R than FccR ExpressionExploring the mechanism of the observed synergy, we examined whether IL-33 pre-incubation altered FccR Lixisenatide expression and/ordownstream processes involved in the expression, biosynthesis, and release of mediators. Reciprocal modulation of FccRII and FccRIII expression is a well-recognized pathway for enhancing the 
responsiveness of cells to immune complexes [44], although we have been unable to confirm that this mechanism is CP21 site active in eitherMast Cell Priming by IL-mouse or human MCs [33]. Exposure of mBMMCs to IL-33 failed to alter surface expression of FccRII or FccRIII (Figure 3A), consistent with the lack of an effect of IL-33 on FcR-mediated MC activation threshold (Figure 2C). Rather, pre-incubation of 
mBMMCs with IL-33 induced a marked accumulation of transcripts that encode numerous pro-inflammatory factors. Most remarkably, the level of IL-1b transcript increased several hundred-fold, an effect that could be observed as low as 3?10 pg/ml of IL-33 (Figure 3B and data not shown). We interpret these results to indicate “priming” of MCs by IL-33, whereby exposure of MC to IL-33 alters the state of the cells to enable markedly enhanced production of pro-inflammatory mediators upon subsequent stimulation via FccRIII.indicating an ST2-dependent MC-FLS pro-inflammatory loop. Whereas MCs have recently been identified as a potential source of IL-33 [15], we assessed IL-33 mRNA from co-cultured mBMMCs in two experiments and found it to be either low (,0.03 vs. GAPDH) or absent (,0.0002 vs. GAPDH), indicating that FLS are the most likely source of IL-33 in our system. Of note, neutralizing antibodies against IL-6 and IL-1b failed to abrogate the loop (data not shown). Therefore, the identity of the MCderived soluble factor(s) mediating IL-33 mRNA up-regulation in FLS remains to be determined.DiscussionAmong their many functions, MCs are immune sentinels, residing near epithelial surfaces, blood vessels, and near vulnerable body cavities where they serve to provide surveillance against pathogen invasion, tissue injury, and other insults [13,46]. Upon activation, MCs can elaborate a range of responses depending not only upon the stimulus but also upon their particular phenotype [4]. MCs from different tissue sites express distinct surface receptors, intracellular proteases, and other effector molecules. These phenotypic changes are mediated by the local environment, though the detailed pathways involved are incompletely defined. Here, we identify a new role for IL-33 and its receptor ST2 in IgG-mediated MC activation. We previously showed that MCs activated via FccRIII elaborate IL-1b, and that this pathway is required for MCs to “jump start” IgG-mediated K/BxN murine arthritis [35]. However, the quantity of IL-1b found to be elaborated by cultured MCs stimulated in vitro via FccRIII was smaller than might have been expected given the prominent in vivo role of this cytokine. The present work helps to 12926553 bridge this gap. We now show that exposure of MCs to IL-33 dramatically increased their production of IL-1b upon FccRIII ligation, and that this effect could be mimicked by co-culture with primary fibroblasts derived from mouse synovium. Further, we found that thisIL-33 and ST2 Mediate Mast Cell Priming by FibroblastsWhereas IL-33 may be elaborated by synovial fibroblasts [21,30], we explored the possibility that this cytokine could be pivotal for MC-fibroblast interactions. For these experiments, we co-cultured mBMMCs and FL.R than FccR ExpressionExploring the mechanism of the observed synergy, we examined whether IL-33 pre-incubation altered FccR expression and/ordownstream processes involved in the expression, biosynthesis, and release of mediators. Reciprocal modulation of FccRII and FccRIII expression is a well-recognized pathway for enhancing the responsiveness of cells to immune complexes [44], although we have been unable to confirm that this mechanism is active in eitherMast Cell Priming by IL-mouse or human MCs [33]. Exposure of mBMMCs to IL-33 failed to alter surface expression of FccRII or FccRIII (Figure 3A), consistent with the lack of an effect of IL-33 on FcR-mediated MC activation threshold (Figure 2C). Rather, pre-incubation of mBMMCs with IL-33 induced a marked accumulation of transcripts that encode numerous pro-inflammatory factors. Most remarkably, the level of IL-1b transcript increased several hundred-fold, an effect that could be observed as low as 3?10 pg/ml of IL-33 (Figure 3B and data not shown). We interpret these results to indicate “priming” of MCs by IL-33, whereby exposure of MC to IL-33 alters the state of the cells to enable markedly enhanced production of pro-inflammatory mediators upon subsequent stimulation via FccRIII.indicating an ST2-dependent MC-FLS pro-inflammatory loop. Whereas MCs have recently been identified as a potential source of IL-33 [15], we assessed IL-33 mRNA from co-cultured mBMMCs in two experiments and found it to be either low (,0.03 vs. GAPDH) or absent (,0.0002 vs. GAPDH), indicating that FLS are the most likely source of IL-33 in our system. Of note, neutralizing antibodies against IL-6 and IL-1b failed to abrogate the loop (data not shown). Therefore, the identity of the MCderived soluble factor(s) mediating IL-33 mRNA up-regulation in FLS remains to be determined.DiscussionAmong their many functions, MCs are immune sentinels, residing near epithelial surfaces, blood vessels, and near vulnerable body cavities where they serve to provide surveillance against pathogen invasion, tissue injury, and other insults [13,46]. Upon activation, MCs can elaborate a range of responses depending not only upon the stimulus but also upon their particular phenotype [4]. MCs from different tissue sites express distinct surface receptors, intracellular proteases, and other effector molecules. These phenotypic changes are mediated by the local environment, though the detailed pathways involved are incompletely defined. Here, we identify a new role for IL-33 and its receptor ST2 in IgG-mediated MC activation. We previously showed that MCs activated via FccRIII elaborate IL-1b, and that this pathway is required for MCs to “jump start” IgG-mediated K/BxN murine arthritis [35]. However, the quantity of IL-1b found to be elaborated by cultured MCs stimulated in vitro via FccRIII was smaller than might have been expected given the prominent in vivo role of this cytokine. The present work helps to 12926553 bridge this gap. We now show that exposure of MCs to IL-33 dramatically increased their production of IL-1b upon FccRIII ligation, and that this effect could be mimicked by co-culture with primary fibroblasts derived from mouse synovium. Further, we found that thisIL-33 and ST2 Mediate Mast Cell Priming by FibroblastsWhereas IL-33 may be elaborated by synovial fibroblasts [21,30], we explored the possibility that this cytokine could be pivotal for MC-fibroblast interactions. For these experiments, we co-cultured mBMMCs and FL.