Figure 5. PTX3 enhances MZ B cell proliferation and CSR from IgM to IgG in response to encapsulated bacteria. (A) IFA of spleens from WT mice stained for MMP9 (green), IgM (red), and MOMA-1 (blue) before (control [Ctrl]) and after (d3) i.v. injection of live SP. Bars, 100 µm. (B) Confocal microscopy of mouse splenic tissue stained for MMP9 (green), B220 (red), and DNA (blue) after i.v. injection (d3) of live SP. The right image corresponds to the magnified boxed tissue area from the left image, which depicts B cell–interacting NBh cells. Arrowheads point to B220 captured by an MMP9+ NBh cell. Bars: (left) 10 µm; (right) 3 µm. (C) ELISA of serum PTX3 from WT mice after i.v. injection of live SP. (D) Flow cytometric quantification of NBh cells from WT and Ptx3−/− mice after i.v. injection of live SP. (E and F) Flow cytometric quantification of frequency of proliferating Ki-67+ MZ B cells, B-1a cells, and B-1b cells (E) and absolute number of total MZ B cells, B-1a cells, and B-1b cells (F) from spleens of WT and Ptx3−/− mice after i.v. injection (d5) of live SP. Dashed lines show the mean frequency or absolute number in nonimmunized WT mice. Similar values were obtained in nonimmunized Ptx3−/− mice. FSC, forward side scatter. (G) qRT-PCR of mRNAs for AID (Aicda), Iγ2b-Cγ2b, and Iγ2c-Cγ2c transcripts in MZ B cells from WT and Ptx3−/− mice after i.v. injection (d5) of live SP. Results are normalized to mRNA for HPRT and presented as relative expression (RE) compared with MZ B cells from WT mice. (H) Absolute numbers of class-switched B cells expressing IgG2b, IgG2c, or IgG3 from spleens of WT and Ptx3−/− mice after i.v. injection (d5) of live SP. Dashed lines show the mean frequency or absolute number in nonimmunized WT mice. Similar values were obtained in nonimmunized Ptx3−/− mice. Data show one representative experiment of at least three with similar results (A and B) or summarize two experiments with at least three mice in each experimental group (C–H). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t test).
Figure 5.

PTX3 enhances MZ B cell proliferation and CSR from IgM to IgG in response to encapsulated bacteria. (A) IFA of spleens from WT mice stained for MMP9 (green), IgM (red), and MOMA-1 (blue) before (control [Ctrl]) and after (d3) i.v. injection of live SP. Bars, 100 µm. (B) Confocal microscopy of mouse splenic tissue stained for MMP9 (green), B220 (red), and DNA (blue) after i.v. injection (d3) of live SP. The right image corresponds to the magnified boxed tissue area from the left image, which depicts B cell–interacting NBh cells. Arrowheads point to B220 captured by an MMP9+ NBh cell. Bars: (left) 10 µm; (right) 3 µm. (C) ELISA of serum PTX3 from WT mice after i.v. injection of live SP. (D) Flow cytometric quantification of NBh cells from WT and Ptx3−/− mice after i.v. injection of live SP. (E and F) Flow cytometric quantification of frequency of proliferating Ki-67+ MZ B cells, B-1a cells, and B-1b cells (E) and absolute number of total MZ B cells, B-1a cells, and B-1b cells (F) from spleens of WT and Ptx3−/− mice after i.v. injection (d5) of live SP. Dashed lines show the mean frequency or absolute number in nonimmunized WT mice. Similar values were obtained in nonimmunized Ptx3−/− mice. FSC, forward side scatter. (G) qRT-PCR of mRNAs for AID (Aicda), Iγ2b-Cγ2b, and Iγ2c-Cγ2c transcripts in MZ B cells from WT and Ptx3−/− mice after i.v. injection (d5) of live SP. Results are normalized to mRNA for HPRT and presented as relative expression (RE) compared with MZ B cells from WT mice. (H) Absolute numbers of class-switched B cells expressing IgG2b, IgG2c, or IgG3 from spleens of WT and Ptx3−/− mice after i.v. injection (d5) of live SP. Dashed lines show the mean frequency or absolute number in nonimmunized WT mice. Similar values were obtained in nonimmunized Ptx3−/− mice. Data show one representative experiment of at least three with similar results (A and B) or summarize two experiments with at least three mice in each experimental group (C–H). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t test).

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