Figure 1.
Protection of lymphocytes from ROS-induced apoptosis by an ERK pathway inhibitor.
MACS-purified human CD8+ T cells or NK cells were preincubated with the ERK1/2 inhibitor PD98059 (25 µM) (filled triangle) for 1 h at 37°C. The T cells and NK cells were then incubated overnight in the presence of PD98059 with H2O2 at indicated concentrations (A–B) or with ROS-producing monocytes (MØ) at indicated MØ:NK ratios (C–D). Lymphocyte viability was assessed using the Live/Dead Fixable Violet Dead Cell Stain kit. ERK inhibitor-equivalent concentrations of DMSO were used as control (open square). Results obtained using DMSO did not significantly differ from PBS. Data are the mean ± SEM of results obtained using blood from 3–7 donors. *P<0.05, **P<0.01 and ***P<0.001.
Figure 2.
Induction of phosphorylated ERK in ROS-exposed lymphocytes.
PBMCs were treated with H2O2 (500 µM) (filled circle) or PBS (open square) at 37°C and analyzed for pERK by flow cytometry at indicated time points. Graphs show the percentage of pERK-positive cells (A) and pERK MFI (B) in gated lymphocytes (mean ± SEM, results from 4–6 donors). (C) Lymphocytes were treated with 250 µM H2O2 (1–10 min), PBS (10 min) or 50 ng/ml PMA (40 min) at 37°C. pERK1/2 was detected in whole cell lysates by Western blot. Total ERK1/2 was measured as loading control in parallel wells. A representative blot of 3 is shown. D. Inhibition of pERK formation by the ERK1/2 pathway inhibitor (PD98059, 25 µM) in gated lymphocytes after exposure to 500 µM H2O2 (10 min) shown as percent positive cells. E. Inhibition of pERK by PD98059 in NK cells exposed to PMA-stimulated monocytes shown as percent positive cells. Panels D and E are the mean ± SEM of results obtained using 5 donors. *P<0.05, **P<0.01 and ***P<0.001.
Figure 3.
Oxidant-induced poly ADP-ribose accumulation: role of the ERK pathway.
(A–B) Lymphocytes were preincubated with or without the ERK1/2 inhibitor PD98059 (25 µM) or the PARP-1 inhibitor PJ34 (2 µM) for 1 hr at 37°C before exposure to 500 µM H2O2 for 20 min. PAR accumulation was analyzed in whole cell lysates. (A) Representative Western blot and (B) mean ± SEM of results from 4 donors (O.D., optical density). β-tubulin was utilized as loading control and H2O2-treated HELA cells served as positive controls. (C) Flow cytometry analysis of PAR accumulation following exposure to H2O2 (500 µM, filled circle) or PBS (open square). (D) Inhibition of PAR formation in lymphocytes after preincubation with PD98059 (25 µM) or PJ34 (2 µM). PAR accumulation was measured after 20 min exposure to 500 µM H2O2 by flow cytometry. Data are the MFI of PAR in gated lymphocytes (mean ± SEM of results obtained in 4–6 donors) *P<0.05, **P<0.01 and ***P<0.001).
Figure 4.
Inhibition of ERK phosphorylation in lymphocytes exposed to oxygen radicals by ERK pathway inhibitor but not by PARP inhibitor.
PBMCs or NK cells were preincubated in presence or absence of ERK1/2 inhibitor PD98059 (25 µM) or PARP-1 inhibitor PJ34 (2 µM) for 1 h at 37°C. (A) A Representative dot plots of pERK+ NK cells after 10 min exposure to PMA-stimulated monocytes.is shown. (B) Mean ± SEM of pERK positive cells in gated lymphocytes after 10 min exposure to H2O2. (C) Mean ± SEM of pERK positive NK cells after 10 min exposure to PMA-stimulated monocytes. B–C: mean ± SEM of 4–6 experiments. *P<0.05, **P<0.01 and ***P<0.001.
Figure 5.
Retained cytotoxicity of NK cells rescued from ROS induced cell death by an ERK1/2 inhibitor.
NK cells were co-incubated with monocytes at Mo:NK ratios of 0.25∶1, 0.5∶1 and 1∶1 overnight in the presence or absence of the ERK1/2 inhibitor PD98059 (25 μM). Anti-CD20 (rituximab) and CFSE-labeled 221 target cells were subsequently added at an effector to target cell ratio of 1∶1 as per the NK cell count prior to incubation. Lysis of 221 cells by ADCC was estimated in a 4 hour assay using the Live/Dead Fixable Far Red Dead Cell Stain kit and flow cytometry. Panel (A) shows contour density plots from a representative experiment displaying an inverse relationship between NK cell and target cell viability. Panel (B) shows NK cell cytotoxicity (mean ± SEM) of seven donors using the lowest Mo:NK cell ratio in which PD98059 rescued NK cells from apoptosis. *P<0.05, **P<0.01 and ***P<0.001.