Caspase-3 activity and expression of Bcl-2 family in human neutrophils by Helicobacter pylori water-soluble proteins.
BACKGROUND
Persistent infiltration of neutrophils is an almost invariable feature of Helicobacter pylori-infected gastric mucosa. A prolongation of neutrophil life-span could contribute to the pathogenesis of H. pylori infection. Recently, we have demonstrated that H. pylori water extracts (HPWE) inhibited neutrophil apoptosis. To elucidate the regulation of intracellular apoptotic signals by HPWE, we examined the activity of caspase-8, -3 and expression of Bcl-2 family in neutrophils.


METHODS
Human neutrophils were obtained from the peripheral blood of healthy volunteers by density gradient separation. HPWE was prepared from a supernatant of the H. pylori suspension in distilled water. After neutrophils were incubated with HPWE, expression of Bcl-2 family [antiapoptotic (Bcl-2, Bcl-XL and Mcl-1) and proapoptotic (Bax, Bak and Bcl-XS)] was determined by RT-PCR and Western blotting, respectively. Western blot for Bcl-2 family also performed in neutrophilic differentiated HL-60 cells by all-trans-retinoic acid. The activity of caspase-8, -3 was measured by the detection of p-nitroanilide after cleavage from labeled substrate.


RESULTS
HPWE suppressed the activation of caspase-8 and -3, and upregulated the expression of Bcl-XL mRNA and proteins in neutrophils. The expression of Bax and Bak was upregulated and Bcl-2, Bcl-XL and Mcl-1 downregulated in HL-60 cells during neutrophilic differentiation.


CONCLUSIONS
Bcl-XL may have an important role in the neutrophilic development and inhibition of neutrophil apoptosis by H. pylori.
Kim
J S
JS
Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Kim
J M
JM
Jung
H C
HC
Song
I S
IS
D001426:Bacterial Proteins / Q000276:immunology / Q000502:physiology*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis; D019254:Genes, bcl-2; D018922:HL-60 Cells; D016480:Helicobacter pylori / Q000276:immunology* / Q000472:pathogenicity; D006801:Humans; D018375:Neutrophil Activation; D009504:Neutrophils / Q000201:enzymology / Q000276:immunology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000235:genetics; D012995:Solubility; D015854:Up-Regulation
D001426:Bacterial Proteins; D004792:Enzyme Precursors; D019253:Proto-Oncogene Proteins c-bcl-2; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases