Original magnification, ��200. … Given that R115777 mast cell activation is higher in Il1rl1?/? mice, we studied their degranulation potential by measuring tryptase in the supernatants of BMMCs generated from WT and Il1rl1?/? mice. Flow cytometry analysis of isolated BMMCs confirmed their mast cell phenotype (c-kit+ Fc��RI+) and the absence of ST2 expression on BMMCs isolated from Il1rl1?/? mice (Figure 5C). Degranulation was induced by calcium ionophore and was inhibited by protamine (used as a negative internal control). After stimulation, Il1rl1?/? mast cells exhibited higher tryptase concentrations in supernatants, compared with WT cells [96.8 ��g/mL (range, 44.9 to 145.6) versus 15.1 (range, 4.1 to 29.3); P < 0.05] (Figure 5D), confirming ST2 involvement in the mast cell degranulation process.

In Mice, IL-33, the ST2 Ligand, Is Expressed in the Pancreas at Baseline and Is Released during AP Both WT and Il1rl1?/? pancreas exhibited constitutive IL-33 mRNA expression (Figure 4A) without any quantitative difference. Immunohistochemistry confirmed constitutive pancreatic IL-33 expression localized in the cytoplasm of acinar cells (Figure 6A). Furthermore, after isolation and sonication of 5 �� 106 acinar cells/mL, we measured IL-33 in the lysed cell suspension by ELISA at 37 pg/mL, confirming acinar cell IL-33 production at the protein level. Figure 6 Pancreatic and plasma expression of IL-33. A: Immunohistochemical expression of IL-33 in the pancreas of control WT mice. IL-33 (left) and control isotype (right) immunostaining reveals acinar cell IL-33 expression.

Original magnification, ��200. … After 48 and 72 hours of the CDE diet leading to AP in mice, IL-33 concentrations rose significantly in the serum [control diet, 43.3 pg/mL (range, 0 to 169.2); 48-hour CDE diet, 128.6 pg/mL (range, 53.1 to 397.3); 72-hour CDE diet, 427 pg/mL (range, 18 to 1707); P < 0.001 versus control] (Figure 6B). Recent reports suggest a regulatory role for IL-33 in the mast cell degranulation process.24,25 In the present in vitro experiments, however, we could not observe this effect, because incubation of BMMCs with IL-33 did not modify tryptase levels detected in supernatants either in Il1rl1?/? BMMCs or in WT BMMCs (data not shown). Nevertheless, because degranulation and production of proinflammatory cytokines by stimulated BMMCs are two independent processes,9,26 we confirmed their normal activity by using LPS or IL-33 as stimulators and positive controls.

In agreement with previous findings,26 LPS did induce IL-6 and IL-13 secretion by mast cells independently of ST2. Moreover, as Moulin et al27 reported, IL-33 alone was able to induce proinflammatory cytokine production by WT BMMCs, but not by Il1rl1?/? BMMCs (Figure 6, C and D). Discussion The present study reports Brefeldin_A for the first time the involvement of the ST2 receptor in the severity of acute pancreatitis.