N-wasp is essential for the negative regulation of B cell receptor signaling

Abstract

Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR) signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell-specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.

Figure 1. N-WASP is activated following WASP activation upon antigen stimulation.

(A–D) TIRFM and IRM analysis of pWASP and pN-WASP in the B-cell contact zone of mouse splenic B cells and human PBMC B cells that were incubated with membrane-tethered AF546–mB-Fab′–anti-mouse or human IgG+M at 37°C for indicated times. (E and G) The MFI of pWASP or pN-WASP in the B-cell contact zone was quantified using TIRFM images and Andor iQ software. (F and H) The MFI of pWASP or pN-WASP in mouse splenic and human PBMC B cells incubated with soluble Fab′–anti-IgG+M plus streptavidin at 37°C for indicated times were analyzed by flow cytometry. Shown are representative images and the average MFI (±SD) from three independent experiments. Bar, 2.5 µm.

Figure 2. Antigen-induced BCR clustering and B-cell spreading depend on both WASP and N-WASP.

(A–C) TIRFM and IRM analysis of mouse splenic B cells that were incubated with membrane-tethered transferrin (Tf) or Fab′–anti-Ig (A), A20 B cells that were transfected with control or WASP/N-WASP siRNA (B), and human B cells that were pretreated with or without wiskostatin (Wis) and stimulated with membrane-tethered Fab′–anti-Ig (C). Shown are representative images from 7 min. Bar, 2.5 µm. (D–I) The average values (±SD) of the TFI of Fab′–anti-Ig in the B-cell contact zone (D, F, and H) and of the B-cell contact area (E, G, and I) were determined using TIRFM and IRM images from >300 individual cells of 18 mice for each data point including littermate controls (D–E) or of three individual experiments (F–G and H–I). *p<0.01, compared to B cells from littermate control mice, transfected with control siRNA or treated with DMSO.

Figure 3. Differential effects of WASP and/or N-WASP KO on BCR signaling.

(A–E) TIRFM and IRM analysis of pY staining in the contact zone of mouse splenic B cells incubated with membrane-tethered Fab′–anti-Ig. Shown are representative images (A–D) and the MFI (±SD) of pY in the B-cell contact zone (E) from three independent experiments. Bars, 2.5 µm. (F and G) The FIRs of pY to the BCR were plotted versus the TFI of the BCR in individual BCR clusters. Each open symbol represents a BCR cluster. The simulated values (solid symbol) were generated by LOSSE nonlinear regression using the Stat software. (H and I) TIRFM analysis of phosphorylated Btk (pBtk) and SHIP-1 (pSHIP) in the contact zone of mouse splenic B cells stimulated with membrane-tethered Fab′–anti-Ig. Shown are the average MFI (±SD) of pBtk and pSHIP in the B-cell contact zone from three independent experiments. *p<0.01, compared to littermate control B cells. (J) Ca²⁺ flux analysis of splenic B cells activated with soluble mB-Fab′–anti-Ig plus streptavidin using flow cytometry. Shown are representative results from three independent experiments.

Figure 4. The serum levels of anti-nuclear and anti-dsDNA antibody are elevated in cNKO mice.

(A) Representative images from immunofluorescence microscopic analysis of anti-nuclear antibody in the serum of littermate control and cNKO mice at 6 mo old (n = 4). (B) ELISA quantification of anti-dsDNA antibody in the serum of littermate control and cNKO mice at 6 and 9 mo old (n = 15). Each dot represents an individual mouse. *p<0.01.

Figure 5. WASP promotes and N-WASP inhibits the F-actin accumulation in the B-cell contact zone.

