The lineage-c-Kit+Sca-1+ cell response to Escherichia coli bacteremia in Balb/c mice

Abstract

During bacterial infection, the bone marrow hematopoietic activity shifts toward granulocyte production, which is critical for host defenses. Along with this enhancement of granulopoiesis, the bone marrow also increases its release of hematopoietic precursors. At the present time, little is known about the commitment of hematopoietic precursor cells, including hematopoietic stem cells and progenitors, in this response. To investigate the hematopoietic precursor cell response to bacterial infection, bacteremia was established in Balb/c mice by i.v. injection of Escherichia coli. Bacteremia caused a 10-fold increase in the number of lineage (lin)-c-kit+Sca-1+ cells in the bone marrow. This dramatic expansion of the lin-c-kit+Sca-1+ cell pool resulted from both increased mitosis of these cells and inversion from lin-c-kit+Sca-1- cell phenotype. Lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6 were potent factors capable of mediating phenotypic inversion of lin-c-kit+Sca-1- cells. Cells in the expanded lin-c-kit+Sca-1+ cell pool contained more colony-forming unit-granulocyte/macrophage. Mobilization of lin-c-kit+Sca-1+ cells into the circulation was significantly enhanced following bacteremia. These results demonstrate that the lin-c-kit+Sca-1+ cell population in the bone marrow constitutes a key component of the host defense response to bacteremia. Functional modifications of these primitive hematopoietic precursors are critical for enhancing granulocyte production following bacterial infection.

Figure 1

Changes in cell populations in bone marrow following bacteremia and representative dot plots of c-kit-APC vs. Sca-1-PE-Cy7 of lineage negative bone marrow cells. N=5∼9. BMCs: bone marrow cells; Bars with different letters in each panel are statistically different (p<0.05).

Figure 2

Changes in 5-bromo-2-deoxyuridine (BrdU) positive marrow lin-c-kit+Sca-1+ and lin-c-kit+Sca-1- cells 12 h (A, B) and 24 h (C, D) following bacteremia. N= 3∼4. BMCs: bone marrow cells; *: p<0.05 vs. Saline group. Panels E and F are representative histograms of BrdU incorporation into marrow lin-c-kit+Sca-1+ cells 24 h after intravenous saline and E. coli, respectively.

Figure 3

Changes in plasma cytokine levels following bacteremia. N=4∼5. Bars with different letters in each panel are statistically different (p<0.05).

Figure 4

Phenotypic changes of marrow lin-c-kit+Sca-1- cells following 24 h culture. N = 5 in each group. Panel A: Culture with 50% plasma of saline or E. coli challenged mice; *: p < 0.05 vs. Saline group. Panel B: Culture with different cytokines and growth factors; CGF: cocktail of growth factors; Bars with different letters are statistically different (p < 0.05). Panel C: Representative flow cytometric dot plots of isotype control antibody stained cells cultured with No. 1: plasma from saline treated mice; No. 4: medium only; and representative flow cytometric dot plots of anti-c-kit and anti-Sca-1 stained cells cultured with: No. 2: plasma from saline treated mice; No. 3: plasma from mice with E. coli bacteremia; No. 5: medium only; No. 6: granulocyte colony-stimulating factor (G-CSF); No. 7: cocktail of growth factors (CGF); No. 8: lipopolysaccharide (LPS); No. 9: tumor necrosis factor-α (TNF-α); No. 10: interleukin-6 (IL-6); No. 11: interferon-γ (IFN-γ); No. 12: TNF-α/IL-6/IFN-γ.

Figure 5

Colony-forming unit (CFU) activity of isolated bone marrow lin-c-kit+Sca-1+ (Panel A, N=3) and lin-c-kit+Sca-1- cells (Panel B, N=5) from mice at 24 h following i.v. challenge with saline or E. coli and CFU activity of isolated bone marrow lin-c-kit+Sca-1+ cells from control mice versus lin-c-kit+Sca-1+ derived from conversion of lin-c-kit+Sca-1- following culture for 24 h with TNF-α and IFN-γ (Panel C, N=5). Cells were cultured on Methocult GF M3434 and Methocult GF M3534 media for 7 days. *: p<0.05 vs. Saline group (Panel A), or p<0.05 vs. Control group (Panel C).

Figure 6

Colony-forming unit (CFU) activity of isolated circulating lin-c-kit+Sca-1+ and lin-c-kit+Sca-1- cells from mice at 24 h following i.v. challenge with saline or E. coli. Cells were cultured on Methocult GF M3434 and Methocult GF M3534 media for 7 days. N=4∼5, *: p<0.05 vs. Saline group.

Figure 7

Changes in the number of lin-c-kit+Sca-1+ and lin-c-kit+Sca-1- cells in peripheral blood mononuclear cells (PBMCs) following bacteremia and representative dot plots of c-kit-APC vs. Sca-1-PE-Cy7 of lineage negative PBMCs. N=4∼6. Bars with different letters in each panel are statistically different (p<0.05).