Pentraxin 3 Facilitates Shrimp-Allergic Responses in IgE-Activated Mast Cells

Figure 6

Inhibitory effect of the RI37 peptides on the allergic responses. (a) RBL-2H3 cells were treated with the RI37 peptide (300 ng/ml) for 1, 2, or 3 days. Cell viability was determined by MTT assay. (b, c) RBL-2H3 cells were incubated with DNP-IgE overnight. IgE-sensitized cells were preincubated with RI37 (300 ng/ml) for 30 min and then stimulated with PTX3 (300 ng/ml) plus DNP-BSA for 30 min. Histamine content and β-hexosaminidase activity were determined as described in the Materials and Methods. (d) Effects of RI37 on the mRNA levels of TNF-α, IL-4, IL-6, IL-13, and COX-2 in PTX3-treated RBL-2H3 cells were determined by qPCR. The mRNA level of β-actin was used as an internal control. (e) RBL-2H3 cells were preincubated with RI37 (150 and 300 ng/ml) for 30 min and then stimulated with PTX3 (300 ng/ml) for 30 min. Total lysates were harvested for western blot analysis using antibodies as indicated. The quantitative analysis of phosphorylated proteins is shown in the graph. (f) Serum-sensitized RBL-2H3 cells were preincubated with RI37 (150 and 300 ng/ml) for 30 min and further stimulated with shrimp extract for 15 min. Total lysates were harvested for western blot analysis using antibodies as indicated. The quantitative analysis of phosphorylated proteins is shown in the graph. (g, h) RBL-2H3 cells were preincubated with RI37 (300 ng/ml) for 30 min and further stimulated with PTX3 or allergic serum for 30 min. Total lysates were harvested for western blot analysis using antibodies as indicated. (i) Three-to-four-week-old female BALB/c mice ( per group) were intraperitoneally sensitized and challenged with PBS or shrimp extract. Using Al(OH)₃ as an adjuvant, shrimp allergy mice received shrimp extract plus Al(OH)₃ in PBS i.p. on days 1, 4, 7, 10, 13, 16, 19, and 22. For the RI37 therapy experiment, group 2 and group 4 mice were intraperitoneally immunized three times on days 19, 22, and 30 with 0.1 mg of RI37 adsorbed to PBS plus shrimp extract and shrimp extract alone, respectively. Blood was collected 2 h after the challenge on day 30 for the determination of IgE levels.

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