ATP levels in neutrophils and PBMC after treatment with respiratory chain inhibitors.

<p>A–F) Neutrophils (▪,□) and PBMC (•,○) were incubated with (▪, •) or without (□, ○) 5 mM glucose. After 2 hours of pre-incubation at 37°C, the cells were incubated for an additional 2 hours in the presence of various concentrations of the mitochondrial uncoupler CCCP (A), or inhibitors for the OXPHOS complexes I-V(F₁), rotenone (B), 3-nitropropionate (3NP; C), antimycin A (D), KCN (E), or aurovertin B (F), repectively. ATP levels were determined with a luciferase-based assay. Data represent the means (±SEM) of three independent experiments performed in duplicate.</p

Supercomplex organisation of OXPHOS complexes in PBMC and neutrophils.

<p>A–B) Mitochondria isolated from PBMC (A) and neutrophils (B) were solubilized in digitonin, and 200 µg of mitochondrial protein was separated by BN-PAGE. Strips from the first dimension were excised and used for second dimension SDS-(12%)PAGE, transferred to PVDF membrane and immuno-detected with anti-complex I (20 kDa), complex II (30 kDa), complex III (Core 2, 47 kDa), complex IV (COX II, 26 kDa) and complex V (α, 55 kDa) subunit antibodies. Blots are representative of three independent experiments. High molecular weight purified proteins were used as molecular mass markers in the first dimension BN-PAGE: thyroglobulin, 669 kDa; ferritin monomer, 440 kDa; catalase 232 kDa; lactate dehydrogenase, 140 kDa.</p

HL-60 cells loose supercomplex organisation and acquire neutrophil-like properties during differentiation.

<p>HL-60 cells were differentiated to neutrophil-like cells in 9 days. To demonstrate that HL-60 cells gain a neutrophil-like morphology and abilities during differentiation, the expression of the neutrophil markers gp91^PHOX and EMR3 was determined by flowcytometry (A) and their ability to produce a respiratory burst was determined by an Amplex Red assay (B). On day 9 of differentiation, approximately 60% of the cells stained positive for both neutrophil markers, while responding to all three stimuli (PMA, fMLP and the combination of PAF and fMLP) by producing a respiratory burst. Plots are representative of four independent experiments. C) Supercomplex organisation in HL-60 mitochondria before (Day 0) and after (Day 9) differentiation. Respiratory chain complexes were detected on Western blots of 2D-native/reduced SDS-PAGE gels with anti-complex I (20 kDa), complex II (30 kDa), complex III (Core 2, 47 kDa), complex IV (COX II, 26 kDa) and complex V (α, 55 kDa) subunit antibodies. Blots are representative of four independent experiments. During differentiation, HL-60 cells gain the metabolic properties of neutrophils (D and E). Undifferentiated (▪,□) and differentiated (•,○) HL-60 cells were incubated with (▪, •) or without (□, ○) 5 mM glucose. After 2 hours of pre-incubation at 37°C, the cells were incubated for an additional 2 hours in the presence of various concentrations of the complex-I inhibitor rotenone (D) or the complex-III inhibitor antimycin A (E) as indicated. Afterwards, ATP (left panels) and lactate (right panels) levels were determined. Data represent the means (±SEM) of three independent experiments performed in duplicate.</p

Lactate levels produced by neutrophils and PBMC after treatment with respiratory chain inhibitors.

<p>A–F) Neutrophils (▪,□) and PBMC (•,○) were incubated with (▪, •) or without (□, ○) 5 mM glucose. After 2 hours of pre-incubation at 37°C, the cells were incubated for an additional 2 hours in the presence of various concentrations of the mitochondrial uncoupler CCCP (A), or inhibitors for the OXPHOS complexes I-V(F₁), rotenone (B), 3-nitropropionate (3NP; C), antimycin A (D), KCN (E), or aurovertin B (F), respectively. Lactate levels were determined with an enzymatic assay. Data represent the means (±SEM) of three independent experiments performed in duplicate.</p

Mitochondrial membrane potential in neutrophils and PBMC.

<p>A–D) Neutrophils (▪,□) and PBMC (•,○) were incubated with (▪, •) or without (□, ○) 5 mM glucose. After 2 hours of pre-incubation at 37°C, the cells were incubated for an additional 2 hours in the presence of various concentrations of respiratory chain enzyme inhibitors. Afterwards, the cells were stained with the fluorescent dye JC-1, and fluorescence in Fl-1 and Fl-2 was determined with by flowcytometry. The ratio (Fl-2/Fl-1) represents the coupling efficiency (Δψ_m) of the mitochondria as expressed as a percentage of the control (2 hour incubation without inhibitor). The average initial value for neutrophils was 3.32±0.85 (SD) and for PBMC 3.41±0.75 (SD). The F₀ inhibitor oligomycin (A) and the F₁ inhibitor aurovertin B (B) had a protective effect in all cells, demonstrating that reversal of complex V does not maintain Δψ_m in neutrophils. The complex III inhibitor antimycin A (C) completely reduced Δψ_m in all cells, while the complex-I inhibitor rotenone (D) did not affect neutrophils cultured with glucose. Data represent the means (±SEM) of five independent experiments performed in duplicate.</p

Mitochondrial content and respiratory chain enzyme activity in neutrophils, PBMC and HL-60 cells.

<p>A) Neutrophils (PMN) contain significantly less mitochondria as expressed per unit citrate synthase activity. Data represent the mean (±SD) of 13 (PMN), 6 (HL-60) or 16 (PBMC) different experiments performed in duplicate. B) Neutrophils retain full complex II and V activity while the activity of the remaining complexes is significantly reduced. Data represent the mean (±SD) of 10 different experiments performed in duplicate. Values were corrected for mitochondrial protein content and citrate synthase activity. C) Crucial subunits of the respiratory chain can be detected in neutrophil lysates on Western blot. 20 µg of mitochondrial protein was separated by SDS-PAGE and immuno-detected with anti-complex I (20 kDa), complex II (30 kDa), complex III (Core 2, 47 kDa), complex IV (COX II, 26 kDa) and complex V (α, 55 kDa) subunit antibodies. Blots exposed for 15 sec and 5 minutes are representative of three independent experiments.</p