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

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

Membrane potential of isolated neutrophil mitochondria.

<p>Isolated neutrophil mitochondria were analysed by flow cytometry after staining with Mitotracker Green, to differentiate intact mitochondria from cellular debris, and TMRM to determine Δψ_m. A) Scatter plot displaying intact mitochondria, defined as Mitotracker-Green-positive events (FITC channel) with high side scatter in Q2. B) Intact mitochondria gained TMRM staining (Δψ_m, PE channel) after incubation with mitochondrial substrates. The complex-I substrates glutamate/malate (green) and the complex-II substrate succinate (yellow) induced a lower Δψ_m than glycerol phosphate (blue). Graphs are representative of three independent experiments. C) Graphic representation of the pooled experiments. Relative Δψ_m is expressed as a percentage of the mean fluorescence intensity (MFI) in the PE channel of the mitochondria population (Q2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002013#pone-0002013-g007" target="_blank">Figure 7A</a>). The MFI of glycerol phosphate (GlycP) treated mitochondria was set at 100% while the MFIs of the untreated (C-), glutamate/malate (Glut/Mal) and succinate (Succ) treated cells were expressed as a percentage of this value. The average original value for C- was 1701±498 (SEM), for Glut/Mal 4442±2689 (SEM), for Succ 5481±3106 (SEM) and for GlycP 5838±3139 (SEM). Bars represent the means (±SEM) of three independent experiments.</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

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

Neutrophil effector mechanisms.

<p>The mechanisms neutrophils employ to fight infections include phagocytosis, the release of various granule components into the extracellular space or into the phagosome (mainly proteases, oxidants, antimicrobial peptides), and the formation of neutrophil extracellular traps (NETs).</p

Extensive Variation in Gene Copy Number at the Killer Immunoglobulin-Like Receptor Locus in Humans

<div><p>Killer immunoglobulin-like receptors (KIRs) are involved in the regulation of natural killer cell cytotoxicity. Within the human genome seventeen KIR genes are present, which all contain a large number of allelic variants. The high level of homology among KIR genes has hampered KIR genotyping in larger cohorts, and determination of gene copy number variation (CNV) has been difficult. We have designed a multiplex ligation-dependent probe amplification (MLPA) technique for genotyping and CNV determination in one single assay and validated the results by next-generation sequencing and with a KIR gene-specific short tandem repeat assay. In this way, we demonstrate in a cohort of 120 individuals a high level of CNV for all KIR genes except for the framework genes <i>KIR3DL3</i> and <i>KIR3DL2</i>. Application of our MLPA assay in segregation analyses of families from the Centre d’Etude du Polymorphisme Humaine, previously KIR-genotyped by classical techniques, confirmed an earlier reported duplication and resulted in the identification of a novel duplication event in one of these families. In summary, our KIR MLPA assay allows rapid and accurate KIR genotyping and CNV detection, thus rendering improved transplantation programs and oncology treatment feasible, and enables more detailed studies on the role of KIRs in human (auto)immunity and infectious disease.</p></div

Validation of the KIR MLPA method with an independent short tandem repeat (STR) assay.

<p>A primer set was used to amplify a stretch of repeats that occurs in intron 4 of most KIR genes. Next-generation sequencing (NGS) was used to sequence the amplification products based on both specific sequence and number of repeats. The graphs show the percentage of reads in NGS, aligned against the reference sequence of that KIR, in donors with a certain number of copies of that gene as determined by MLPA. The number of reads of framework gene <i>KIR3DL2</i> was used as a reference to adjust for the total number of genes per donor. <i>KIR2DL1</i> and <i>KIR2DS1</i> have the same number of STR and no SNPs to distinguish one from the other and have therefore been combined. Several SNPs within the repeat region of <i>KIR2DL3</i> and <i>KIR3DL3</i> confirm the copy numbers as found with the MLPA method.</p