The B cell antigen receptor controls integrin activity through Btk and PLCgamma2

Abstract

Integrin-mediated adhesion and B cell antigen receptor (BCR) signaling play a critical role in B cell development and function, including antigen-specific B cell differentiation. Here we show that the BCR controls integrin alpha4beta1 (VLA-4)-mediated adhesion of B cells to vascular cell adhesion molecule-1 and fibronectin. Molecular dissection of the underlying signaling mechanism by a combined biochemical, pharmacological, and genetic approach demonstrates that this BCR-controlled integrin-mediated adhesion requires the (consecutive) activation of Lyn, Syk, phosphatidylinositol 3-kinase, Bruton's tyrosine kinase (Btk), phospholipase C (PLC)gamma2, IP3R-mediated Ca2+ release, and PKC. In contrast, activation of mitogen-activated protein kinase kinase (MEK) or extracellular signal-regulated kinase (ERK) is not required, and simultaneous activation of MEK, ERK, and PKB is not sufficient either. Furthermore, Btk is also involved in the control of integrin-mediated adhesion of preB cells. The control of integrin alpha4beta1-mediated B cell adhesion by the BCR involves cytoskeletal reorganization and integrin clustering. These results reveal a novel function for the BCR and Btk, i.e., regulation of integrin alpha4beta1 activity, thereby providing new insights into the control of B cell development and differentiation, as well as into the pathogenesis of the immunodeficiency disease X-linked agammaglobulineamia (XLA).

Figures

Figure 1.

BCR activation induces integrin α4β1-mediated B cell adhesion to VCAM-1 and FN. (A) Primary tonsillar B cells were not stimulated (C) or stimulated with (Fab)2-fragments of anti-IgM (a-IgM) or PMA and plated for 20 min on a surface coated with either VCAM-1 or FN, as indicated. (B) Mouse splenic B cells were not stimulated (C) or stimulated with (Fab)2-fragments of anti-IgM (a-IgM), or PMA and plated on a surface coated with 0.3 μg/ml VCAM-1. (C) Namalwa cells were not stimulated (C) or stimulated with anti-IgM (a-IgM) or PMA and plated on a surface coated with either VCAM-1 or FN, as indicated. (D) DT40 cells were not stimulated (C) or stimulated with anti-IgM (a-IgM) or PMA and plated on a surface coated with VCAM-1. (E) Namalwa cells were preincubated for 1 h at 4°C with medium alone (none), 10 μg/ml of an IgG1 isotype control (IgG1), or antibody TS1/22 blocking LFA-1 (TS1/22), Act-1 blocking α4β7 (Act-1), HP2/1 blocking α4β1 (HP2/1), or antibody TS2/16 activating integrin α4β1 (TS2/16). Subsequently, cells were not stimulated (C) or stimulated with anti-IgM (a-IgM), as indicated, and plated on a surface coated with VCAM-1. (F) DT40 cells were preincubated for 1 h at 4°C with medium alone (none), or 10 μg/ml of an IgG2b isotype control (IgG2b) or antibody CSAT against integrin subunit β1 (CSAT). Subsequently, cells were stimulated with anti-IgM and plated on a surface coated with VCAM-1. (A-D) The adhesion is presented as absolute adhesion, with input being determined by adhesion of all cells to PLL (= 100% adhesion). (E and F) The adhesion was normalized to 100% for the anti-IgM-stimulated cells.

Figure 2.

BCR-controlled adhesion is dependent on Lyn and Syk and requires activation of PI3K, but not of MEK or ERK. (A) WT or Lyn/Syk double-deficient DT40 cells (Lyn−/Syk−) were not stimulated (C) or stimulated with anti-IgM (a-IgM) and plated on a surface coated with VCAM-1. (B) Activation of PKB and ERK in WT or Lyn/Syk double-deficient DT40 cells (Lyn−/Syk−) after stimulation with anti-IgM for the indicated period of time (min). The immunoblots are probed with anti-phospho-PKB (P-PKB) and anti-phospho-MAPK (P-ERK) and reprobed with anti-ERK2 and anti-PKB, as indicated. (C) Activation of PKB and ERK in Namalwa cells (left) or DT40 cells (right) which were pretreated with 20 μM LY294002 (LY), 100 nM Wortmannin (WM), 50 μM PD98059 (PD), or left untreated (−) for 30 min, and stimulated with anti-IgM (a-IgM) or not (C) for 5 min, as indicated. The blots are probed as in B. (D) Namalwa cells (left) or DT40 cells (right) were pretreated with 50 μM LY294002 (LY), 100 nM Wortmannin (WM), 50 μM PD98059 (PD), or left untreated for 30 min, and not stimulated (C) or stimulated with anti-IgM (a-IgM) and plated on a surface coated with VCAM-1.

Figure 3.

Btk is required for BCR-controlled adhesion. (A) WT, Btk-deficient (Btk−), or Btk-deficient DT40 cells reconstituted with Btk (Btk+) were not stimulated (C) or stimulated with anti-IgM (a-IgM) or PMA and plated on a surface coated with VCAM-1. The inset shows expression of Btk in the Btk-reconstituted DT40 cell-line as detected by immunoblotting with anti-T7. (B) Activation of PKB and ERK in WT or Btk-deficient (Btk−) DT40 cells after stimulation with anti-IgM for the indicated period of time (min). The blots are probed as in Fig. 2 B. (C) B cells from WT or Btk-deficient (Btk−) mice were not stimulated (C) or stimulated with anti-IgM (a-IgM) or PMA and plated for 15 min on a surface coated with 0.3 μg/ml VCAM-1. The experiment shown is representative for two independent experiments. (D) Nalm6 (circles), DT40 (squares), or Namalwa (triangles) cells were not stimulated (open symbols) or stimulated with PMA (closed symbols) and plated for 20 min on a surface coated with different concentrations of VCAM-1. The adhesion was normalized to 100% for the PMA-stimulated cells plated on 1 μg/ml VCAM-1. The experiment shown is representative for two independent experiments. (E) Pre-B cells from WT or Btk-deficient (Btk−) mice were not stimulated (C) or stimulated with PMA and plated for 15 min on a surface coated with 0.3 μg/ml VCAM-1. The adhesion was normalized to 100% for the unstimulated WT preB cells. The bars represent the means ±SD of 2 WT and 2 Btk-deficient mice, each assayed in triplicate. In a total of three independent experiments (6 WT and 6 Btk-deficient mice), the basal adhesion of the Btk-deficient mice was 54% ± 10% in comparison to WT preB cells.

Figure 4.

BCR-controlled adhesion requires activation of PLCγ2, IP3R-mediated Ca2+ release, and PKC. (A) Namalwa cells (left) or DT40 cells (right) were pretreated with 1 or 2.5 μM U73122 (U), respectively, or left untreated for 30 min, and subsequently not stimulated (C) or stimulated with anti-IgM (a-IgM) and plated on a surface coated with VCAM-1. (B) WT, PLCγ2-deficient (PLCγ2−), or PLCγ2-deficient DT40 cells reconstituted with PLCγ2 (PLCγ2+) were not stimulated (C) or stimulated with anti-IgM (a-IgM) or PMA and plated on a surface coated with VCAM-1. The inset shows expression of PLCγ2 in the PLCγ2-reconstituted DT40 cell-line as detected by immunoblotting with anti-T7. (C) WT or IP3R-deficient DT40 cells (IP3R−) were not stimulated (C) or stimulated with anti-IgM (a-IgM) and plated on a surface coated with VCAM-1. (D) Activation of PKB and ERK in WT, PLCγ2(PLCγ2−)-, or IP3R(IP3R−)-deficient DT40 cells after stimulation with anti-IgM for the indicated period of time (min). The blots are probed as in Fig. 2 B. (E) Namalwa cells (left) or DT40 cells (right) were pretreated with 5 or 10 μM Chelerythrine (CE), respectively, or left untreated for 30 min, and subsequently not stimulated (C) or stimulated with anti-IgM (a-IgM) and plated on a surface coated with VCAM-1.

Figure 5.

BCR-controlled integrin α4β1-mediated adhesion involves integrin clustering and cytoskeletal reorganization. (A) DT40 cells were incubated without (open circles) or with 10 μg/ml anti-IgM (closed squares) or 3 mM MnCl2 (closed triangles) and 0, 2, or 10 μg/ml soluble VCAM-1-Fc, as indicated. Binding of soluble VCAM-1-Fc was measured by FACS analysis. Mean fluorescence intensity (MFI) values of a representative experiment are depicted. (B) DT40 cells, either WT or Btk-deficient (Btk−), were not stimulated (C) or stimulated for 15 or 30 min with anti-IgM (a-IgM) or PMA, as indicated. Clustering of integrin α4β1 was analyzed by means of confocal microscopy. For each condition, representative images of optical midheight sections (1st and 3rd row), and corresponding whole cell projections along the z-axis using maximum fluorescence values (2nd and 4th row), are shown. (C) Namalwa cells (left) or DT40 cells (right) were pretreated for 30 min with 1 μg/ml Cytochalasin D (CD), 1 μM Jasplakinolide (JP), 100 μg/ml Calpeptin (CP), or left untreated, and subsequently not stimulated (C) or stimulated with anti-IgM (a-IgM) and plated on a surface coated with VCAM-1.

Figure 6.

Molecular dissection of the signaling cascade underlying BCR-controlled integrin α4β1-mediated adhesion. A schematic representation of the signaling pathway underlying the inside-out activation mechanism of integrin α4β1 upon stimulation of the BCR is shown. The proteins encoded for by the inactivated genes in the DT40 cells and the pharmacological inhibitors used in this study are boxed and italicized, respectively. For sake of clarity, the role of actin cytoskeleton (re)organization is not included. See the Discussion section for further detail.