A functional granulocyte colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation

Abstract

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is widely used to treat neutropenia. In addition to stimulating polymorphonuclear neutrophil (PMN) production, G-CSF may have significant effects on PMN function. Because G-CSF receptor (G-CSFR)-deficient mice do not have the expected neutrophilia after administration of human interleukin-8 (IL-8), we examined the effect of the loss of G-CSFR on IL-8-stimulated PMN function. Compared with wild-type PMNs, PMNs isolated from G-CSFR-deficient mice demonstrated markedly decreased chemotaxis to IL-8. PMN emigration into the skin of G-CSFR-deficient mice in response to IL-8 was also impaired. Significant chemotaxis defects were also seen in response to N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, or macrophage inflammatory protein-2. The defective chemotactic response to IL-8 does not appear to be due to impaired chemoattractant receptor function, as the number of IL-8 receptors and chemoattractant-induced calcium influx, actin polymerization, and release of gelatinase B were comparable to those of wild-type PMNs. Chemoattractant-induced adhesion of G-CSFR-deficient PMNs was significantly impaired, suggesting a defect in beta2-integrin activation. Collectively, these data demonstrate that selective defects in PMN activation are present in G-CSFR-deficient mice and indicate that G-CSF plays an important role in regulating PMN chemokine responsiveness.

Figure 1

Granulocyte colony-stimulating factor receptor (G-CSFR)–deficient PMN chemotaxis. PMN chemotaxis was determined in modified Boyden chambers with bone marrow–purified PMNs (a) or thioglycollate-elicited peritoneal PMNs (b). Buffer alone (HBSS), N-formyl-methionyl-leucyl-phenylalanine (fMLP; 0.1 mM), zymosan-activated serum (ZAS; 5% vol/vol), or the indicated amount of human interleukin-8 (IL-8) was used as chemoattractant. The average number of PMNs per high-power field (PMN per HPF) ± SE of triplicate samples is shown. Data are representative of three separate experiments. PMN, polymorphonuclear neutrophil.

Figure 2

IL-8 receptor orthologue expression. Bone marrow–purified PMNs were incubated with anti–murine IL-8 receptor orthologue antibody or control antibody and analyzed by flow cytometry. Results are gated for Gr-1–positive (granulocytic) cells. Data are representative of three separate experiments.

Figure 3

Chemoattractant-induced calcium influx. Bone marrow–purified PMNs were loaded with the calcium sensitive dye fluo-3 and stimulated (at time 0) with buffer alone (control) or the indicated stimulant. Changes in cytoplasmic free Ca²⁺ were measured continuously by flow cytometry. Data are representative of three separate experiments.

Figure 4

Superoxide generation. Superoxide dismutase–inhibitable cytochrome c reduction was assessed after stimulation with PMA (100 ng/ml) by measuring OD at 550 nm (OD₅₅₀). Data represent the mean ± SD of triplicate determinations. Data are representative of three separate experiments.

Figure 5

Degranulation. (a) Gelatinase B release. Bone marrow–purified PMNs were stimulated with buffer alone (lanes 1 and 6), fMLP (0.1 mM; lanes 2 and 7), IL-8 (0.1 μg/ml; lanes 3 and 8), IL-8 (3 μg/ml; lanes 4 and 9), or IL-8 (30 μg/ml; lanes 5 and 10), and the amount of gelatinase B released into the supernatant was analyzed by gelatin zymography. Zones of enzymatic activity appear as unstained bands. (b) CD11b upregulation. Bone marrow–purified PMNs were stimulated with buffer alone, interleukin-8 (IL-8; 3 μg/ml), or N-formyl-methionyl-leucyl-phenylalanine (fMLP; 0.1 mM), and the expression of CD11b on Gr-1–positive cells was measured by flow cytometry. Data are representative of three separate experiments.

Figure 6

Adhesion. An equal number of calcein-AM–loaded bone marrow–purified PMNs were layered onto FBS-coated 96-well plates and stimulated with buffer alone (HBSS), N-formyl-methionyl-leucyl-phenylalanine (fMLP; 0.1 mM), ZAS (0.5% vol/vol), IL-8 (3 μg/ml), or PMA (100 ng/ml) for 3 min at 37°C. Fluorescence (at 538 nm) was measured before and after washing to remove nonadherent PMNs. The adhesion index is the fluorescence after washing divided by the fluorescence before washing (×100) and represents the percentage of adherent cells. Results were corrected for the percentage of PMNs present in the starting sample. Data represent the mean ± SD of triplicate determinations and are representative of four separate experiments.

Figure 7

IL-8–induced F-actin polymerization. Bone marrow–purified PMNs were stimulated with buffer alone or interleukin-8 (IL-8; 3 μg/ml) for the indicated times, and the F-actin content of Gr-1–positive (granulocytic) cells was measured by flow cytometry. Data are representative of three separate experiments.

Figure 8

IL-8–induced PMN emigration into the skin. Interleukin-8 (IL-8; 50 ng) or buffer alone was injected intradermally into wild-type or G-CSFR–deficient mice (G-CSFR^–/–). Four hours later, the skin at the injection site was excised and homogenized, and protein extracts were analyzed for MPO activity as described in Methods. MPO content is expressed as the relative MPO activity (OD₄₅₀) per milligram of protein. Six wild-type and nine G-CSFR–deficient mice were analyzed. Data represent the mean ± SD. *P <0.05 compared with buffer alone injected skin. MPO, myeloperoxidase.