Actin-binding protein 1 links B-cell antigen receptors to negative signaling pathways

Abstract

Prolonged or uncontrolled B-cell receptor (BCR) signaling is associated with autoimmunity. We previously demonstrated a role for actin in BCR signal attenuation. This study reveals that actin-binding protein 1 (Abp1/HIP-55/SH3P7) is a negative regulator of BCR signaling and links actin to negative regulatory pathways of the BCR. In both Abp1(-/-) and bone marrow chimeric mice, in which only B cells lack Abp1 expression, the number of spontaneous germinal center and marginal zone B cells and the level of autoantibody are significantly increased. Serum levels of T-independent antibody responses and total antibody are elevated, whereas T-dependent antibody responses are markedly reduced and fail to undergo affinity maturation. Upon activation, surface BCR clustering is enhanced and B-cell contraction delayed in Abp1(-/-) B cells, concurrent with slow but persistent increases in F-actin at BCR signalosomes. Furthermore, BCR signaling is enhanced in Abp1(-/-) B cells compared with wild-type B cells, including Ca(2+) flux and phosphorylation of B-cell linker protein, the mitogen-activated protein kinase kinase MEK1/2, and ERK, coinciding with reductions in recruitment of the inhibitory signaling molecules hematopoietic progenitor kinase 1 and SH2-containing inositol 5-phosphatase to BCR signalosomes. Our results indicate that Abp1 negatively regulates BCR signaling by coupling actin remodeling to B-cell contraction and activation of inhibitory signaling molecules, which contributes to the regulation of peripheral B-cell development and antibody responses.

Keywords: B-lymphocytes; actin cytoskeleton; signal transduction.

Fig. 1.

B-cell–specific Abp1 deficiency is sufficient to increase the differentiation of MZ and spontaneous GC B cells. (A–C) Peripheral B-cell subsets in Abp1^−/− and wt mice. Cells from spleens (A) and PerC (B) were labeled for surface markers of transitional 1 (T1), transitional 2 (T2), follicular (FO), marginal zone (MZ), isotype switched (IS) B cells or B1a and B1b B cells and were analyzed by flow cytometry. Shown are average numbers (+SD) of cells per spleen (A) (n = 5) or in the PerC (B) (n = 4). (C) Average numbers of GC B cells per spleen by flow cytometry (+SD; n = 4). (D) Immunofluorescent staining images of spleen sections from mice (6 mo old). (Scale bar, 100 μm.) n = 12 sections/4 wt or Abp1^−/− mice. (E) Average numbers of PNA⁺ GCs per spleen section and average length of GC (μm × 20) (+SD; n = 12 sections/4 wt or Abp1^−/− mice). (F) Average percentages of MZ and GC B cells in B220⁺ splenic B cells of wt-Ch and Abp1^−/−-Ch by flow cytometry (+SD; n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Production of autoAb, T-independent Ab responses, and total Ab increases while T-dependent high-affinity Ab responses decrease in Abp1^−/− mice. (A) Immunofluorescence microscopy analysis of antinuclear Ab in the serum of nonimmunized wt and Abp1^−/− mice (n = 4∼9) at different ages. (B) ELISA quantification of anti-dsDNA Ab in the serum of wt and Abp1^−/− mice of 1.5 and 6 mo of age, and wt-Ch and Abp1^−/−-Ch mice at 1 and 4 mo posttransplant. Dots represent individual mice. (C–H) 6∼8-wk-old mice (n = 4∼5) were immunized with NP-Ficoll (C and D) or NP-KLH (E–H). NP-specific IgM (C and E), total IgM (D and F), NP-specific IgG (G), or total IgG (H) in the serum (µg/mL) determined by ELISA. (I) Relative affinity of NP-specific IgG in NP-KLH-immunized mice assessed as the concentration ratio of IgG bound to NP₄ versus NP₃₀ by ELISA (+SD; n = 4∼5). *P < 0.05; **P > 0.01; ***P < 0.001.

Fig. 3.

Abp1 knockout augments B-cell spread and BCR clustering. (A–D) IRM and TIRF analysis of wt and Abp1^−/− splenic B cells incubated with Fab’-anti-Ig-tethered lipid bilayers. Representative images. (Scale bar, 5 µm; n = 3) (A). Average contact area (B), TFI (C), and MFI (D) of labeled BCRs from >50 cells per time point (n = 3), quantified using Andor iQ software. (E and F) Confocal analysis of splenic B cells stained with AF546-mB-Fab’–anti-IgG+M and then streptavidin to activate. Representative images at 1 min (E) and average percentage (+SD) of cells showing polarized BCR caps (F) quantified from >100 cells per time point (n = 3; scale bar, 5 µm). *P < 0.05.

Fig. 4.

BCR activation induces recruitment of Abp1 to the outer edge of the spreading B cell. (A) TIRF and IRM analysis of Abp1 in the contact zone of splenic B cells incubated with Fab’-anti-Ig-tethered lipid bilayers. Representative images (n = 3; scale bar, 5 µm). (B) Relative intensity of IRM and fluorescence intensity of BCRs and Abp1 across the blue line in cell at 7 min (A). (C) Average MFI of Abp1 (±SD) from >50 cells for each point quantified using Andor iQ (n = 3).

Fig. 5.

BCR signaling is enhanced in Abp1^−/− B cells. (A–E) Splenic B cells from wt and Abp1^−/− mice were activated with F(ab’)₂-goat anti-mouse IgG+M, fixed, permeabilized, labeled for phosphotyrosine (pY) (A), pBLNK (B), pMEK1/2 (C), pErk (D), and pJnk (E) and analyzed by flow cytometry (average MFI ± SD; n = 3). (D and E) Upstream Erk and Jnk inhibitor controls (INH) at indicated times. (F) Ca²⁺ flux in splenic B cells activated with F(ab’)₂-goat anti-mouse IgG+M, using flow cytometry (n = 3). *P < 0.05.

Fig. 6.

Abp1 is required for recruitment of the inhibitory signaling molecules, HPK1 and SHIP-1, but not pSyk to the B-cell contact zone. TIRF analysis of HPK1 (A and B), phosphorylated SHIP-1 (pSHIP-1) (C and D) and Syk (pSyk) (E and F) in the contact zone of wt and Abp1^−/− B cells incubated with Fab’-anti-Ig-tethered lipid bilayers. Shown are representative images and MFI (±SD) in the contact zone (50 cells per time point; n = 3; scale bar, 5 µm). *P < 0.01.

Fig. 7.

Abp1 regulates actin remodeling by modulating activation of the actin nucleation promoting factors, WASP and N-WASP. (A–F) Splenic B cells from wt and Abp1^−/− mice were activated with Fab’-anti-Ig-tethered lipid bilayers, fixed, permeabilized, and stained with AF488 phalloidin for F-actin (A), p-WASP (pWA) (C), or pN-WASP (pN-WA) (E). Representative images at 7 min (n = 3; Scale bar, 5 µm). The MFI (± SD) of F-actin (B), p-WASP (pWA) (D), and pN-WASP (pN-WA) (F) in the contact zone quantified in >50 cells per time point (n = 3). *P < 0.05.