Severe exercise and exercise training exert opposite effects on human neutrophil apoptosis via altering the redox status

Abstract

Neutrophil spontaneous apoptosis, a process crucial for immune regulation, is mainly controlled by alterations in reactive oxygen species (ROS) and mitochondria integrity. Exercise has been proposed to be a physiological way to modulate immunity; while acute severe exercise (ASE) usually impedes immunity, chronic moderate exercise (CME) improves it. This study aimed to investigate whether and how ASE and CME oppositely regulate human neutrophil apoptosis. Thirteen sedentary young males underwent an initial ASE and were subsequently divided into exercise and control groups. The exercise group (n = 8) underwent 2 months of CME followed by 2 months of detraining. Additional ASE paradigms were performed at the end of each month. Neutrophils were isolated from blood specimens drawn at rest and immediately after each ASE for assaying neutrophil spontaneous apoptosis (annexin-V binding on the outer surface) along with redox-related parameters and mitochondria-related parameters. Our results showed that i) the initial ASE immediately increased the oxidative stress (cytosolic ROS and glutathione oxidation), and sequentially accelerated the reduction of mitochondrial membrane potential, the surface binding of annexin-V, and the generation of mitochondrial ROS; ii) CME upregulated glutathione level, retarded spontaneous apoptosis and delayed mitochondria deterioration; iii) most effects of CME were unchanged after detraining; and iv) CME blocked ASE effects and this capability remained intact even after detraining. Furthermore, the ASE effects on neutrophil spontaneous apoptosis were mimicked by adding exogenous H(2)O(2), but not by suppressing mitochondrial membrane potential. In conclusion, while ASE induced an oxidative state and resulted in acceleration of human neutrophil apoptosis, CME delayed neutrophil apoptosis by maintaining a reduced state for long periods of time even after detraining.

Figure 1. Effects of ASE on neutrophil apoptosis.

Blood specimens were obtained from all subjects at rest and immediately after ASE. Apoptosis assays were carried out using neutrophils cultured in vitro for 0, 4 and 10 h. The neutrophil ΔΨm was quantified by JC-1 aggregate FI (A–C), Ann-V binding by the fraction of Ann-V⁺ cells (D–F), and mtROS by the fraction of MitoSOX⁺ cells (G–I). Data in (C, F, I) were analyzed by two-way ANOVA with repeated measures followed by Bonferroni post-test. * p<0.05, after ASE vs resting; # p<0.05 compared with corresponding specimens at 0 h; n = 13.

Figure 2. Effects of ASE on neutrophil redox status.

Redox-related parameters of freshly isolated neutrophils at rest were compared with those immediately after the ASE. Neutrophil basal cytosolic ROS and PMA-stimulated cytosolic ROS were measured by DCF-DA fluorescence intensities before and after 10-min PMA stimulation, respectively (A, B). Neutrophil intracellular redox capacity was indicated by the total GSH amount (C). Neutrophil intracellular oxidation level was indicated by the GSSG/GSH ratio (D). Data were analyzed by paired t-test. * p<0.05, after ASE vs resting; n = 13.

Figure 3. Effects of CME and DT on redox status in resting neutrophils.

At various time points of the CME-DT paradigm, neutrophils were freshly isolated from subjects at rest to determine the redox-related parameters, i.e. basal cytosolic ROS, PMA-stimulated cytosolic ROS, total GSH, and GSSG/GSH ratio. Data were analyzed by one-way ANOVA with repeated measures followed by Bonferroni post-test. * p<0.05, compared with initial values. No differences between exercise (n = 8) and sedentary control (n = 5) groups were found at the beginning (analyzed by unpaired t-tests). There was no time-dependent effect in the sedentary control group.

Figure 4. Effects of CME and DT on apoptosis in resting neutrophils.

At various time points of the CME-DT paradigm, neutrophils isolated under resting conditions were subsequently cultured in vitro for up to 10 h to determine the apoptosis-related parameters (ΔΨm, Ann-V binding, and mtROS). (A): The kinetics of neutrophil apoptosis-related parameters before and after 2-month CME. (B): The CME and DT effects on apoptosis-related parameters measured after 10 h incubation in vitro. Data in (A) were analyzed by two-way ANOVA with repeated measures followed by Bonferroni post-test. Data in (B) were analyzed by one-way ANOVA with repeated measures followed by Bonferroni post-test. * p<0.05, compared with initial values; # p<0.05 compared with corresponding specimens at 0 h; n = 8. No differences between exercise (n = 8) and sedentary control (n = 5) groups were found at the beginning (analyzed by unpaired t-tests). There was no time-dependent effect in the sedentary control group.

Figure 5. Effects of exogenous H₂O₂ application on resting neutrophil redox status and apoptosis.

After 30-min H₂O₂ exposure, redox-related parameters of neutrophils were analyzed immediately (A–E), whereas the apoptosis-related parameters were determined in neutrophils cultured for 10 h (F–H). (A): neutrophil basal cytosolic ROS levels in response to different concentrations of H₂O₂ (0, 100, and 1000 µM). (B–E): effects of 100 µM H₂O₂ exposure on neutrophil basal cytosolic ROS, PMA-stimulated cytosolic ROS, total GSH, and GSSG/GSH. (F–H): effects of 100 µM H₂O₂ exposure on ΔΨm, Ann-V binding, and mtROS. Data in (A) were analyzed by one-way ANOVA with repeated measures followed by Bonferroni post-test. Data in other panels were analyzed by paired t-test. * p<0.05, H₂O₂ exposure vs untreated control; n = 9.

Figure 6. Effects of ΔΨm reduction on ROS levels and apoptosis in resting neutrophils.

Freshly isolated neutrophils were incubated with FCCP (0, 10, and 100 nM) for 20 min (A) or 10 h (B) before ΔΨm determination. Neutrophils were incubated with 10 nM of FCCP for 20 min before measuring basal cytosolic ROS and PMA-stimulated cytosolic ROS (C, E). Neutrophils were incubated with 10 nM of FCCP for 10 h before measuring Ann-V binding and mtROS (D, F). Data in (A, B) were analyzed by one-way ANOVA with repeated measures followed by Bonferroni post-test. Data in (C–F) were analyzed by paired t-test. * p<0.05, FCCP vs untreated control; n = 6.