Hematopoietic stem and progenitor cells are differentially mobilized depending on the duration of Flt3-ligand administration.

BACKGROUND: Flt3-ligand is a cytokine that induces relatively slow mobilization of hematopoietic cells in animals and humans in vivo. This provides a time-frame to study hematopoietic stem and progenitor cell migration kinetics in detail. DESIGN AND METHODS: Mice were injected with Flt3-ligand (10 microg/day, intraperitoneally) for 3, 5, 7 and 10 days. Mobilization of hematopoietic stem and progenitor cells was studied using colony-forming-unit granulocyte/monocyte and cobblestone-area-forming-cell assays. The radioprotective capacity of mobilized peripheral blood mononuclear cells was studied by transplantation of 1.5 x 10(6) Flt3-ligand-mobilized peripheral blood mononuclear cells into lethally irradiated (9.5 Gy) recipients. RESULTS: Hematopoietic progenitor cell mobilization was detected from day 3 onwards and prolonged administration of Flt3-ligand produced a steady increase in mobilized progenitor cells. Compared to Flt3-ligand administration for 5 days, the administration of Flt3-ligand for 10 days led to a 5.5-fold increase in cobblestone-area-forming cells at week 4 and a 5.0-fold increase at week 5. Furthermore, transplantation of peripheral blood mononuclear cells mobilized by 5 days of Flt3-ligand administration did not radioprotect lethally irradiated recipients, whereas peripheral blood mononuclear cells mobilized by 10 days of Flt3-Ligand administration did provide 100% radioprotection of the recipients with significant multilineage donor chimerism. Compared to the administration of Flt3-ligand or interleukin-8 alone, co-administration of interleukin-8 and Flt3-ligand led to synergistic enhancement of hematopoietic stem and progenitor cell mobilization on days 3 and 5. CONCLUSIONS: These results indicate that hematopoietic stem and progenitor cells show different mobilization kinetics in response to Flt3-ligand, resulting in preferential mobilization of hematopoietic progenitor cells at day 5, followed by hematopoietic stem cell mobilization at day 10.

CD97 is differentially expressed on murine hematopoietic stem-and progenitor-cells.

BACKGROUND: CD97 is a member of the epidermal growth factor-seven transmembrane (EGF-TM7) family of adhesion receptors and is broadly expressed on hematopoietic cells. The aim of this study was to investigate the expression of CD97 on hematopoietic stem- and progenitor cells (HSC/HPC). DESIGN AND METHODS: CD97 expression on hematopoietic stem- and progenitor cells was studied in BALB/c, C57BL/6 and DBA/1 mice using flow cytometry. Functional hematopoietic stem- and progenitor cell characteristics were investigated in vitro and in vivo by progenitor cell assays, cobblestone area forming cell assays and bone marrow cell transplantation. RESULTS: Analysis of CD97 expression on murine bone marrow cells showed three major populations i.e. CD97(HI), CD97(INT) and CD97(NEG) cells. Functional studies revealed that radioprotective capacity and cobblestone area forming cell day 28-35 activity resides in the CD97(INT) bone marrow cell fraction while CFU-GM colony-forming capacity mainly resides in the CD97(NEG) population in all strains. In C57BL/6 and DBA/1 mice CD97(NEG) and CD97(HI) bone marrow cells show hematopoietic stem cell characteristics as well. Further functional analysis of BALB/c CD97(INT) bone marrow cells revealed that c-Kit(HI)CD97(INT) bone marrow cells exhibit HSC activity and are 1.5-fold enriched for cobblestone area forming cell-day 35 activity compared to c-Kit(HI) bone marrow cells. Moreover, phenotypical analysis showed that BALB/c and C57BL/6 HSC are CD97(INT), while DBA/1 HSC are CD97(HI). CONCLUSIONS: CD97 is differentially expressed on hematopoietic stem cells and hematopoietic progenitor cells. Committed progenitor cell activity is largely comprised in the CD97(NEG) fraction, while the CD97(INT) population contains hematopoietic stem cell activity. In BALB/c mice, CD97 expression can be applied to almost completely separate colony-forming cells and cells exhibiting radioprotective capacity. In addition we propose that the CD97(INT)c-Kit(HI) phenotype allows simple and rapid purification of murine hematopoietic stem cells.

Differential role of CD97 in interleukin-8-induced and granulocyte-colony stimulating factor-induced hematopoietic stem and progenitor cell mobilization.

CD97 is broadly expressed on hematopoietic cells and is involved in neutrophil migration. Since neutrophils are key regulators in HSC/HPC mobilization, we studied a possible role for CD97 in interleukin-8 and granulocyte-colony stimulating factor-induced HSC/HPC mobilization. Mobilization was absent in mice receiving CD97 mAb followed by interleukin-8, while granulocyte-colony stimulating factor-induced mobilization remained unaltered following anti-CD97 administration. Furthermore, combined administration of CD97 mAb and IL-8 induced a significant reduction in the neutrophilic compartment. We hypothesize that the absence of interleukin-8-induced HSC/HPC mobilization after CD97 mAb administration is due to its effect on neutrophil function.

