The coordinated action of G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute inflammation

Abstract

In this study, we have identified a unique combinatorial effect of the chemokines KC/MIP-2 and the cytokine granulocyte colony-stimulating factor (G-CSF) with respect to the rapid mobilization of neutrophils from the bone marrow in a model of acute peritonitis. At 2 hours following an intraperitoneal injection of thioglycollate, there was a 4.5-fold increase in blood neutrophil numbers, which was inhibited 84% and 72% by prior administration of blocking mAbs against either the chemokines KC/MIP-2 or G-CSF, respectively. An intraperitoneal injection of G-CSF acted remotely to stimulate neutrophil mobilization, but did not elicit recruitment into the peritoneum. Further, in vitro G-CSF was neither chemotactic nor chemokinetic for murine neutrophils, and had no priming effect on chemotaxis stimulated by chemokines. Here, we show that, in vitro and in vivo, G-CSF induces neutrophil mobilization by disrupting their SDF-1alpha-mediated retention in the bone marrow. Using an in situ perfusion system of the mouse femoral bone marrow to directly assess mobilization, KC and G-CSF mobilized 6.8 x 10(6) and 5.4 x 10(6) neutrophils, respectively, while the infusion of KC and G-CSF together mobilized 19.5 x 10(6) neutrophils, indicating that these factors act cooperatively with respect to neutrophil mobilization.

Figure 1

KC/MIP-2 and G-CSF but not GM-CSF contribute to blood and tissue neutrophilia in TG-induced peritonitis. Neutralizing antibodies against KC (50 μg), MIP-2 (20 μg), KC and MIP-2, G-CSF (150 μg), GM-CSF (50 μg), or rat isotype-control mAb (mixture of control isotype IgGs) were administered intraperitoneally 20 minutes before intraperitoneal injection of 3% thioglycollate (TG). The number of neutrophils per milliliter of blood (A) and the number of neutrophils per milliliter of peritoneal lavage fluid (B) were determined 2 hours following TG injection. Results are expressed as means plus or minus SEM. Data from 6 independent experiments are combined (n = 4-15 mice). **P < .01 (1-way ANOVA and Dunnett test).

Figure 2

Intravenous administration of KC, MIP-2, and G-CSF stimulates the mobilization of neutrophils from the bone marrow into the blood. Mice injected intravenously with either 100 μL of PBS, KC, MIP-2, or G-CSF (660 nM each). The number of neutrophils per milliliter of blood (A) and the number of bone marrow neutrophils (per femur) (B) were determined 2 hours following injection. Results are expressed as means plus or minus SEM (n = 4 mice). *P < .05; **P < .01 (1-way ANOVA and Dunnett test).

Figure 3

Mobilization of neutrophils from the perfused femoral bone marrow in mice following the infusion of KC and G-CSF alone or in combination. The number of neutrophils (Gr-1^high) mobilized from the bone marrow after infusion of PBS, G-CSF (15 nM), KC (15 nM), or G-CSF and KC are shown. Results are expressed as means plus or minus SEM (n = 4 mice). *P < .05; **P < .01 (1-way ANOVA and Dunnett test).

Figure 4

G-CSF and MIP-2 both mobilize neutrophils from the bone marrow, while MIP-2 but not G-CSF stimulates recruitment into tissue. Mice were injected intraperitoneally with either 200 μL of PBS, MIP-2, or G-CSF (330 nM each). The number of neutrophils per milliliter of blood (A) and the number of neutrophils per milliliter of peritoneal lavage fluid (B) were determined 2 hours following injection. Results are expressed as means plus or minus SEM (n = 3 mice). *P < .05; **P < .01 (1-way ANOVA and Dunnett test).

Figure 5

G-CSF does not stimulate the chemotaxis or chemokinesis of murine neutrophils. Freshly isolated murine bone marrow neutrophils were used in a chemotaxis assay. Neutrophils were placed on top of a chemotaxis plate in the presence or absence of G-CSF (10 nM). KC (1 nM), MIP-2 (1 nM), or G-CSF (10 nM) were added to the bottom chamber as indicated. Migration of neutrophils is expressed as the mean number of cells counted per bottom well, mean plus or minus SD. A representative figure of 3 independent experiments is shown (n = 3). ***P < .001 (1-way ANOVA and Dunnett test).

Figure 6

G-CSF blocks the retention of neutrophils by SDF-1α. Freshly isolated murine bone marrow neutrophils were used in a chemotaxis assay and then placed on top of the chemotaxis plate in the presence or absence of G-CSF (10 nM) as indicated. Neutrophils were preincubated in the presence or absence of SDF-1α (50 nM) for 15 minutes at 37°C. KC (1 nM) was added to the bottom chamber as indicated. Migration of neutrophils is expressed as the mean number of cells counted per bottom well, mean plus or minus SD. A representative figure of 4 independent experiments is shown (n = 3). **P < .01 (1-way ANOVA and Dunnett test).

Figure 7

Inhibition of neutrophil mobilization by blockade of G-CSF is abrogated by administration of AMD3100. Anti–G-CSF–blocking antibody (150 μg) and AMD3100 (100 μg) alone or in combination were administered intraperitoneally 20 minutes before intraperitoneal injection of 3% TG. The number of neutrophils per milliliter of blood (A) and the number of neutrophils per milliliter of peritoneal lavage fluid (B) were determined 2 hours following TG injection. Results are expressed as means plus or minus SEM (n = 3). *P < .05; **P < .005 (1-way ANOVA and Dunnett test).