Post-transcriptional stabilization by interleukin-1β of interleukin-6 mRNA induced by c-kit ligand and interleukin-10 in mouse bone marrow-derived mast cells

Abstract

We demonstrate that a specific combination of cytokines elicits high levels of interleukin (IL)-6 gene expression in mast cells and define the cellular mechanisms of the exogenous cytokine action. The addition of c.kit ligand (KL) and IL-10 to IL-3-derived mouse bone marrow mast cells (BMMC) elicited an ~2-fold increase in steady-state IL-6 mRNA levels that peaked after 0.5 h and was followed by the release of ~0.2 ng of IL-6/106 cells by 5-7 h. The addition of IL-1β to KL + IL-10 elicited a prolonged ~12-fold increase in the level of IL-6 mRNA by 3-5 h and an ~50-fold increase in the level of IL-6 protein released by 7 h. As determined by nuclear run-on analysis, KL + IL-10 stimulated IL-6 gene transcription within 0.5 h, and the addition of IL-1β did not increase transcription. Instead, IL-1β slowed by ~8-fold the decay of IL-6 mRNA as compared to its decay in BMMC stimulated with KL + IL-10 alone. The exposure of BMMC to cycloheximide 0.5 h before the addition of the three exogenous cytokines inhibited by ~50% the level of IL- 6 mRNA generated but did not inhibit the effects of KL + IL-10, indicating that IL-1β induces the synthesis of a protein that stabilizes IL-6 mRNA. The stabilization of IL-6 mRNA was inhibited by the addition of actinomycin D at 0.5 but not 3 h after BMMC were stimulated with IL-1β in combination with KL + IL-10, suggesting that once transcribed, the stabilizing protein is long- lived. The addition of cycloheximide to BMMC after stimulation with KL + IL- 10 with or without IL-1β increased the levels of steady-state IL-6 mRNA compared to levels in cells without drug, indicating that in addition to stimulating IL-6 transcription, KL + IL-10 induces a protein factor that destabilizes IL-6 mRNA. Thus, there exists a novel Fcε receptor type I- independent mechanism by which a mast cell can provide substantial amounts of IL-6 protein in response to the synergistic action of KL and IL-10 to induce IL-6 gene transcription, and IL-1β to stabilize otherwise short-lived IL-6 transcripts.