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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 increases neutrophils in peritoneum following CLP.Mice were treated once by i.p. injection with 1 µg of rhBCL2A1 or rhBim at 18 hours prior to CLP. Peritoneal lavage fluid was collected at 24 hours following CLP. Panels A and B show the total number of leukocytes and neutrophils (PMNs) in lavage fluid. Mice treated with rhBCL2A1 showed an increase in peritoneal PMNs following CLP as compared to mice that did not receive CLP or mice that were treated with rhBim. Panel C shows the viability of peritoneal neutrophils at 24 hours following CLP as assessed by staining with annexin V and 7-amino-actinomycin D. Treatment with rhBCL2 significantly increased the number of viable neutrophils compared to treatment with saline or rhBCL2A1. *p<0.01.
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pone-0014729-g005: Treatment with rhBCL2A1 increases neutrophils in peritoneum following CLP.Mice were treated once by i.p. injection with 1 µg of rhBCL2A1 or rhBim at 18 hours prior to CLP. Peritoneal lavage fluid was collected at 24 hours following CLP. Panels A and B show the total number of leukocytes and neutrophils (PMNs) in lavage fluid. Mice treated with rhBCL2A1 showed an increase in peritoneal PMNs following CLP as compared to mice that did not receive CLP or mice that were treated with rhBim. Panel C shows the viability of peritoneal neutrophils at 24 hours following CLP as assessed by staining with annexin V and 7-amino-actinomycin D. Treatment with rhBCL2 significantly increased the number of viable neutrophils compared to treatment with saline or rhBCL2A1. *p<0.01.

Mentions: In separate experiments, leukocyte populations in peritoneal lavage were evaluated. Mice received rhBim, rhBCL2A1, or no treatment followed 18 hours later by CLP. Additionally, some mice received either rhBim or rhBCL2A1, but did not have CLP. In those animals not subjected to CLP, there was no significant accumulation of total leukocytes or neutrophils in animals treated with rhBCL2A1 (Fig. 5 A, B). In contrast, in animals subjected to CLP there was a significant increase in the total number of leukocytes in the peritoneal lavage fluid in mice treated with rhBCL2A1 as compared to the other treatment groups (Fig. 5A). This increase in total cells was due specifically to an increase in neutrophils in the peritoneal lavage fluid (Fig. 5B). We did not observe any changes in circulating neutrophil counts, and G-CSF levels in peritoneal fluid and G-CSF mRNA expression in spleen (Fig. S1) were similar in the rhBCL2A1-treated mice compared to rhBim-treated mice following CLP, making it less likely that the increase in peritoneal neutrophils following CLP was due solely to increased production. However, annexin V-staining of peritoneal cells showed that there were more viable neutrophils in the peritoneal lavage of rhBCL2A1-treated animals following CLP (Fig. 5C), consistent with a reduction in apoptosis.


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 increases neutrophils in peritoneum following CLP.Mice were treated once by i.p. injection with 1 µg of rhBCL2A1 or rhBim at 18 hours prior to CLP. Peritoneal lavage fluid was collected at 24 hours following CLP. Panels A and B show the total number of leukocytes and neutrophils (PMNs) in lavage fluid. Mice treated with rhBCL2A1 showed an increase in peritoneal PMNs following CLP as compared to mice that did not receive CLP or mice that were treated with rhBim. Panel C shows the viability of peritoneal neutrophils at 24 hours following CLP as assessed by staining with annexin V and 7-amino-actinomycin D. Treatment with rhBCL2 significantly increased the number of viable neutrophils compared to treatment with saline or rhBCL2A1. *p<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3044724&req=5

pone-0014729-g005: Treatment with rhBCL2A1 increases neutrophils in peritoneum following CLP.Mice were treated once by i.p. injection with 1 µg of rhBCL2A1 or rhBim at 18 hours prior to CLP. Peritoneal lavage fluid was collected at 24 hours following CLP. Panels A and B show the total number of leukocytes and neutrophils (PMNs) in lavage fluid. Mice treated with rhBCL2A1 showed an increase in peritoneal PMNs following CLP as compared to mice that did not receive CLP or mice that were treated with rhBim. Panel C shows the viability of peritoneal neutrophils at 24 hours following CLP as assessed by staining with annexin V and 7-amino-actinomycin D. Treatment with rhBCL2 significantly increased the number of viable neutrophils compared to treatment with saline or rhBCL2A1. *p<0.01.
Mentions: In separate experiments, leukocyte populations in peritoneal lavage were evaluated. Mice received rhBim, rhBCL2A1, or no treatment followed 18 hours later by CLP. Additionally, some mice received either rhBim or rhBCL2A1, but did not have CLP. In those animals not subjected to CLP, there was no significant accumulation of total leukocytes or neutrophils in animals treated with rhBCL2A1 (Fig. 5 A, B). In contrast, in animals subjected to CLP there was a significant increase in the total number of leukocytes in the peritoneal lavage fluid in mice treated with rhBCL2A1 as compared to the other treatment groups (Fig. 5A). This increase in total cells was due specifically to an increase in neutrophils in the peritoneal lavage fluid (Fig. 5B). We did not observe any changes in circulating neutrophil counts, and G-CSF levels in peritoneal fluid and G-CSF mRNA expression in spleen (Fig. S1) were similar in the rhBCL2A1-treated mice compared to rhBim-treated mice following CLP, making it less likely that the increase in peritoneal neutrophils following CLP was due solely to increased production. However, annexin V-staining of peritoneal cells showed that there were more viable neutrophils in the peritoneal lavage of rhBCL2A1-treated animals following CLP (Fig. 5C), consistent with a reduction in apoptosis.

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