Figure 2

Mast cells up-regulate the Bcl-6 expression in Th2 cells.

IgE-sensitized BMMC were cultured with CD4⁺ T cells (isolated from OTII mouse spleen; 10⁶ cells/ml; BMMC:T cell = 1:10) in the presence of specific antigen OVA and DC (T cell:DC = 10:1) for 0–6 h with the following conditions: (1) with or without the addition of anti-mMCP-6 antibody (Ab) [isotype IgG (cAb) was used as a control]. (2) CD4⁺ T cells were knocked down the PAR2 gene by PAR2 RNAi (shRNA); control shRNA (shCon) was used as a control. (3) In some experiments, mast cell mediator antagonists (C and D; see Table S1 for detail) were added to the culture. (4) The CD4⁺ T cells were treated with PAR2 active peptides (AP) (or control peptides; CP). (5) CD4⁺ T cells were treated with BSA and DC. (6) CD4⁺ T cells were cultured alone (T cell). The CD4⁺ T cells were isolated by MACS and processed for qRT-PCR and Western blotting or ELISA. (A), the bars indicate the Bcl-6 mRNA levels. (B), the immune blots indicate the Bcl-6 protein levels. The bars below the blots indicate the summarized integrated density of the immune blots. (C–D), OTII mice were fed with OVA (5 mg/mouse; using BSA as a control) for 48 h. OVA-specific CD4⁺ T cells were isolated from OTII mouse spleen by MACS and cultured in the presence of recombinant mMCP-6 at the indicated doses for 6 days. The cells were collected, RNA and protein were extracted and analyzed by qRT-PCR (C) and Western blotting (D). The bars indicate the levels of Bcl-6 at mRNA level (C) and protein level (D). *, p < 0.01, compared with group “0 h” in (A–B), or the dose “0” group in (C–D). The data were presented as mean ± SD from 3 experiments.