Figure 2.

Involvement of B-1a cells in the innate immune response triggered by acrolein-modified proteins.A, acrBSA-induced Ca²⁺ flux in PerC B cells in vitro. a, representative Ca²⁺ signaling images in PerC B cells from CD19-Cre/YC3.60^fl/fl mice. Ratiometric images (YFP/CFP excitation at 458 nm) are shown. acrBSA in PBS (final concentration: 10 μg/ml) was added to the cell culture at the indicated time point. A rainbow parameter indicates the relative Ca²⁺ concentrations. b, the indicated cells were measured fluorescence intensities of the YFP/CFP ratio upon excitation at 458 nm. c, time course for fluorescence intensities of the YFP/CFP ratio upon excitation at 458 nm in the cells. B, production of IgM by acrBSA in the PerC B cells. B cell subpopulations (B220⁺) were isolated from the PerC of BALB/c mice by MACS MicroBeads UltraPure, stimulated with BSA or acrBSA, and the culture supernatants were tested for the production of IgM. Differences were analyzed by a paired Student's t test; ∗∗∗p < 0.001. C, differentiation of B cells into the IgM-secreting plasma cells by acrBSA. D, identification of B cell subsets involved in the production of IgM by acrBSA. A total of the 10,000 events were counted and analyzed. E, production of IgM by acrBSA in the B-1a cells. The PerC B-1a cells were stimulated with BSA or acrBSA, and the culture supernatants were tested for the production of IgM. In panels B–E, the cells were treated with 100 μg/ml protein samples (BSA or acrBSA) for 48 h (B, C, and E) or 72 h (D). Differences were analyzed by the paired Student's t test (B and E) or by Dunnett's test (C and D); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; versus N/C (C); versus N/C within each B cell subset (D). acrBSA, acrolein-modified BSA; IgM, immunoglobulin M; MACS, magnetic-activated cell sorting; N/C, negative control; PerC, peritoneal cavity; YC3.60, yellow cameleon 3.60.