PTX3 binds to MZ B cells independently of TLR4 and FcγRs. (A) IFA of mouse spleen stained for MMP9 (green), Ly6G (red), and IgM or DNA (blue). FO, follicle; PALS, periarteriolar lymphoid sheath. Bars: (top) 100 µm; (inset) 10 µm. (B) Confocal microscopy of mouse tissue stained for MMP9 (green), B220 (red), and DNA (blue). Bar, 3 µm. (C) qRT-PCR of RNAs for BAFF (Tnfsf13b), APRIL (Tnfsf13), CD40L (Cd40lg), IL-10, and IL-6 in mouse neutrophils from bone marrow and spleen. Results are normalized to mRNA for HPRT and presented as relative expression compared with bone marrow neutrophils. The color code indicates high (red), mean (black), and low (green) expression. (D) qRT-PCR of mRNA for PTX3 from mouse NBh cells stimulated with LPS for 4 h. Results are normalized to mRNA for HPRT and presented as relative expression (RE) compared with NBh cells incubated with medium alone. (E) ELISA of serum PTX3 from WT and MyD88^−/−Trif^−/− mice. (F) ELISA of serum PTX3 from MyD88^−/−Trif^−/− mice before (control [Ctrl]) and after adoptive transfer of NBh cells from WT mice. (G) Flow cytometric analysis of PTX3 or BSA binding to MZ and FO B cells from WT mice. MFI, mean fluorescence intensity. (H and I) Flow cytometric analysis of PTX3-binding to splenic B220⁺AA4-1⁺ transitional B cells (T B cell), including B220⁺AA4-1⁺CD23⁻IgM^hi T1 B cells, B220⁺AA4-1⁺CD23⁺IgM^hi T2 B cells, and B220⁺AA4-1⁺CD23⁺IgM^lo T3 B cells (H) and of PTX3 or BSA binding to MZ B cells from WT, FcγR^−/−, or Tlr4^−/− mice (I). Data show one representative experiment of at least four with similar results (A, B, H, and I) or one to two experiments with at least three samples in each experimental group (C–G). Error bars, SEM. *, P < 0.05; ***, P < 0.001 (two-tailed Student’s t test).