Figure 7
Figure 7. Pretreatment with SF1670 enhances the function and increases the efficacy of transfusion of G-CSF–mobilized neutrophils. (A) G-CSF (GCSF) induced neutrophil mobilization. A wild-type C57Bl/6 mouse was subcutaneously injected with G-CSF once daily at 250 μg/kg for 5 days. Peripheral blood (PB) was collected by cardiopuncture. White blood cells were counted using a Hematology Analyzer (Hemavet). (B) PTEN inhibitor SF1670 (SF) enhances fMLP-induced PtdIns(3,4,5)P3 signaling in both G-CSF–mobilized peripheral blood neutrophils and bone marrow–derived neutrophils (BM). Mouse peripheral blood samples were mixed with 2% dextran sulfate/PBS and 3 mg/mL EDTA/PBS at the ratio of 1:1:1 and incubated at 37°C for 20 minutes to allow red blood cells to settle. The leukocyte-containing layer was removed, and neutrophils were isolated by negative selection as described here. Isolated neutrophils were treated with SF1670 (500nM) or DMSO at 37°C for 30 minutes in PBS (0.2% bovine serum albumin), washed twice with HBSS, and then stimulated with 500nM fMLP for 4 minutes. The levels of phosphorylated AKT were assessed by Western blotting with specific antibody. (C) PTEN inhibitor SF1670 enhances fMLP-induced ROS production in G-CSF–mobilized peripheral blood neutrophils. Neutrophils were treated with SF1670 (500nM) or DMSO at 37°C for 30 minutes in PBS (0.2% BSA), washed twice with HBSS, and then stimulated with 2μM fMLP. ROS production was monitored in the presence of 50μM isoluminol and 0.8 U of horseradish peroxidase in a luminometer at 37°C. Chemiluminescence (arbitrary light units) was recorded at indicated time points. (D) Pretreatment with SF1670 increases the efficacy of transfusion of G-CSF–mobilized neutrophils. G-CSF–mobilized mouse peripheral blood neutrophils were pretreated with DMSO or SF1670 (250nM) for 30 minutes at 37°C, washed twice with PBS, and then intravenously injected into neutropenic mice that had been challenged with 105 CFU of E coli for 5 hours. Mice not receiving granulocyte transfusion were used as control. Histologic analysis of lungs reveals bacterial colonies (white arrow) in the pulmonary parenchyma. The number of bacterial colony was calculated. Pulmonary edema formation was quantified as the percentage of edema in the total parenchymal region using IPLab imaging software as described previously.29. *P < .05, **P < .01 vs control; ##P < .01 vs DMSO.

Pretreatment with SF1670 enhances the function and increases the efficacy of transfusion of G-CSF–mobilized neutrophils. (A) G-CSF (GCSF) induced neutrophil mobilization. A wild-type C57Bl/6 mouse was subcutaneously injected with G-CSF once daily at 250 μg/kg for 5 days. Peripheral blood (PB) was collected by cardiopuncture. White blood cells were counted using a Hematology Analyzer (Hemavet). (B) PTEN inhibitor SF1670 (SF) enhances fMLP-induced PtdIns(3,4,5)P3 signaling in both G-CSF–mobilized peripheral blood neutrophils and bone marrow–derived neutrophils (BM). Mouse peripheral blood samples were mixed with 2% dextran sulfate/PBS and 3 mg/mL EDTA/PBS at the ratio of 1:1:1 and incubated at 37°C for 20 minutes to allow red blood cells to settle. The leukocyte-containing layer was removed, and neutrophils were isolated by negative selection as described here. Isolated neutrophils were treated with SF1670 (500nM) or DMSO at 37°C for 30 minutes in PBS (0.2% bovine serum albumin), washed twice with HBSS, and then stimulated with 500nM fMLP for 4 minutes. The levels of phosphorylated AKT were assessed by Western blotting with specific antibody. (C) PTEN inhibitor SF1670 enhances fMLP-induced ROS production in G-CSF–mobilized peripheral blood neutrophils. Neutrophils were treated with SF1670 (500nM) or DMSO at 37°C for 30 minutes in PBS (0.2% BSA), washed twice with HBSS, and then stimulated with 2μM fMLP. ROS production was monitored in the presence of 50μM isoluminol and 0.8 U of horseradish peroxidase in a luminometer at 37°C. Chemiluminescence (arbitrary light units) was recorded at indicated time points. (D) Pretreatment with SF1670 increases the efficacy of transfusion of G-CSF–mobilized neutrophils. G-CSF–mobilized mouse peripheral blood neutrophils were pretreated with DMSO or SF1670 (250nM) for 30 minutes at 37°C, washed twice with PBS, and then intravenously injected into neutropenic mice that had been challenged with 105 CFU of E coli for 5 hours. Mice not receiving granulocyte transfusion were used as control. Histologic analysis of lungs reveals bacterial colonies (white arrow) in the pulmonary parenchyma. The number of bacterial colony was calculated. Pulmonary edema formation was quantified as the percentage of edema in the total parenchymal region using IPLab imaging software as described previously.29 . *P < .05, **P < .01 vs control; ##P < .01 vs DMSO.

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