Abstract
We demostrate 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 approximately 2-fold increase in steady-state IL-6 mRNA levels that peaked after 0.5 h and was followed by the release of approximately 0.2 ng of IL-6/10(6) cells by 5-7 h. The addition of IL-1beta to KL + IL-10 elicited a prolonged approximately 12-fold increase in the level of IL-6 mRNA by 3-5 h and an approximately 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-1beta did not increase transcription. Instead, IL-1beta slowed by approximately 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 approximately 50% the level of IL-6 mRNA generated but did not inhibit the effects of KL + IL-10, indicating that IL-1beta 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-1beta 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-1beta 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 Fcepsilon 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-1beta to stabilize otherwise short-lived IL-6 transcripts.
Highlights
Mast cells are the major source of IL-6 in tissues undergoing certain types of allergic inflammation
We have shown that a particular combination of exogenous cytokines acts synergistically to induce high levels of IL-6 expression in mouse bone marrow mast cells (BMMC) and that these cytokines act at discrete steps in the pathway of IL-6 generation
Nuclear run-on analysis showed that approximately the same level of IL-6 gene transcription was induced 0.5 h after BMMC were exposed to kit ligand (KL) ϩ IL-10 with or without IL-1 (Fig. 3)
Summary
Mast cells are the major source of IL-6 in tissues undergoing certain types of allergic inflammation. Cytokine-stimulated Production of IL-6 mRNA and Protein in BMMC—When BMMC, generated in 50% WEHI-3 cell-conditioned medium as a source of IL-3, were cultured for up to 48 h in either KL (50 ng/ml) ϩ IL-10 (20 units/ml) alone or with IL-1 (5 ng/ml), steady-state IL-6 mRNA levels rapidly increased in the cells by 0.5 h, as shown on typical autoradiograms (Fig. 1, A and B) and by the 18 S RNA-adjusted data derived by Betascope analysis from multiple experiments (Fig. 1C).
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