Figure 2. PTX3 from NBh cells binds to B cells and enhances CSR from IgM to IgG. (A) IFA of human splenic tissue stained for IgD (green), PTX3 (red), and DNA (blue). Boxes and arrowheads highlight IgD+ B cells proximal to NBh cells, including areas of IgD-PTX3 colocalization. Bars: (top) 25 µm; (bottom insets) 5 µm. (B) Flow cytometric analysis of PTX3 and control BSA bound to human circulating CD19+ B cells. (C and D) Flow cytometric analysis of fluorochrome-labeled PTX3 bound to human circulating naive, MZ, and memory B cells before (C) and after (D) preincubation with unlabeled PTX3 or control (Ctrl) IgG. MFI, mean fluorescence intensity. (E) Flow cytometric analysis of PTX3 bound to human MZ B cells after exposure to NC cells incubated with medium alone (control), LPS, GM-CSF, or both LPS and GM-CSF for 4 h. The dashed line indicates mean fluorescence intensity of PTX3 binding to MZ B cells cultured with medium alone. (F) Flow cytometric analysis of PTX3 bound to human splenic MZ B cells stimulated with medium alone (control) or CpG DNA for 1 d. (G) Flow cytometric analysis of PTX3 binding to human circulating IgD+ B cells in the presence or absence of C1q. Binding of BSA is shown as the control. (H) IFA of human splenic tissue stained for C1q (green), PTX3 (red), and IgD (blue). CA, central arteriole; FO, follicle; PFZ, perifollicular zone. Bar, 100 µm. (I) Southern blotting of Iμ-Cμ, Iγ1-Cγ1, Iγ2-Cγ2, and Iα1-Cα1 germline transcripts (GTs) and Iγ-Cμ and switch Iα-Cμ circle transcripts (CTs) amplified by standard reverse transcription PCR from human circulating IgD+ B cells cultured with or without PTX3 for 2 d (germline transcripts) or 4 d (circle transcripts). Iμ-Cμ was the loading control. (J) qRT-PCRs of mRNAs for AID (AICDA) and BLIMP-1 from human circulating IgD+ B cells exposed to PTX3 for 2 d. Results are normalized to mRNA for β-actin and presented as relative expression (RE) compared with B cells incubated with medium alone. (K) ELISA of total IgG from human circulating IgD+ B cells cultured for 4 d with medium alone (control), BAFF, or IL-10 in the presence or absence of PTX3. Data show one representative experiment of at least four with similar results (A, B, D, H, and I) or one to two experiments with at least three samples in each experimental group (C, E–G, and I–K). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t test).
Figure 2.

PTX3 from NBh cells binds to B cells and enhances CSR from IgM to IgG. (A) IFA of human splenic tissue stained for IgD (green), PTX3 (red), and DNA (blue). Boxes and arrowheads highlight IgD+ B cells proximal to NBh cells, including areas of IgD-PTX3 colocalization. Bars: (top) 25 µm; (bottom insets) 5 µm. (B) Flow cytometric analysis of PTX3 and control BSA bound to human circulating CD19+ B cells. (C and D) Flow cytometric analysis of fluorochrome-labeled PTX3 bound to human circulating naive, MZ, and memory B cells before (C) and after (D) preincubation with unlabeled PTX3 or control (Ctrl) IgG. MFI, mean fluorescence intensity. (E) Flow cytometric analysis of PTX3 bound to human MZ B cells after exposure to NC cells incubated with medium alone (control), LPS, GM-CSF, or both LPS and GM-CSF for 4 h. The dashed line indicates mean fluorescence intensity of PTX3 binding to MZ B cells cultured with medium alone. (F) Flow cytometric analysis of PTX3 bound to human splenic MZ B cells stimulated with medium alone (control) or CpG DNA for 1 d. (G) Flow cytometric analysis of PTX3 binding to human circulating IgD+ B cells in the presence or absence of C1q. Binding of BSA is shown as the control. (H) IFA of human splenic tissue stained for C1q (green), PTX3 (red), and IgD (blue). CA, central arteriole; FO, follicle; PFZ, perifollicular zone. Bar, 100 µm. (I) Southern blotting of Iμ-Cμ, Iγ1-Cγ1, Iγ2-Cγ2, and Iα1-Cα1 germline transcripts (GTs) and Iγ-Cμ and switch Iα-Cμ circle transcripts (CTs) amplified by standard reverse transcription PCR from human circulating IgD+ B cells cultured with or without PTX3 for 2 d (germline transcripts) or 4 d (circle transcripts). Iμ-Cμ was the loading control. (J) qRT-PCRs of mRNAs for AID (AICDA) and BLIMP-1 from human circulating IgD+ B cells exposed to PTX3 for 2 d. Results are normalized to mRNA for β-actin and presented as relative expression (RE) compared with B cells incubated with medium alone. (K) ELISA of total IgG from human circulating IgD+ B cells cultured for 4 d with medium alone (control), BAFF, or IL-10 in the presence or absence of PTX3. Data show one representative experiment of at least four with similar results (A, B, D, H, and I) or one to two experiments with at least three samples in each experimental group (C, E–G, and I–K). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (two-tailed Student’s t test).

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