Regulation of cytokine expression in mast cells: pro- and antiinflammatory potential

Abstract

Mast cells are tissue-dwelling cells that are predominantly located at the interfaces of the organism and the exterior, such as skin, gut mucosal membranes and lung. They contain cytoplasmic granules: an organelle that contains highly bioactive mediators and that can be rapidly released upon stimulation. Nevertheless, it is often observed that mast cells secrete bioactive compounds, including cytokines, without full degranulation. The mechanisms responsible for selective release of granule cargo without the full degranulation remain largely unclear. Investigating TNF trafficking in mast cells we employed a strategy of expression of constructs coding for TNF-EGFP fusion protein transiently expressed in rat and human mast cells. The protein containing the full TNF was sorted into cytoplasmic secretory granules with the use of the ER/Golgi secretory pathway. Ectopically expressed TNF-EGFP fusion protein was not only sorted to mast cell granules, but also efficiently released. In rodent cells TNF is retrieved from the secretory pathway at the late Golgi. This retrieval seems to be mediated by a mannose-6-phosphate receptor system. Mutation of N-glycosylated residue (N86S) resulted in abrogation of granular sorting. In human mast cells, however, carbohydrate-dependent trafficking does not seem important: N86 is not conserved and TNF is not sialylated in these cells. We showed that residues 21-46 are critical for ER entry of human TNF and its sorting, while majority of its cytoplasmic tail is expendable for these processes. Surface biotinylation showed that TNF is exposed on the outer membrane and re-endocytosed on its way to the granules in a process dependent on the extracellular part of the molecule. These important differences in modes of TNF trafficking between rodent and human systems stress potential weaknesses of rodent models when TNF biology in mast cells is considered. Until recently mast cells were considered the cells that, when activated, will augment the development of inflammatory state. Recent reports, however, indicate that mast cells may also contribute to the resolution of an inflammatory state. This prompted our research concerning the regulation of IL-4, a potentially anti-inflammatory cytokine expression in mast cells. We show that in hypoxic conditions IL-4 (and IL-13) expression and release is induced, as opposed to IL-6, TNF and IFN-gamma. This induction does not correlate with stabilization of HIF-1 alpha, a major transducer of hypoxic signalling. We show that regulation of IL-4 promoter activity in hypoxia is mediated by Akt/GSK-3 and calcineurin pathways that converge at the level of NFAT. Treatment with mercuric ions is used as a model system for systemic autoimmune inflammation including arthritis, vasculitis and glomerulonephritis. The involvement of mast cell-derived IL-4 in elicitation of these conditions has been suggested but the molecular mechanism underlying mercury-induced IL-4 expression is not known. We show that in murine mast cells Hg activates IL-4 production and this effect is dose-dependent, additive to IgE/antigen stimulation and critically depends on P1 NFAT binding site in IL-4 promoter. We also demonstrate that calcineurin is involved in the regulation of mercury-induced IL-4 expression.