(A–E) TIRFM analysis of F-actin staining in the contact zone of splenic B cells incubated with membrane-tethered Fab′–anti-Ig. Shown are representative images (A–D) and the average MFI (±SD) of F-actin staining in the B-cell contact zone (E) from three independent experiments. (F and I) TIRFM analysis of Arp2 staining at the contact zone of splenic B cells incubated with membrane-tethered Fab′–anti-Ig. The MFI of Arp2 staining in the B-cell contact zone was quantified. (G, H, and J) TIRFM analysis of the spatial relationship of Arp2 with pWASP (G) or pN-WASP (H) in the contact zone of splenic B cells incubated with membrane-tethered Fab′–anti-Ig for 5 min. The colocalization coefficients between Arp2 and pWASP or pN-WASP staining were determined using Zeiss LSM software (J). Shown are representative images (F–H) and the average MFI (I) or colocalization coefficients (±SD) (J) from ∼50 individual cells of three independent experiments. Bars, 2.5 µm. *p<0.01, compared to B cells from littermate control mice.

Figure 6. N-WASP plays a dominant role in BCR internalization.

(A–C) Immunofluorescence microscopic analysis of BCR internalization. Colocalization coefficients between the surface-labeled BCR with LAMP-1 in splenic B cells (A and C) or A20 B cells transfected with WASP/N-WASP siRNA (B and C) were measured using the Zeiss LSM software. The surface BCR were labeled with AF546-mB-Fab′–anti-Ig plus soluble streptavidin and warmed to 37°C for 0 or 30 min. Shown are representative images (A and B) and the average correlation coefficients (±SD) (C) from >300 cells of three independent experiments. Bars, 2.5 µm. (D) Flow cytometry analysis of BCR internalization by quantifying the percentage of biotin-F(ab′)₂–anti-Ig–labeled BCR remaining on the cell surface after the 37°C chase. Shown are the average percentages (±SD) from three independent experiments. *p≤0.05, compared to B cells from littermate control mice.

Figure 7. WASP and N-WASP negatively regulates each other.

(A–D) TIRFM analysis of pWASP and pN-WASP in the contact zone of splenic B cells stimulated with membrane-tethered Fab′–anti-Ig (A and B). The MFI of pWASP (C) or pN-WASP (D) in the B-cell contact zone was quantified. (E–G) Flow cytometry analysis of the cellular MFI of pWASP or pN-WASP in splenic B cells from WKO and littermate control mice (F), and mouse splenic (E) and human PBMC B cells (G) that were treated with or without Wis and soluble mB-Fab′–anti-Ig plus streptavidin. (H) Flow cytometry analysis of the cellular MFI of pN-WASP in PBMC B cells from WAS patients and age-matched healthy donors that were incubated with or without soluble mB-Fab′–anti-Ig plus streptavidin for 2 min. Shown are representative images at 7 min and the average MFI (±SD) from three independent experiments. Bars, 2.5 µm. * p<0.01, compared to B cells from wt or littermate control mice, without Wis treatment or healthy donors.

Figure 8. WASP and N-WASP are inversely regulated by Btk and SHIP-1.

(A–B and D–E) TIRFM analysis of pN-WASP in the contact zone of splenic B cells from wt, xid, control, and B-cell–specific SHIP-1^−/− mice that were incubated with membrane-tethered Fab′–anti-Ig. The MFI of pN-WASP in the B-cell contact zone was determined (B and E). (C and F) Flow cytometry analysis of the cellular MFI of pN-WASP in splenic B cells incubated with soluble mB-Fab′–anti-Ig plus streptavidin. Shown are representative images at 7 min and the average MFI (±SD) from three independent experiments. Bars, 2.5 µm. * p<0.01, compared to B cells from wt or littermate control mice.

Figure 9. A working model for the coordination of N-WASP with WASP in the regulation of BCR signaling.

(A) Antigen binding to the BCR induces an early activation of Btk. (B) Activated Btk in turn activates and translocates WASP to the cell surface. Activated WASP (W) stimulates actin polymerization and reorganization by binding to Arp2/3 and suppresses N-WASP activation, which drives B-cell spreading and facilitates BCR clustering and signaling. (C) The activation of SHIP-1 induced by the BCR after initial signaling inhibits Btk and WASP activation, consequently releasing N-WASP from WASP suppression. Active N-WASP decreases actin accumulation at the B-cell contact zone, which enables B-cell contraction and promotes the coalescence of BCR microclusters into a central cluster and BCR internalization. The formation of BCR central clusters and BCR endocytosis lead to the down-regulation of BCR signaling at the cell surface.