Reduced expression of flavocytochrome b558, a component of the NADPH oxidase complex, in neutrophils from patients with myelodysplasia.
OBJECTIVE
Patients with myelodysplasia (MDS) show a disturbed production of ROS in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) in granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils. Because generation of ROS is mediated by the NADPH oxidase complex, a component of which is flavocytochrome b558, we investigated whether the expression of flavocytochrome b558 in neutrophils from MDS patients is affected.


METHODS
Neutrophils were stimulated with fMLP and GM-CSF, and plasma membrane expression of flavocytochrome b558 and specific granule markers were assessed by fluorescence-activated cell sorting analysis. Protein levels of the flavocytochrome b558 subunits gp91phox and p22phox in whole neutrophil lysates were detected by Western blotting.


RESULTS
Stimulation of neutrophils with GM-CSF and fMLP increased the flavocytochrome b558 plasma membrane expression. The fMLP-induced translocation of flavocytochrome b558 was reduced in neutrophils from MDS patients (140%+/-9% vs 180%+/-13%, p<0.05). Analysis of cell surface expression of markers of flavocytochrome b558 containing granules (CD35 and CD66b) indicated that exocytosis of these granules in response to fMLP stimulation was not affected in MDS patients. Western blot analysis demonstrated a decreased protein expression level of the flavocytochrome b558 subunits gp91phox and p22phox in neutrophils from MDS patients.


CONCLUSIONS
Our results indicate both a lower basal protein level and a disturbed fMLP-induced increase in plasma membrane expression of flavocytochrome b558 in neutrophils from MDS patients, which together might play a role in decreased ROS production.
Fuhler
Gwenny M
GM
Division of Hematology, Department of Medicine, University Hospital Groningen, Groningen, The Netherlands.
Hooijenga
Floris
F
Drayer
A Lyndsay
AL
Vellenga
Edo
E
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D003573:Cytochrome b Group / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D008297:Male; D008875:Middle Aged; D009190:Myelodysplastic Syndromes / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000235:genetics; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000201:enzymology*; D017382:Reactive Oxygen Species / Q000378:metabolism
D003573:Cytochrome b Group; D017382:Reactive Oxygen Species; D009240:N-Formylmethionine Leucyl-Phenylalanine; C049813:N-formylmethionyl-leucyl-phenylalanyl-lysine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; C036888:cytochrome b558; D019255:NADPH Oxidases