Sustained activation of cell adhesion is a differentially regulated process in B lymphopoiesis

Abstract

It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12, previously called stromal cell-derived factor 1, are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However, this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and, strikingly, is absent in circulating mature B cells, which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK), a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation, whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover, sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage-specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways.

Figure 1.

CXCL12 induces rapid and transient adhesion of peripheral blood as well as bone marrow B cells to VCAM-1 using the short-term assay conditions (refer to Materials and Methods). (A) REH cells were incubated in VCAM-1–coated wells for 28 min and then stimulated with different concentrations of CXCL12 for 2 min followed by the removal of nonadherent cells and quantitation of adhesion as described in Materials and Methods. (B and C) Kinetics of CXCL12-induced transient adhesion of REH pro-B and peripheral blood B cells to VCAM-1 is shown. Cells were incubated in VCAM-1– or BSA-coated wells for 25–29 min and after that time 1.0 μM CXCL12 was added for 5 to 1 min followed by the removal of nonadherent cells and quantitation of adhesion as described in Materials and Methods. Data represent the mean ± SD of three separate experiments, each performed in triplicate. (D and E) Comparison of transient CXCL12-induced adhesion of bone marrow and peripheral blood B cells to VCAM-1. The adhesion assay was conducted using 1 μM CXCL12 as described in A. Data represent the mean ± SD of five (bone marrow) or six (peripheral blood) separate experiments, each performed in triplicate. *, **, and ***, statistical significance as compared with negative control and assessed as P < 0.05, P < 0.01, and P < 0.005, respectively.

Figure 2.

CXCL12 induces sustained adhesion to VCAM-1 of bone marrow but not peripheral blood B cells using long-term assay conditions (refer to Materials and Methods). (A) REH cells were pretreated with different concentrations of CXCL12 for 30 min and then incubated in VCAM-1–coated wells for 30 min followed by the removal of nonadherent cells and quantitation of adhesion as described in Materials and Methods. (B and C) Cells were pretreated with 1.0 μM CXCL12 for 1–30 min (time shown as a first number on x axis) and then incubated in VCAM-1– or BSA-coated wells for 30 min (time shown as a second number on x axis) followed by the removal of nonadherent cells and quantitation of adhesion as described in Materials and Methods. CXCL12 was present throughout the incubation period. Kinetics of CXCL12-induced sustained adhesion of REH pro-B cells to VCAM-1 is shown in B and the comparison of sustained CXCL12-induced adhesion of REH, bone marrow, and peripheral blood CD19⁺ cells to VCAM-1 is shown in C. Data represent the mean ± SD of three (REH cells), four (bone marrow), and five (peripheral blood) independent experiments, each performed in triplicate. *, **, and ***, statistical significance as compared with negative control and are assessed as P < 0.05, P < 0.01, and P < 0.005, respectively.

Figure 3.

Decreasing response of B cells to CXCL12-induced adhesion to VCAM-1 during maturation. Sustained adhesion responses by developmental B cell subsets are shown in A and B. Transient adhesion responses by B cell subsets are shown in C. Long-term adhesion assay (A and B) was conducted as described in Fig. 2 and short-term adhesion assay (C) was conducted as described in Fig. 1. Data represent the mean ± SD of four (bone marrow) and five (peripheral blood) experiments, each performed in triplicate. *, **, and ***, statistical significance between adhesion of CXCL12- and medium-stimulated cells (negative control) and are assessed as P < 0.05, P < 0.01, and P < 0.005, respectively. The difference in CXCL12-induced adhesion response between pro-/pre-B cells and mature bone marrow B cells was statistically significant for sustained adhesion (P < 0.0005) as well as for transient adhesion (P < 0.05).

Figure 4.

CXCL12-mediated adhesion of B cells to VCAM-1 is integrin VLA-4 dependent. (A–C) CXCL12-mediated adhesion to VCAM-1 is blocked by anti-α4 monoclonal antibody. Before the adhesion assay, REH pro-B cells (A and B) or peripheral blood CD19⁺ cells (C) were incubated with 5 μg/ml anti–VLA-4 monoclonal antibody or with matching isotype control antibody for 30 min. Cells were then subjected to either long-term (A) or short-term (B and C) adhesion assays as described in Materials and Methods. Data represent the mean ± SD of three independent experiments, each performed in triplicate. *, statistical significance and is assessed as P < 0.01. (D and E) Expression of α₄ integrin chain does not change during B cell development. Expression of α₄ integrin chain on bone marrow B cell subpopulations (D) and total peripheral blood B cells (E) is shown. The black line represents cells stained with the isotype control and the shaded part of the histogram represents cells stained with anti-α₄ antibody. The values of MCF represent the average of three independent experiments ± SD. The differences between MCF values of different subsets are not statistically significant.

Figure 5.

CXCL12-mediated adhesion of B cells to VCAM-1 is Gα_i protein–coupled receptor and PKC dependent. (A and B) REH pro-B cells or (C) peripheral blood CD19⁺ cells were incubated with or without 100 ng/ml PTX for 2 h at 37°C. Cells were then subjected to either long-term (A) or short-term (B and C) adhesion assays as described in Materials and Methods. Otherwise, after incubation with PTX, separate cell aliquots were stimulated with 100 ng/ml PMA for 10 min and then placed into VCAM-1–coated wells for 30 min. (D) Peripheral blood CD19⁺ cells were either left untreated or were incubated with different concentrations of BIM-I, PKC inhibitor, or its negative control BIM-V for 2 h at 37°C. Cells were then subjected to short-term adhesion assay as described in Materials and Methods. (E and F) REH cells were either left untreated or were incubated with 1 μM BIM-I or BIM-V for 2 h at 37°C followed by short-term (F) or long-term (E) adhesion assay as described in Materials and Methods. Separate cell aliquots were stimulated with 100 ng/ml PMA for 10 min after incubation with BIM-I/BIM-V and before being placed onto VCAM-1–coated wells for 30 min. Results are shown as specific adhesion (background adhesion in the absence of a stimulus was subtracted from CXCL12- or PMA-induced adhesion). Data represent the mean ± SD of three independent experiments, each performed in triplicate. *, **, and ***, statistical significance between adhesion of PTX-treated or untreated cells (A–C) or between BIM-V– and BIM-I–treated cells (D–F) and are assessed as P < 0.05, P < 0.02, and P < 0.01, respectively.

Figure 6.

CXCL12-induced adhesion of B cells to VCAM-1 is PI3-K but not MEK dependent. REH cells were pretreated with the carrier (DMSO) or with different concentrations of wortmannin for 45 min (A and B) or PD98059 for 2 h (C and D). Cells were then subjected to either long-term (A and C) or short-term (B and D) adhesion assays as described in Materials and Methods. Results are shown as specific adhesion (background adhesion in the absence of a stimulus was subtracted from CXCL12-induced adhesion). Data represent the mean ± SD of three independent experiments, each performed in triplicate. * and **, statistical significance between adhesion of wortmannin- and carrier-treated cells and are assessed as P < 0.05 and P < 0.02, respectively.

Figure 7.

CXCL12-induced FAK phosphorylation is prolonged in early B cells but not in mature B cells. (A) CXCL12-stimulated FAK phosphorylation of REH pro-B cells is concentration dependent. REH cells were stimulated with indicated CXCL12 concentrations for 3 min. Immunoprecipitation (IP) with anti-FAK antibody was then performed followed by Western blot (WB) with antiphosphotyrosine antibody (4G10). The membranes were then stripped and reblotted (RB) with anti-FAK antibody to ensure equal protein loading (as shown in the right panels). Numbers under each lane are based on densitometry values and indicate the fold increase of phosphorylated FAK, expressed as multiples of the control (assigned as a value of 1). (B) REH, 697, and HS Sultan cells or (C) isolated bone marrow or peripheral blood B cells were stimulated with 100 nM CXCL12 for the indicated times and FAK phosphorylation was evaluated as described in A. (D) CXCL12-induced FAK phosphorylation is Gα_i protein–coupled receptor dependent. REH cells were incubated for 1 h with the indicated concentrations of PTX and then stimulated with 100 nM CXCL12 for 3 min followed by the evaluation of FAK phosphorylation as described in Fig. 7 A. Each experiment was repeated three times from which the representative blots are shown.

Figure 8.

CXCL12-induced FAK phosphorylation of early B cells is PKC dependent. REH cells were exposed to different concentrations of BIM-I or its negative control BIM-V and then stimulated with 100 nM CXCL12 for 20 (A and B) or 3 min (C and D). Immunoprecipitation (IP) with anti-FAK antibody was performed followed by Western blot (WB) with antiphosphotyrosine antibody (4G10). The membranes were then stripped and reblotted (RB) with anti-FAK antibody to verify equal protein loading (as shown in the bottom panels). Numbers under each lane indicate the fold increase of phosphorylated FAK based on densitometry values. Data are representative of three independent experiments.