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TNF-alpha mediates eosinophil cationic protein-induced apoptosis in BEAS-2B cells.

Chang KC, Lo CW, Fan TC, Chang MD, Shu CW, Chang CH, Chung CT, Fang SL, Chao CC, Tsai JJ, Lai YK - BMC Cell Biol. (2010)

Bottom Line: Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha).Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan.

ABSTRACT

Background: Eosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis.

Results: To address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.

Conclusion: In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.

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Related in: MedlinePlus

Effect of rECP on the viability of BEAS-2B cells. BEAS-2B cells (5 × 103) were incubated in a 96-well plate and treated with various concentrations of rECP as indicated for 48 h. Cell viability was determined by the MTT assay. (A) Cells were treated with rECP (up to 50 μM) for 48 h in the presence or absence of the general caspase inhibitor Z-VAD-FMK. (B) Morphology of the cells treated with serial concentrations of rECP ranging from 0 to 50 μM (concentrations shown below each panel). (C) Nuclei of BEAS-2B cells were stained with Hoechst 33342. Cells were treated or untreated with 20 μM rECP for 48 h. Stained nuclei were visualized by fluorescence microscopy. The chromatin condensation is indicated by bright blue spots shown by white arrows. (D) BEAS-2B cells were incubated in the presence or absence of 20 μM rECP for 24 h. The cells were stained with annexin-V-FITC and analyzed by FACS. Intact cells are located in the lower left quadrant. The apoptotic cells stained by annexin-V-FITC are located in the lower right quadrants, respectively. All data represent the arithmetic mean ± SEM.
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Figure 1: Effect of rECP on the viability of BEAS-2B cells. BEAS-2B cells (5 × 103) were incubated in a 96-well plate and treated with various concentrations of rECP as indicated for 48 h. Cell viability was determined by the MTT assay. (A) Cells were treated with rECP (up to 50 μM) for 48 h in the presence or absence of the general caspase inhibitor Z-VAD-FMK. (B) Morphology of the cells treated with serial concentrations of rECP ranging from 0 to 50 μM (concentrations shown below each panel). (C) Nuclei of BEAS-2B cells were stained with Hoechst 33342. Cells were treated or untreated with 20 μM rECP for 48 h. Stained nuclei were visualized by fluorescence microscopy. The chromatin condensation is indicated by bright blue spots shown by white arrows. (D) BEAS-2B cells were incubated in the presence or absence of 20 μM rECP for 24 h. The cells were stained with annexin-V-FITC and analyzed by FACS. Intact cells are located in the lower left quadrant. The apoptotic cells stained by annexin-V-FITC are located in the lower right quadrants, respectively. All data represent the arithmetic mean ± SEM.

Mentions: The effect of rECP on BEAS-2B cell viability was determined by the MTT assay. The rECP was added to the cell culture at concentrations of 0, 10, 20, 30, 40 and 50 μM at 37°C for 48 h. rECP inhibited cell viability with an IC50 of 21.03 μM, and cell viability was rescued by general caspase inhibitor, Z-VAD-FMK (Figure 1A). After co-incubation with rECP, shrinkage and unattachment of the cells from culture plate were observed (Figure 1B). BEAS-2B is a human bronchial epithelial cell line which is quite similar to primary cell. To determine whether such cell death was related to apoptosis, the nuclei were stained with Hoechst 33342 to monitor condensation of nuclear chromatin. Bright spots in the rECP-treated cells indicated nuclei undergoing chromatin condensation, strongly suggesting that BEAS-2B cells underwent apoptosis (Figure 1C). Here, apoptosis was also evaluated by staining with annexin V, a reagent commonly used to detect early apoptosis. BEAS-2B cells were treated with 20 μM rECP for 24 h. The treated BEAS-2B cells showed 14.5 ± 0.1% apoptosis (Figure 1D). Besides, the characteristic DNA fragmentation upon treatment with rECP was observed (Additional file 1). In comparison with untreated cells, the data indicated that BEAS-2B cells underwent early apoptosis after treatment with rECP.


TNF-alpha mediates eosinophil cationic protein-induced apoptosis in BEAS-2B cells.

Chang KC, Lo CW, Fan TC, Chang MD, Shu CW, Chang CH, Chung CT, Fang SL, Chao CC, Tsai JJ, Lai YK - BMC Cell Biol. (2010)

Effect of rECP on the viability of BEAS-2B cells. BEAS-2B cells (5 × 103) were incubated in a 96-well plate and treated with various concentrations of rECP as indicated for 48 h. Cell viability was determined by the MTT assay. (A) Cells were treated with rECP (up to 50 μM) for 48 h in the presence or absence of the general caspase inhibitor Z-VAD-FMK. (B) Morphology of the cells treated with serial concentrations of rECP ranging from 0 to 50 μM (concentrations shown below each panel). (C) Nuclei of BEAS-2B cells were stained with Hoechst 33342. Cells were treated or untreated with 20 μM rECP for 48 h. Stained nuclei were visualized by fluorescence microscopy. The chromatin condensation is indicated by bright blue spots shown by white arrows. (D) BEAS-2B cells were incubated in the presence or absence of 20 μM rECP for 24 h. The cells were stained with annexin-V-FITC and analyzed by FACS. Intact cells are located in the lower left quadrant. The apoptotic cells stained by annexin-V-FITC are located in the lower right quadrants, respectively. All data represent the arithmetic mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2819994&req=5

Figure 1: Effect of rECP on the viability of BEAS-2B cells. BEAS-2B cells (5 × 103) were incubated in a 96-well plate and treated with various concentrations of rECP as indicated for 48 h. Cell viability was determined by the MTT assay. (A) Cells were treated with rECP (up to 50 μM) for 48 h in the presence or absence of the general caspase inhibitor Z-VAD-FMK. (B) Morphology of the cells treated with serial concentrations of rECP ranging from 0 to 50 μM (concentrations shown below each panel). (C) Nuclei of BEAS-2B cells were stained with Hoechst 33342. Cells were treated or untreated with 20 μM rECP for 48 h. Stained nuclei were visualized by fluorescence microscopy. The chromatin condensation is indicated by bright blue spots shown by white arrows. (D) BEAS-2B cells were incubated in the presence or absence of 20 μM rECP for 24 h. The cells were stained with annexin-V-FITC and analyzed by FACS. Intact cells are located in the lower left quadrant. The apoptotic cells stained by annexin-V-FITC are located in the lower right quadrants, respectively. All data represent the arithmetic mean ± SEM.
Mentions: The effect of rECP on BEAS-2B cell viability was determined by the MTT assay. The rECP was added to the cell culture at concentrations of 0, 10, 20, 30, 40 and 50 μM at 37°C for 48 h. rECP inhibited cell viability with an IC50 of 21.03 μM, and cell viability was rescued by general caspase inhibitor, Z-VAD-FMK (Figure 1A). After co-incubation with rECP, shrinkage and unattachment of the cells from culture plate were observed (Figure 1B). BEAS-2B is a human bronchial epithelial cell line which is quite similar to primary cell. To determine whether such cell death was related to apoptosis, the nuclei were stained with Hoechst 33342 to monitor condensation of nuclear chromatin. Bright spots in the rECP-treated cells indicated nuclei undergoing chromatin condensation, strongly suggesting that BEAS-2B cells underwent apoptosis (Figure 1C). Here, apoptosis was also evaluated by staining with annexin V, a reagent commonly used to detect early apoptosis. BEAS-2B cells were treated with 20 μM rECP for 24 h. The treated BEAS-2B cells showed 14.5 ± 0.1% apoptosis (Figure 1D). Besides, the characteristic DNA fragmentation upon treatment with rECP was observed (Additional file 1). In comparison with untreated cells, the data indicated that BEAS-2B cells underwent early apoptosis after treatment with rECP.

Bottom Line: Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha).Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan.

ABSTRACT

Background: Eosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis.

Results: To address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.

Conclusion: In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.

Show MeSH
Related in: MedlinePlus