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

Spaargaren M, Beuling EA, Rurup ML, Meijer HP, Klok MD, Middendorp S, Hendriks RW, Pals ST - J. Exp. Med. (2003)

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Dept. of Pathology, Academic Medical Center, Meibergdreef 9 1105 AZ Amsterdam, Netherlands. marcel.spaargaren@amc.uva.nl

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).

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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.
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fig1: 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.

Mentions: Adhesion assays were done in triplicate on round-bottom 96-well plates (Costar) coated overnight at 4°C with PBS containing, unless otherwise indicated, 1 μg/ml sVCAM-1, 10 μg/ml Fibronectin, 4% BSA, or for 15 min at 37°C with 1 mg/ml poly-l-lysine (PLL), and blocked for 2 h at 37°C with 4% BSA in RPMI 1640. Cells were pretreated with the pharmacological inhibitors at 37°C or anti-integrin antibodies TS1/22 (1:1000), Act-1 (1:100), TS2/16 (1:5), and HP2/1 (1:250) at 4°C for 30 min in RPMI with 1% BSA. Subsequently, cells were stimulated with either 10 μg/ml mouse anti-human IgM MH15, goat anti-chicken IgM, (Fab)2 fragments of anti-human or anti-mouse IgM, anti-mouse IgM microbeads (1:300) or 50 ng/ml PMA, and 1.5 × 105 Namalwa or DT40 cells, or 3 × 105 primary B cells, were plated in 100 μl/well and incubated at 37°C (39.5°C for DT40) for 30 min, unless otherwise indicated. After extensive washing of the plate with RPMI containing 1% BSA to remove nonadhering cells, the adherent cells were fixed for 10 min with 10% glutaraldehyde in PBS and stained for 45 min with 0.5% crystal violet in 20% methanol. After extensive washing with water, the dye was eluted in methanol and absorbance was measured after 5 to 40 min at 570 nm on a spectrophotometer (Microplate Reader 450, Biorad). Background absorbance (no cells added) was subtracted. Absorbance due to nonspecific adhesion, as determined in wells coated with 4% BSA, was always less than 10% of the absorbance of anti-IgM-stimulated cells. Maximal (100%) adhesion was determined by applying the PMA-stimulated cells to wells coated with the positively charged PLL, resulting in nonspecific adhesion of all cells, without washing the wells before fixation (see also Fig. 1, A-D) . Unless otherwise indicated, adhesion of the nonpretreated anti-IgM-stimulated cells was normalized to 100% and the bars represent the means ±SD of a triplicate experiment representative of at least three independent experiments.


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

Spaargaren M, Beuling EA, Rurup ML, Meijer HP, Klok MD, Middendorp S, Hendriks RW, Pals ST - J. Exp. Med. (2003)

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.
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Related In: Results  -  Collection

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fig1: 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.
Mentions: Adhesion assays were done in triplicate on round-bottom 96-well plates (Costar) coated overnight at 4°C with PBS containing, unless otherwise indicated, 1 μg/ml sVCAM-1, 10 μg/ml Fibronectin, 4% BSA, or for 15 min at 37°C with 1 mg/ml poly-l-lysine (PLL), and blocked for 2 h at 37°C with 4% BSA in RPMI 1640. Cells were pretreated with the pharmacological inhibitors at 37°C or anti-integrin antibodies TS1/22 (1:1000), Act-1 (1:100), TS2/16 (1:5), and HP2/1 (1:250) at 4°C for 30 min in RPMI with 1% BSA. Subsequently, cells were stimulated with either 10 μg/ml mouse anti-human IgM MH15, goat anti-chicken IgM, (Fab)2 fragments of anti-human or anti-mouse IgM, anti-mouse IgM microbeads (1:300) or 50 ng/ml PMA, and 1.5 × 105 Namalwa or DT40 cells, or 3 × 105 primary B cells, were plated in 100 μl/well and incubated at 37°C (39.5°C for DT40) for 30 min, unless otherwise indicated. After extensive washing of the plate with RPMI containing 1% BSA to remove nonadhering cells, the adherent cells were fixed for 10 min with 10% glutaraldehyde in PBS and stained for 45 min with 0.5% crystal violet in 20% methanol. After extensive washing with water, the dye was eluted in methanol and absorbance was measured after 5 to 40 min at 570 nm on a spectrophotometer (Microplate Reader 450, Biorad). Background absorbance (no cells added) was subtracted. Absorbance due to nonspecific adhesion, as determined in wells coated with 4% BSA, was always less than 10% of the absorbance of anti-IgM-stimulated cells. Maximal (100%) adhesion was determined by applying the PMA-stimulated cells to wells coated with the positively charged PLL, resulting in nonspecific adhesion of all cells, without washing the wells before fixation (see also Fig. 1, A-D) . Unless otherwise indicated, adhesion of the nonpretreated anti-IgM-stimulated cells was normalized to 100% and the bars represent the means ±SD of a triplicate experiment representative of at least three independent experiments.

Bottom Line: 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.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).

View Article: PubMed Central - PubMed

Affiliation: Dept. of Pathology, Academic Medical Center, Meibergdreef 9 1105 AZ Amsterdam, Netherlands. marcel.spaargaren@amc.uva.nl

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).

Show MeSH
Related in: MedlinePlus