Regulation Of Cytokine Gene Expression In Mast Cells

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Abstract

In immunity, reactive oxygen species (ROS) and nitric oxide (NO) are important antimicrobial agents and regulators of cell signaling and activation pathways. Our aim was to determine the regulatory roles of such chemical species on mast cell activation. To examine this question, we set out to determine whether NO inhibits mast cell cytokine production, and, if so, whether it also alters FceRI-dependent signal transduction. As hypothesized, the radical inhibited IgE/Ag-induced IL-4, IL-6, and TNF production. Although NO did not influence phosphorylation of JNK, p38 MAPK, or p44/42 MAPK, it did inhibit phosphorylation of phospholipase C 1 and the AP-1 transcription factor protein c-Jun, but not NF- B or CREB. NO further completely abrogated IgE/Ag-induced DNA-binding activity of the nuclear AP-1 proteins Fos and Jun. These results show that NO is capable of inhibiting FceRI-dependent mast cell cytokine production at the level of gene regulation, and suggest too that NO may contribute to resolution of allergic inflammation.[unreadable] [unreadable] We similarly investigated the enzymatic source of FceRI-dependent production of ROS and the potential role of these species in mast cell secretory responses. In addition, the ability of mast cells to generate ROS in response to other stimuli was examined. 5-lipoxygenase (5LO) was the primary enzymatic source of ROS in human and mouse mast cells following FceRI aggregation. 5LO-deficient mouse mast cells had greatly reduced FceRI-dependent DCF fluorescence compared to wild type, gp91phox- or p47phox- deficient mast cells. A minor role for cyclooxygenase-1 (COX1) was also demonstrated. Complete abrogation of FceRI-dependent ROS production in mast cells had marginal effects on degranulation and cytokine secretion. In response to classic NADPH oxidase stimuli, mast cells produced negligible levels of ROS. IgG-coated latex beads, however, did stimulate production of ROS in human mast cells. 5LO and COX1 were the enzymatic sources of ROS since AA861, zileuton and FR122047 but not DPI reduced IgG-LB-dependent DCF fluorescence. In contrast, only DPI completely abrogated IgG-LB induced ROS production in polymorphonuclear leukocytes. We thus concluded that mast cells generate ROS by 5LO and COX1 in response to either FcR aggregation and such ROS appear to have marginal roles in FceRI-dependent secretory responses.[unreadable] [unreadable] The neurotransmitter serotonin (5-hydroxytryptamine [5-HT]) is implicated in enhancing inflammatory reactions of skin, lung and gastrointestinal tract. To determine if 5-HT acts in part through mast cells, we established that mast cells express mRNA for multiple 5-HT receptors. We next determined the effect of 5-HT on mast cell degranulation, adhesion and chemotaxis. We found no evidence that 5-HT degranulates mast cells or modulates IgE dependent activation. 5-HT did induce mast cell adherence to fibronectin and immature and mature mast cell migration. Chemotaxis was accompanied by actin polymerization. Using receptor antagonists and pertussis toxin we identified 5-HT1A as the principal receptor mediating the effects of 5-HT on mast cells. Further, mast cells from the 5-HT1A receptor knockout mouse (5-HT1A R -/-) did not respond to 5-HT. 5-HT did induce accumulation of mst cells in the dermis of 5-HT1A R +/+ mice, but not in 5-HT1A R -/- mice. These studies are the first to demonstrate an effect of 5-HT on mast cells and through the 5-HT1A receptor.[unreadable] [unreadable] In a series of experiments now completed, we examined the effect of phagocytosis of bacteria by mast cells. Results showed a re-programming of mast cells responses. The high affinity receptor was down-regulated and mast cells responded less to IgE nmediated signals. However, mast cells did release inflammatory cytokines following the phagocytic stimulus and up-regulated genes associated with innate immune responses.