Quantification of Bax and Bcl2 in polymorphonuclear leukocytes from haemodialysis patients: relation to hydrogen peroxide.
BACKGROUND
Bax and Bcl2 are two apoptosis-related molecules that play an important role in determining cell fate following oxidative injury. In the present study, we explored the relation of hydrogen peroxide (H2O2) generation by polymorphonuclear cells (PMNs) to the cytosolic expression of Bax and Bcl2 proteins and apoptosis in haemodialysis (HD) patients.


METHODS
Cytosolic generation of H2O2 by PMNs from control subjects and HD patients was measured by flow cytometry using the dichlorofluorescin diacetate assay. Bax and Bcl2 expression was detected by flow cytometry using FITC-conjugated antibodies. Apoptosis was quantified by flow cytometry using propidium iodide nuclear staining. To examine the effect of H2O2 on Bcl2 and Bax expression, PMNs from control subjects were briefly exposed to H2O2 (0.1-100 microM) for 10 min and then washed and cultured for 6 h, with or without catalase, a H2O2 detoxifying molecule. Bcl2 and Bax expression was determined by Western blot analysis.


RESULTS
Basal H2O2 generation by resting PMNs was significantly higher in HD patients compared with control subjects (211 +/- 115 vs. 23 +/- 5 MFI; P=0.002). However, PMNs from HD patients did not undergo accelerated programmed cell death compared with control subjects (58 +/- 7% vs. 46 +/- 5; P=0.14). Polymorphonuclear cells cytosolic Bcl2 was undetected in control subjects but detected in 25% of HD patients, and Bax was more frequently detected in PMNs from HD patients (75% vs. 67%; P=0.04). In the HD patients with detectable cytosolic Bax and Bcl2 proteins, the Bax to Bcl2 ratio inversely correlated with H2O2 levels (P<0.0001). Finally, brief exposure of PMNs to 0.1-100 microM of H2O2 resulted in a marked increase in Bcl2 expression (P=0.001), which was prevented by catalase (P=0.05). There was no apparent effect on Bax expression.


CONCLUSIONS
This study demonstrates that in HD patients, high-resting cytosolic H2O2 production by PMNs is not associated with accelerated in vitro apoptosis, and that the Bax/Bcl2 system may counter-balance the deleterious effects of reactive oxygen species in human PMNs.
D000328:Adult; D000368:Aged; D017209:Apoptosis; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000097:blood* / Q000494:pharmacology; D007676:Kidney Failure, Chronic / Q000097:blood / Q000628:therapy; D008875:Middle Aged; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000097:blood* / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000097:blood* / Q000187:drug effects; D006435:Renal Dialysis; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D006861:Hydrogen Peroxide