
Methods Cultured murine bone marrow derived mast cells (BMMCs) were stimulated by lipopolysaccharide (LPS) with or without epinephrine at different concentrations from 10-6 M to 10-4 M for 2 hours. Then, SB203580(p38MAPK inhibitor), PD98059 (ERK1/2 MAPK inhibitor), PDTC (NF-κB inhibitor), SR8278 and GSK 4112 (rev-erb α antagonist and agonist) were added into cultures according to the protocol, respectively. Rev-erb α siRNA was transfected in to BMMCs by an nucleofector II device to confirm the role of rev-erb α in modulating pro-inflammatory cytokines production in BMMCs. IL-6, TNF-α and MCP-1 levels in medium were determined by ELISA. And, NF-κB p65, MAPK p38 and ERK1/2 phosphorylation and rev-erb α protein were assessed by western blot. BMMCs viability was assessed by WST-1 kit.
Results The production of IL-6, TNF-α and MCP-1 from LPS challenged BMMCs was significantly enhanced at the epinephrine concentration of 10-4M. Epinephrine increased phosphorylated p38 MAPK and phosphorylated p65 NF-κB expression, and LPS plus epinephrine could further increase the expression of phosphorylated p38, ERK1/2 and p65. SB203580, PD98059 and PDTC inhibited such enhancing effect of epinephrine on IL-6, TNF-α and MCP-1 from LPS challenged BMMCs. LPS increased rev-erb α protein expression in BMMCs, and epinephrine could exert further increasing effect on rev-erb α protein expression in LPS challenged BMMCs. Both GSK4112 and SR8278 significantly inhibited IL-6 production in LPS-challenged BMMCs. But a decreased cell viability was detected in GSK4112 activated BMMCs by WST-1 kit. And epinephrine had no significant enhancing effect on LPS-induced IL-6 release from SR8278 cultured BMMCs. Suppressive siRNA inhibited rev-erb α mRNA expression efficiently, and a decreased IL-6 releasing was observed in siRNA transfected BMMCs, compared to scramble control siRNA transfected BMMCs. No significant enhancing effect of epinephrine on IL-6 production was observed in siRNA transfected BMMCs.
Conclusions Epinephrine could further increase the production of pro-inflammatory cytokines from LPS-challenged BMMCs. MAPK and NF-κB pathway activations were involved in such effects of epinephrine on pro-inflammatory cytokines production. Rev-erb α activation negatively regulated LPS-induced pro-inflammatory cytokines release from BMMCs. The enhancing effect of epinephrine on pro-inflammatory cytokines was rev-erb α dependent.