Figure 1.
The caspase inhibitor Q-VD.OPh profoundly inhibits neutrophil apoptosis.
A,B) Neutrophils were cultured for A) 8 hours, and B) 20 hours with and without the pan-caspase inhibitors zVAD.fmk and Q-VD.OPh with a DMSO vehicle control (n = 4). Q-VD.OPh dramatically decreased neutrophil apoptosis from 10 nM, whereas no effects of zVAD were seen below 100 µM. *P<0.05, **P<0.01, ***p<0.001 for comparison of Q-VD vs zVAD, one way ANOVA with Bonferroni's post-test correction, comparing all values. C) Cytospin preparation of neutrophils in control conditions after 20 hours. Apoptotic cells are clearly visible by their condensed nuclei. D) Cytospin preparation of neutrophils cultured with Q-VD.OPh after 20 hours. No apoptotic cells can be seen and neutrophils exhibit the classic polymorphic nuclear morphology of a healthy neutrophil. Images were taken using a 60× Plan Apo Oil immersion NA1.40, Nikon, with 1.5× magnification on a Nikon TE2000U.
Figure 2.
At early timepoints, Mcl-1 levels fall even in the presence of highly effective caspase inhibition.
Neutrophils were cultured ± caspase inhibitor, Q-VD.OPh (QVD), ± DMSO (vehicle control) for 8 hours. Neutrophil apoptosis rates were determined by cytospin analysis (upper panel), and relative Mcl-1 levels determined using western blotting for 3 independent experiments (middle panel). Q-VD.OPh significantly reduced rates of apoptosis. In the presence of Q-VD.OPh, amounts of Mcl-1 were significantly lower in control neutrophil lysates compared to lysates from GMCSF treated cells (*p<0.001 for control vs GMCSF, one way ANOVA with Bonferroni's post-test correction, n = 3.)
Figure 3.
At late timepoints, Mcl-1 levels are maintained by highly effective caspase inhibition.
Neutrophils were cultured ± caspase inhibitor, Q-VD.OPh (QVD), ± DMSO (vehicle control) for 20 hours. Neutrophil apoptosis rates were determined by cytospin analysis (upper panel), and relative Mcl-1 levels determined using western blotting for 3 independent experiments (middle panel) as previously shown. In the presence of Q-VD.OPh, there was no significant difference in levels of Mcl-1 in control neutrophil lysates compared to lysates from GMCSF treated cells (*p<0.001 for control vs GMCSF, one way ANOVA with Bonferroni's post-test correction, n = 3.)
Figure 4.
GM-CSF increased Mcl-1 levels in standard purity and highly purified neutrophils between 8 and 20 hours.
A. Rates of apoptosis (top) and Mcl-1 expression ± GM-CSF as determined by western blot analysis in neutrophils isolated using the Percoll method. n = 3 independent experiments from different donors. *P<0.05, *** p<0.001, one-way ANOVA, with Bonferroni's post-test correction for multiple comparisons. B. Rates of apoptosis (top) and Mcl-1 expression ± GM-CSF as determined by western blot analysis in neutrophils isolated using the OptiPrep (+NMS) method. n = 3 independent experiments from different donors. *P<0.05 one-way ANOVA, with Bonferroni's post-test correction for multiple comparisons.