G-CSF Induces Alterations in the Bone Marrow Microenvironment That Suppress B Lymphopoiesis,

Abstract 3243

During infectious stress, there is a marked shift in the bone marrow from lymphopoiesis to granulopoiesis. Granulocyte colony-stimulating factor (G-CSF) is the principal cytokine regulating granulopoiesis, and its expression is induced during infection. In this study, we show that G-CSF treatment in mice is associated with a marked suppression of lymphopoiesis in the bone marrow. Specifically, after 5 days of G-CSF treatment (250 μg/kg), the number of B cells in the bone marrow was reduced 8.6 ± 1.3-fold, the number of T cells reduced 14.8 ± 3.8-fold, and the number of NK cells reduced 7.5 ± 1.6-fold. Though modest increases in splenic and blood lymphocytes were observed following G-CSF treatment, this did not account for the loss in the bone marrow. To assess B cell development, modified Hardy fractions were analyzed. All stages of B cell development were significantly reduced by G-CSF, but to different degrees. Fraction A (pre-pro B cells) declined 2.1 ± 0.5-fold; fraction B/C (mostly pro-B cells): 9.4 ± 1.7-fold; fraction D cells (pre-B cells): 5.9 ±1.1-fold; fraction E (immature B cells): 8.1 ± 1.6-fold; and fraction F (mature B cells): 87 ±13-fold. In addition, mature plasma cells declined 1.3 ± 0.07-fold while immature plasmablasts decreased 7.7 ± 1.7-fold. Interestingly, preliminary analysis suggests that there is no significant change in the number of common lymphoid progenitors in the bone marrow. Since there are reports of G-CSF receptor (G-CSFR) expression on certain B cell subsets, we next asked whether G-CSFR signals act in a cell-intrinsic fashion to suppress B lymphopoiesis. Mixed bone marrow chimeras were generated that contain both wild type and G-CSFR^−/− bone marrow cells. G-CSF treatment of these mixed chimeras demonstrated equal suppression of wild type and G-CSFR^−/− B cells. Thus, G-CSF works in a cell-extrinsic fashion to suppress B lymphopoiesis. Certain bone marrow stromal cell populations are known to regulate B lymphopoiesis, including osteoblasts and CXCL12-abundant reticular (CAR) cells. We previously showed that G-CSF treatment results in a loss of mature osteoblasts. To examine CAR cells, we analyzed mice in which green fluorescent protein (GFP) has been knocked-in to the Cxcl12 locus, allowing for CAR cell identification (Tokoyoda et al. 2004). Whereas G-CSF treatment did not alter the number of CAR cells, a significant decrease in GFP expression per CAR cell was observed. Consistent with this observation, we observed a significant decrease in CXCL12 mRNA expression in the bone marrow following G-CSF treatment. Interestingly, we also noted significant decreases in RNA and/or protein expression of a number of B-supportive cytokines, including interleukin-6, interleukin-7, and B cell activating factor (BAFF) protein. In summary, G-CSF treatment results in marked changes in the bone marrow microenvironment that lead to a suppression of B lymphopoiesis. The ability of G-CSF to disrupt homeostatic signals required for B cell maintenance at multiple stages of development suggest that upfront G-CSF treatment may be a novel strategy to sensitize certain B cell malignancies to chemotherapy.