Repeated hematopoietic stem and progenitor cell mobilization without depletion of the bone marrow stem and progenitor cell pool in mice after repeated administration of recombinant murine G-CSF.

Administration of recombinant-human G-CSF (rhG-CSF) is highly efficient in mobilizing hematopoietic stem and progenitor cells (HSC/HPC) from the bone marrow (BM) toward the peripheral blood. This study was designed to investigate whether repeated G-CSF-induced HSC/HPC mobilization in mice could lead to a depletion of the bone marrow HSC/HPC pool with subsequent loss of mobilizing capacity. To test this hypothesis Balb/c mice were treated with a maximum of 12 repeated 5-day cycles of either 10 microg rhG-CSF/day or 0.25 microg rmG-CSF/day. Repeated administration of rhG-CSF lead to strong inhibition of HSC/HPC mobilization toward the peripheral blood and spleen after >4 cycles because of the induction of anti-rhG-CSF antibodies. In contrast, after repeated administration of rmG-CSF, HSC/HPC mobilizing capacity remained intact for up to 12 cycles. The number of CFU-GM per femur did not significantly change for up to 12 cycles. We conclude that repeated administration of G-CSF does not lead to depletion of the bone marrow HSC/HPC pool.

Serpina1 is a potent inhibitor of IL-8-induced hematopoietic stem cell mobilization.

Here, we report that cytokine-induced (granulocyte colony-stimulating factor and IL-8) hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is completely inhibited after low-dose (0.5 Gy) total-body irradiation (TBI). Because neutrophil granular proteases are regulatory mediators in cytokine-induced HSC/HPC mobilization, we considered a possible role for protease inhibitors in the induction of HSC/HPC mobilization. Bone marrow (BM) extracellular extracts that were obtained from murine femurs after 0.5 Gy of TBI contained an inhibitor of elastase. Also, after low-dose TBI, both Serpina1 mRNA and protein concentrations were increased in BM extracts, compared with extracts that were obtained from controls. The inhibitory activity in BM extracts of irradiated mice was reversed by addition of an Ab directed against Serpina1. To further study a possible in vivo role of Serpina1 in HSC/HPC mobilization, we administered Serpina1 before IL-8 injection. This administration resulted in an almost complete inhibition of HSC/HPC mobilization, whereas heat-inactivated Serpina1 had no effect. These results indicate that low-dose TBI inhibits cytokine-induced HSC/HPC mobilization and induces Serpina1 in the BM. Because exogenous administration of Serpina1 inhibits mobilization, we propose that radiation-induced Serpina1 is responsible for the inhibition of HSC/HPC mobilization. Also, we hypothesize that cytokine-induced HSC/HPC mobilization is determined by a critical balance between serine proteases and serine protease inhibitors.

Reduced stem cell mobilization in mice receiving antibiotic modulation of the intestinal flora: involvement of endotoxins as cofactors in mobilization.

Since endotoxins are potent inducers of stem cell mobilization, we hypothesized that their presence in the gut may play a role in cytokine-induced mobilization. To address this possibility we added ciprofloxacin and polymyxin B to the drinking water of Balb/c mice mobilized with either interleukin-8 (IL-8), granulocyte colony-stimulating factor (G-CSF), or flt3 ligand (FL). The yield of colony-forming units (CFUs) was significantly reduced in all mice treated with these antibiotics when compared with controls (IL-8: 192 +/- 61 vs 290 +/- 64, P <.05; G-CSF: 1925 +/- 1216 vs 3371 +/- 1214, P <.05; FL: 562 +/- 213 vs 1068 +/- 528, P <.05). Treatment with ciprofloxacin eliminated only aerobic Gram-negative bacteria from the feces without effect on mobilization. Polymyxin B treatment did not result in decontamination but significantly reduced the number of mobilized hematopoietic progenitor cells (HPCs) most likely due to the endotoxin binding capacity of polymyxin B. More than 90% of the gastrointestinal flora consists of anaerobic bacteria. Elimination of the anaerobic flora by metronidazol led to a significantly reduced number of mobilized HPCs when compared with controls (IL-8: 55 +/- 66 vs 538 +/- 216, P <.05). Germ-free OF1 mice showed a significantly reduced mobilization compared with their wild-type controls (IL-8 controls: 378 +/- 182, IL-8 germ free: 157 +/- 53, P <.05). Finally, we performed reconstitution experiments adding Escherichia coli-derived endotoxins to the drinking water of decontaminated mice. This resulted in partial restoration of the IL-8-induced mobilization (67 +/- 28 vs 190 +/- 98.1, P <.01). Our results indicate that endotoxins serve as cofactors in cytokine-induced mobilization. Modification of the endotoxin content by antibiotic treatment may affect the yield of cytokine-induced mobilization.