Milar results. *P,0.05, **P,0.01, WT versus ST22/2. doi:10.1371/journal.pone.0047252.gWT mBMMCs by 2.4G2 is typically blocked due to engagement of the inhibitory receptor FccRII, IL-33 enabled MC to partially bypass this blockade and respond productively to this immune complex mimic (Figure 2B). Of note, we failed to observe any effect of IL-33 on exocytosis of the granule constituent bhexosaminidase (Figure 2C), a defect that could not be reversed using published methods [23,31], including overnight pre-incubation with IgE (anti-trinitrophenyl, 0.3 mg/ml) or K/BxN mouse serum (1:100-1:1) (data not shown). We also could not identify a synergistic effect of exogenous IL-33 and C5a in our mBMMC, with or without concomitant FccRII/III ligation (data not shown). To understand more fully the effect of cooperative IL-33- and FccRIII-mediated activation of MCs, we employed a multiplex array to evaluate cytokines and chemokines elaborated by 2.4G2stimulated FccRII2/2 mBMMCs, pre-incubated with IL-33 or vehicle for 4 hours. The results confirmed our IL-6 data, andshowed that amplification extended to other mediators as well, most prominently TNF-a and CXCL2 (Figure 2D, quantitated in Figure 2E). The latter finding is particularly interesting, since arthritis is neutrophil Tubastatin A site dependent and the receptor CXCR2 has been implicated in IL-33-mediated neutrophil recruitment [41,42,43]. Selected results were further confirmed by specific ELISAs (Figure 2F). Whereas IL-1b has been shown to be 25837696 a key MC-derived arthritogenic SIS3 cytokine [35], we assayed for IL-1b in lysates of FccRIII-stimulated FccRII2/2 mBMMCs and found a marked increase after IL-33 pre-incubation (Figure 2F). Linear regression demonstrated that the effect of sequential stimulation by IL-33 followed by FccRIII ligation far exceeded an additive effect, confirming synergy (TNF-a P = 0.0005, IL-1b and CXCL2 P,0.0001). Interestingly, in mBMMCs stimulated with IL-33 and subsequently washed to remove released cytokine, accumulation of intracellular cytokine was observed for IL-6 and IL-1b, but not TNF-a (Figure 2G).Mast Cell Priming by IL-Figure 2. IL-33 enhances FccRIII-mediated cytokine production by mast cells. (A ) FccRII2/2 or WT B6 mBMMCs were pre-incubated with or without IL-33 (10 ng/ml) for 4 hours, and then cells in pre-incubation media were spun onto plates pre-coated with anti-FccRII/III Ab (2.4G2). Supernatants were harvested at 16 hours and assayed for IL-6 (A B) and the granule mediator b-hexosaminidase (C). Differences in baseline IL-6 production were reproducibly observed between FccRII2/2 and B6 mBMMCs, and may reflect divergent genetic backgrounds or other factors. (D) FccRII2/2 mBMMCs incubated with or without IL-33 for 4 hours were activated by plate-bound 2.4G2 Ab (10 mg/ml) for 16 hours and assayed by multiplex cytokine array. (E) Quantitation of optical density of 
selected 
mediators from D (mean of 2 dots). (F) Assay of mediators identified in D-E via specific ELISA in separate experiments employing an identical experimental design (IL-1b performed on lysates). (G) To determine whether IL-33 induced intracellular accumulation of cytokine, B6 mBMMCs were stimulated with IL-33 (10 ng/ml) for the intervals indicated, washed62 in ice-cold PBS, and lysed in the presence of protease inhibitors. All results representative of at least 2 independent experiments. *P,0.05, **P,0.01, ***P,0.001. doi:10.1371/journal.pone.0047252.gMast Cell Priming by IL-33 Reflects mRNA Accumulation Rathe.Milar results. *P,0.05, **P,0.01, WT versus ST22/2. doi:10.1371/journal.pone.0047252.gWT mBMMCs by 2.4G2 is typically blocked due to engagement of the inhibitory receptor FccRII, IL-33 enabled MC to partially bypass this blockade and respond productively to this immune complex mimic (Figure 2B). Of note, we failed to observe any effect of IL-33 on exocytosis of the granule constituent bhexosaminidase (Figure 2C), a defect that could not be reversed using published methods [23,31], including overnight pre-incubation with IgE (anti-trinitrophenyl, 0.3 mg/ml) or K/BxN mouse serum (1:100-1:1) (data not shown). We also could not identify a synergistic effect of exogenous IL-33 and C5a in our mBMMC, with or without concomitant FccRII/III ligation (data not shown). To understand more fully the effect of cooperative IL-33- and FccRIII-mediated activation of MCs, we employed a multiplex array to evaluate cytokines and chemokines elaborated by 2.4G2stimulated FccRII2/2 mBMMCs, pre-incubated with IL-33 or vehicle for 4 hours. The results confirmed our IL-6 data, andshowed that amplification extended to other mediators as well, most prominently TNF-a and CXCL2 (Figure 2D, quantitated in Figure 2E). The latter finding is particularly interesting, since arthritis is neutrophil dependent and the receptor CXCR2 has been implicated in IL-33-mediated neutrophil recruitment [41,42,43]. Selected results were further confirmed by specific ELISAs (Figure 2F). Whereas IL-1b has been shown to be 25837696 a key MC-derived arthritogenic cytokine [35], we assayed for IL-1b in lysates of FccRIII-stimulated FccRII2/2 mBMMCs and found a marked increase after IL-33 pre-incubation (Figure 2F). Linear regression demonstrated that the effect of sequential stimulation by IL-33 followed by FccRIII ligation far exceeded an additive effect, confirming synergy (TNF-a P = 0.0005, IL-1b and CXCL2 P,0.0001). Interestingly, in mBMMCs stimulated with IL-33 and subsequently washed to remove released cytokine, accumulation of intracellular cytokine was observed for IL-6 and IL-1b, but not TNF-a (Figure 2G).Mast Cell Priming by IL-Figure 2. IL-33 enhances FccRIII-mediated cytokine production by mast cells. (A ) FccRII2/2 or WT B6 mBMMCs were pre-incubated with or without IL-33 (10 ng/ml) for 4 hours, and then cells in pre-incubation media were spun onto plates pre-coated with anti-FccRII/III Ab (2.4G2). Supernatants were harvested at 16 hours and assayed for IL-6 (A B) and the granule mediator b-hexosaminidase (C). Differences in baseline IL-6 production were reproducibly observed between FccRII2/2 and B6 mBMMCs, and may reflect divergent genetic backgrounds or other factors. (D) FccRII2/2 mBMMCs incubated with or without IL-33 for 4 hours were activated by plate-bound 2.4G2 Ab (10 mg/ml) for 16 hours and assayed by multiplex cytokine array. (E) Quantitation of optical density of selected mediators from D (mean of 2 dots). (F) Assay of mediators identified in D-E via specific ELISA in separate experiments employing an identical experimental design (IL-1b performed on lysates). (G) To determine whether IL-33 induced intracellular accumulation of cytokine, B6 mBMMCs were stimulated with IL-33 (10 ng/ml) for the intervals indicated, washed62 in ice-cold PBS, and lysed in the presence of protease inhibitors. All results representative of at least 2 independent experiments. *P,0.05, **P,0.01, ***P,0.001. doi:10.1371/journal.pone.0047252.gMast Cell Priming by IL-33 Reflects mRNA Accumulation Rathe.