Figure 2.
Figure 2. Developing B cell motility in BM parenchyma is strictly dependent on α4β1 integrin–mediated adhesion to VCAM-1. (A–D) Rag1GFP/+ were injected (i.v.) with integrin α4 or VCAM-1 blocking antibodies, and calvarial BM was imaged before and immediately after treatment. Blood vessels were labeled with 2,000-kD dextran-rhodamine injected i.v. (A, left) Distribution of GFP+ B cells (green) in BM. (middle and right) Movement of GFP+ B cells tracked for 30 min before and after treatment with anti–VCAM-1 antibody. Colored lines represent cell trajectories. (B) Mean motility coefficient of B lineage cells before (blue line) and after VCAM-1 (red) and α4 blockade (green). Lines depict the average mean motility coefficient calculated from three independent experiments. (C) Median velocity (µm/min). (D) Cell axis ratios before (blue) and after treatment with VCAM-1 (red) and α4 (green) blocking antibodies. The data are representative of three independent experiments. (C and D) Lines indicate mean. (E) Mice were injected with 50 µg of 500-kD FITC-conjugated dextran (i.v.), and calvaria BM was imaged before and after treatment. BM vasculature (S) and parenchyma (P) are indicated. White boxes delineate regions of interest used for measurements of dextran perfusion. Time is shown in each panel (mm:ss). (A and E) Bars, 20 µm. (F) FITC-conjugated dextran perfusion of BM parenchyma and vasculature (red fluorescent voxels; y axis) over 30 min (time; x axis). Data are representative of >10 independent experiments. (G) Interstitial fluid flow rate (µm3/s) determined after injection (i.v.) of FITC-dextran (500 kD; green) or BSA–Texas red (red). Lines indicate the mean; symbols represent regions of interest obtained from three mice (FITC-dextran) or from two mice (BSA–Texas red). (H) In vivo staining of pro-B and pre-B cells in BM and of marginal zone (MZB) and follicular (FOB) B cell subsets in spleen with biotin–anti-CD19 antibody administered i.v. for 2 (red) or 10 (blue) min. Saturation with anti-CD19 antibody was determined by ex vivo staining with excess antibody (green). Data are representative of three independent experiments. **, P < 0.005; ***, P < 0.0005 by unpaired Student’s t test.

Developing B cell motility in BM parenchyma is strictly dependent on α4β1 integrin–mediated adhesion to VCAM-1. (A–D) Rag1GFP/+ were injected (i.v.) with integrin α4 or VCAM-1 blocking antibodies, and calvarial BM was imaged before and immediately after treatment. Blood vessels were labeled with 2,000-kD dextran-rhodamine injected i.v. (A, left) Distribution of GFP+ B cells (green) in BM. (middle and right) Movement of GFP+ B cells tracked for 30 min before and after treatment with anti–VCAM-1 antibody. Colored lines represent cell trajectories. (B) Mean motility coefficient of B lineage cells before (blue line) and after VCAM-1 (red) and α4 blockade (green). Lines depict the average mean motility coefficient calculated from three independent experiments. (C) Median velocity (µm/min). (D) Cell axis ratios before (blue) and after treatment with VCAM-1 (red) and α4 (green) blocking antibodies. The data are representative of three independent experiments. (C and D) Lines indicate mean. (E) Mice were injected with 50 µg of 500-kD FITC-conjugated dextran (i.v.), and calvaria BM was imaged before and after treatment. BM vasculature (S) and parenchyma (P) are indicated. White boxes delineate regions of interest used for measurements of dextran perfusion. Time is shown in each panel (mm:ss). (A and E) Bars, 20 µm. (F) FITC-conjugated dextran perfusion of BM parenchyma and vasculature (red fluorescent voxels; y axis) over 30 min (time; x axis). Data are representative of >10 independent experiments. (G) Interstitial fluid flow rate (µm3/s) determined after injection (i.v.) of FITC-dextran (500 kD; green) or BSA–Texas red (red). Lines indicate the mean; symbols represent regions of interest obtained from three mice (FITC-dextran) or from two mice (BSA–Texas red). (H) In vivo staining of pro-B and pre-B cells in BM and of marginal zone (MZB) and follicular (FOB) B cell subsets in spleen with biotin–anti-CD19 antibody administered i.v. for 2 (red) or 10 (blue) min. Saturation with anti-CD19 antibody was determined by ex vivo staining with excess antibody (green). Data are representative of three independent experiments. **, P < 0.005; ***, P < 0.0005 by unpaired Student’s t test.

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