B-lymphopoiesis is stopped by mobilizing doses of G-CSF and is rescued by overexpression of the anti-apoptotic protein Bcl2

Abstract

Osteoblasts are necessary to B lymphopoiesis and mobilizing doses of G-CSF or cyclophosphamide inhibit osteoblasts, whereas AMD3100/Plerixafor does not. However, the effect of these mobilizing agents on B lymphopoiesis has not been reported. Mice (wild-type, knocked-out for TNF-α and TRAIL, or over-expressing Bcl-2) were mobilized with G-CSF, cyclophosphamide, or AMD3100. Bone marrow, blood, spleen and lymph node content in B cells was measured. G-CSF stopped medullar B lymphopoiesis with concomitant loss of B-cell colony-forming units, pre-pro-B, pro-B, pre-B and mature B cells and increased B-cell apoptosis by an indirect mechanism. Overexpression of the anti-apoptotic protein Bcl2 in transgenic mice rescued B-cell colony forming units and pre-pro-B cells in the marrow, and prevented loss of all B cells in marrow, blood and spleen. Blockade of endogenous soluble TNF-α with Etanercept, or combined deletion of the TNF-α and TRAIL genes did not prevent B lymphopoiesis arrest in response to G-CSF. Unlike G-CSF, treatments with cyclophosphamide or AMD3100 did not suppress B lymphopoiesis but caused instead robust B-cell mobilization. G-CSF, cyclophosphamide and AMD3100 have distinct effects on B lymphopoiesis and B-cell mobilization with: 1) G-CSF inhibiting medullar B lymphopoiesis without mobilizing B cells in a mechanism distinct from the TNF-α-mediated loss of B lymphopoiesis observed during inflammation or viral infections; 2) CYP mobilizing B cells but blocking their maturation; and 3) AMD3100 mobilizing B cells without affecting B lymphopoiesis. These results suggest that blood mobilized with these three agents may have distinct immune properties.

Figure 1.

Effect of G-CSF, CYP and AMD3100 on CFU-C mobilization and expression of IL-7, CXCL12 and TNF-α. (A) Mobilization of CFU-C in peripheral blood was measured at indicated time points. Data are means±SD, 4 mice per time point. (B) IL-7 and CXCL12 mRNA were measured in the endosteal region of the BM by RT-qPCR. Data are relative to b2-microglobulin mRNA. Each dot is the result from an individual mouse. Bars are the average of each group. (C) Concentration of TNF-α protein in BM fluids. Each dot is the result from an individual mouse. Bars are the average of each group. ***P<0.001, **P<0.01, and *P<0.05. Gray boxes on kinetics show time period during which HSPC were mobilized into the blood.

Figure 2.

Comparative effect of G-CSF, CYP and AMD3100 on B cells in BM, blood and spleen. At indicated time points, SSC^low CD11b⁻ B220⁺ B cells were measured by flow cytometry in BM, blood and spleen. The charts on the right column show the number of B cells in total BM, blood, spleen per mouse calculated after summation of content in BM, blood and spleen. Data are average±SD of 4 mice per time point per treatment group. ***P<0.001, **P 0.01, and *P<0.05.

Figure 3.

G-CSF induces loss of all B-cell subsets in the BM. (A) Gating strategy to identify B-cell subsets during mobilization. B-cell subsets were defined within the SSC^low CD11b⁻ NK1.1-B220⁺ population as shown in the top two dot-plots. Mature B cells were defined as sIgM⁺ whereas immature B cells were sIgM⁻. Immature B cells were then subdivided in B220⁺ CD19⁻ CD43⁺ pre-pro-B cells, B220^low CD19⁺ pro-B cells and B220⁺ CD19⁺ pre-B cells. Typical B-cell profiles are shown for BM from control saline treated mice (top row) and G-CSF-treated mice (bottom row). Note the strong reduction of all B-cell subsets in G-CSF-mobilized BM. (B) The number of cells in each B-cell subset (pre-pro-B, Pro-B, pre-B and mature B cells) was measured by flow cytometry in BM, blood and spleen at indicated time points of G-CSF treatment. Results per mouse were calculated after summation of content in BM, blood and spleen. The number of CFU-B was measured in B-cell colony assays. (C) Apoptosis in BM cells was measured by flow cytometry. Cells were considered apoptotic when they were positive for annexin-V and/or 7-AAD. Data are average±SD of 4 mice per time-point per treatment group. ***P<0.001, **P<0.01, and *P<0.05.

Figure 4.

Overexpression of Bcl-2 in vavBcl2 mice rescues medullar B lymphopoiesis during G-CSF treatment. (A) CFU-C in blood, BM and spleen were measured in colony assays. (B) The number of cells in each B-cell subset (pre-pro-B, Pro-B, pre-B and mature B cells) was measured by flow cytometry in BM, blood and spleen at Day 6 of G-CSF or saline treatments. Results per mouse were calculated after summation of content in BM, blood and spleen. The number of CFU-B was measured in B-cell colony assays. Data are average±SD of 4 mice per time-point per treatment group. ***P<0.001, **P<0.01, and *P<0.05.