Figure 4.

MCPIP-1 is engaged in the regulation of neutrophil life span by GM-CSF. (A,B) Freshly isolated human neutrophils were incubated with PBS or 20 ng/ml GM-CSF, 10 ng/ml G-CSF, 200 ng/ml IL-8, 2 μg/ml C5a for 2 or 4 h, respectively. (A) MCPIP-1 was estimated 2 h post cells stimulation using qPCR. Data shows the percent of mean control values ± SD. N = 3–7 (B) The level of MCPIP-1 protein in the cells was determined 4 h post stimulation by Western blot. Densitometry analysis demonstrating MCPIP-1 protein fold change was normalized to GAPDH level. A representative result is depicted above. (C) Neutrophils were incubated in the presence of 20 ng/ml GM-CSF for 24 h. Cells were double-stained with Annexin V and PI and analyzed using flow cytometry. The histogram shows mean values ± SD; n = 3. A representative histogram depicts immunostaining analysis. (D) Kinetic changes of MCPIP-1 mRNA in PMNs cultivated with or w/o GM-CSF. Data obtained from a representative donor was presented ± SEM (left). The level of MCPIP-1 protein after incubation of cells with GM-CSF for 3 h was determined by Western blot analysis (right). A representative result is depicted. (E) Kinetic changes of MCL-1 mRNA in PMNs cultivated with or w/o GM-CSF. Data obtained from three independent donors presented as the percent of control ± SD. (F) Neutrophils were exposed for 1 h to GM-CSF previously incubated or not for 15 min with neutralizing or isotypic anti-GM-CSF antibodies. Control cells were treated with PBS or antibodies alone. After isolation of total RNA MCPIP-1 was measured using qPCR. A representative result out of 3 was presented ± SEM. p-value of < 0.05 (*); p-value of < 0.01 (**); p-value of < 0.001 (***); ns for non-significant.