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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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Effects of TNF-α on rECP-induced apoptosis. BEAS-2B cells were treated with 20 μM rECP. (A) TNF-α was measured in cell lysates by rECP treatment for ranging from 0 to 48 h. (B) TNF-α was measured in cultured supernatant medium by rECP treatment for 48 h. All the TNF-α measurements were determined by ELISA assay. (C) To investigate the role of TNF-α in rECP-induced apoptosis, BEAS-2B cells were pretreated with 5 μM anti-TNF-α Ab for 4 h before rECP treatment. The addition of IgG Ab to the medium of cells was used as controls in neutralization studies. All data represent the arithmetic mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 7: Effects of TNF-α on rECP-induced apoptosis. BEAS-2B cells were treated with 20 μM rECP. (A) TNF-α was measured in cell lysates by rECP treatment for ranging from 0 to 48 h. (B) TNF-α was measured in cultured supernatant medium by rECP treatment for 48 h. All the TNF-α measurements were determined by ELISA assay. (C) To investigate the role of TNF-α in rECP-induced apoptosis, BEAS-2B cells were pretreated with 5 μM anti-TNF-α Ab for 4 h before rECP treatment. The addition of IgG Ab to the medium of cells was used as controls in neutralization studies. All data represent the arithmetic mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: BEAS-2B cells treated with rECP induced TNF-α production and release (Figures 7). Secretion of TNF-α in the culture medium was monitored in BEAS-2B cells treated with rECP for periods from 0 to 48 h (Figure 7A), suggesting that TNF-α production in rECP-treated cells was time-dependent. An ELISA analysis showed that TNF-α accumulation in cell lysate of BEAS-2B cells significantly increased in those treated with rECP after 24 h (49.9 ± 0.5 pg/mg total proteins). The maximum of TNF-α production in the cells reached at 48 h (58.8 ± 0.6 pg/mg total protein). In addition, higher TNF-α level was detected in the supernatant of BEAS-2B cells treated with rECP for 48 h than control cells (16.9 ± 0.1 and 4.0 ± 0.6 pg/ml, respectively) (Figure 7B). In this study, we have found that mutant ECP lacking of RNase activity (mECP) can also induce TNF-α liberation; however, there is no significant increase of TNF-α liberation upon treating with RNase A (Additional file 3).


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)

Effects of TNF-α on rECP-induced apoptosis. BEAS-2B cells were treated with 20 μM rECP. (A) TNF-α was measured in cell lysates by rECP treatment for ranging from 0 to 48 h. (B) TNF-α was measured in cultured supernatant medium by rECP treatment for 48 h. All the TNF-α measurements were determined by ELISA assay. (C) To investigate the role of TNF-α in rECP-induced apoptosis, BEAS-2B cells were pretreated with 5 μM anti-TNF-α Ab for 4 h before rECP treatment. The addition of IgG Ab to the medium of cells was used as controls in neutralization studies. All data represent the arithmetic mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Effects of TNF-α on rECP-induced apoptosis. BEAS-2B cells were treated with 20 μM rECP. (A) TNF-α was measured in cell lysates by rECP treatment for ranging from 0 to 48 h. (B) TNF-α was measured in cultured supernatant medium by rECP treatment for 48 h. All the TNF-α measurements were determined by ELISA assay. (C) To investigate the role of TNF-α in rECP-induced apoptosis, BEAS-2B cells were pretreated with 5 μM anti-TNF-α Ab for 4 h before rECP treatment. The addition of IgG Ab to the medium of cells was used as controls in neutralization studies. All data represent the arithmetic mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: BEAS-2B cells treated with rECP induced TNF-α production and release (Figures 7). Secretion of TNF-α in the culture medium was monitored in BEAS-2B cells treated with rECP for periods from 0 to 48 h (Figure 7A), suggesting that TNF-α production in rECP-treated cells was time-dependent. An ELISA analysis showed that TNF-α accumulation in cell lysate of BEAS-2B cells significantly increased in those treated with rECP after 24 h (49.9 ± 0.5 pg/mg total proteins). The maximum of TNF-α production in the cells reached at 48 h (58.8 ± 0.6 pg/mg total protein). In addition, higher TNF-α level was detected in the supernatant of BEAS-2B cells treated with rECP for 48 h than control cells (16.9 ± 0.1 and 4.0 ± 0.6 pg/ml, respectively) (Figure 7B). In this study, we have found that mutant ECP lacking of RNase activity (mECP) can also induce TNF-α liberation; however, there is no significant increase of TNF-α liberation upon treating with RNase A (Additional file 3).

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