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Extracellular administration of BCL2 protein reduces apoptosis and improves survival in a murine model of sepsis.

Iwata A, de Claro RA, Morgan-Stevenson VL, Tupper JC, Schwartz BR, Liu L, Zhu X, Jordan KC, Winn RK, Harlan JM - PLoS ONE (2011)

Bottom Line: Severe sepsis and septic shock are major causes of morbidity and mortality worldwide.We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2) family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP).We show that intraperitoneal injection of picomole range doses of recombinant human (rh) BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival.

Methodology/principal findings: We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2) family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP). We show that intraperitoneal injection of picomole range doses of recombinant human (rh) BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2- mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo.

Conclusions/significance: Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins.

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Treatment with rhBCL2A1 or rhBCL2 protein improves survival following CLP.Mice were subjected to CLP and followed for 7 days. An assessment table was used as a surrogate marker for death and the animals euthanized according to approved criteria. (A) Mice were treated by i.p. injection of 1 µg rhBCL2A1 or saline 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBCL2A1 conferred significant protection compared to saline. *p = 0.012. (B) Mice were treated by i.p. injection of 1 µg rhBim or saline 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBim did not improve survival compared to saline. (C) Mice were treated by i.p. injection of 1 µg rhBCL2 or 0.5 µg of rhUbiquitin at 18 hours prior to CLP, at time of CLP, and then every 12 hours for 3 consecutive days. Treatment with rhBCL2 conferred significant protection compared to rhUbiquitin *p = 0.003. (D) Mice were treated by i.p. injection of 1 µg rhBCL2A1 or rhBim at 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBCL2A1 conferred significant protection compared to rhBim. *p = 0.005.
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pone-0014729-g001: Treatment with rhBCL2A1 or rhBCL2 protein improves survival following CLP.Mice were subjected to CLP and followed for 7 days. An assessment table was used as a surrogate marker for death and the animals euthanized according to approved criteria. (A) Mice were treated by i.p. injection of 1 µg rhBCL2A1 or saline 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBCL2A1 conferred significant protection compared to saline. *p = 0.012. (B) Mice were treated by i.p. injection of 1 µg rhBim or saline 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBim did not improve survival compared to saline. (C) Mice were treated by i.p. injection of 1 µg rhBCL2 or 0.5 µg of rhUbiquitin at 18 hours prior to CLP, at time of CLP, and then every 12 hours for 3 consecutive days. Treatment with rhBCL2 conferred significant protection compared to rhUbiquitin *p = 0.003. (D) Mice were treated by i.p. injection of 1 µg rhBCL2A1 or rhBim at 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBCL2A1 conferred significant protection compared to rhBim. *p = 0.005.

Mentions: Mice were subjected to CLP and followed for 7 days as described in Methods. An assessment table was used as a surrogate marker for death and the animals euthanized according to approved criteria. In the initial experiments, mice were treated with 1 µg (∼40 picomoles) of rhBCL2A1 or saline vehicle at 18 hours prior to CLP and then every 12 hours for 3 consecutive days for a total of eight doses. Treatment with the BH4-domain protein, rhBCL2A1, was protective (Fig. 1A), whereas treatment with the BH3-domain protein, rhBim, was not (Fig. 1B). In separate experiments, rhBCL2A1 and rhBCL2 were compared directly with other recombinant proteins, which had been prepared similarly in E. coli and were also his-tagged. Treatment with the anti-apoptotic BH4-domain BCL2 proteins conferred a significant increase in survival, compared with treatment with the control recombinant proteins, rhUbiquitin (Fig. 1C) or rhBim (Fig. 1D). Consequently, rhBim was used as control in most experiments.


Extracellular administration of BCL2 protein reduces apoptosis and improves survival in a murine model of sepsis.

Iwata A, de Claro RA, Morgan-Stevenson VL, Tupper JC, Schwartz BR, Liu L, Zhu X, Jordan KC, Winn RK, Harlan JM - PLoS ONE (2011)

Treatment with rhBCL2A1 or rhBCL2 protein improves survival following CLP.Mice were subjected to CLP and followed for 7 days. An assessment table was used as a surrogate marker for death and the animals euthanized according to approved criteria. (A) Mice were treated by i.p. injection of 1 µg rhBCL2A1 or saline 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBCL2A1 conferred significant protection compared to saline. *p = 0.012. (B) Mice were treated by i.p. injection of 1 µg rhBim or saline 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBim did not improve survival compared to saline. (C) Mice were treated by i.p. injection of 1 µg rhBCL2 or 0.5 µg of rhUbiquitin at 18 hours prior to CLP, at time of CLP, and then every 12 hours for 3 consecutive days. Treatment with rhBCL2 conferred significant protection compared to rhUbiquitin *p = 0.003. (D) Mice were treated by i.p. injection of 1 µg rhBCL2A1 or rhBim at 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBCL2A1 conferred significant protection compared to rhBim. *p = 0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044724&req=5

pone-0014729-g001: Treatment with rhBCL2A1 or rhBCL2 protein improves survival following CLP.Mice were subjected to CLP and followed for 7 days. An assessment table was used as a surrogate marker for death and the animals euthanized according to approved criteria. (A) Mice were treated by i.p. injection of 1 µg rhBCL2A1 or saline 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBCL2A1 conferred significant protection compared to saline. *p = 0.012. (B) Mice were treated by i.p. injection of 1 µg rhBim or saline 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBim did not improve survival compared to saline. (C) Mice were treated by i.p. injection of 1 µg rhBCL2 or 0.5 µg of rhUbiquitin at 18 hours prior to CLP, at time of CLP, and then every 12 hours for 3 consecutive days. Treatment with rhBCL2 conferred significant protection compared to rhUbiquitin *p = 0.003. (D) Mice were treated by i.p. injection of 1 µg rhBCL2A1 or rhBim at 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBCL2A1 conferred significant protection compared to rhBim. *p = 0.005.
Mentions: Mice were subjected to CLP and followed for 7 days as described in Methods. An assessment table was used as a surrogate marker for death and the animals euthanized according to approved criteria. In the initial experiments, mice were treated with 1 µg (∼40 picomoles) of rhBCL2A1 or saline vehicle at 18 hours prior to CLP and then every 12 hours for 3 consecutive days for a total of eight doses. Treatment with the BH4-domain protein, rhBCL2A1, was protective (Fig. 1A), whereas treatment with the BH3-domain protein, rhBim, was not (Fig. 1B). In separate experiments, rhBCL2A1 and rhBCL2 were compared directly with other recombinant proteins, which had been prepared similarly in E. coli and were also his-tagged. Treatment with the anti-apoptotic BH4-domain BCL2 proteins conferred a significant increase in survival, compared with treatment with the control recombinant proteins, rhUbiquitin (Fig. 1C) or rhBim (Fig. 1D). Consequently, rhBim was used as control in most experiments.

Bottom Line: Severe sepsis and septic shock are major causes of morbidity and mortality worldwide.We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2) family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP).We show that intraperitoneal injection of picomole range doses of recombinant human (rh) BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival.

Methodology/principal findings: We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2) family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP). We show that intraperitoneal injection of picomole range doses of recombinant human (rh) BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2- mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo.

Conclusions/significance: Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins.

Show MeSH
Related in: MedlinePlus