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A nanoconjugate Apaf-1 inhibitor protects mesothelial cells from cytokine-induced injury.

Santamaría B, Benito-Martin A, Ucero AC, Aroeira LS, Reyero A, Vicent MJ, Orzáez M, Celdrán A, Esteban J, Selgas R, Ruíz-Ortega M, Cabrera ML, Egido J, Pérez-Payá E, Ortiz A - PLoS ONE (2009)

Bottom Line: Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells.Specific caspase-8 and caspase-3 inhibitors were similarly effective.Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration.

View Article: PubMed Central - PubMed

Affiliation: Dialysis Unit, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Instituto Reina Sofía de Investigación Nefrológica, Madrid, Spain.

ABSTRACT

Background: Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization.

Methodology: Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma. Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice.

Conclusion: Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.

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Cooperation of cytokines induces apoptosis in mesothelial cells.A) Representative flow cytometry of DNA content diagrams. Note the increase in the hypodiploid, apoptotic population in HPMC cultured for 48 h in presence of TNFα/IFNγ. B) Quantification of apoptosis in HPMC by flow cytometry. The combination of TNFα/IFNγ induces apoptosis. When not specified, 300 U/mL IFNγ and 250 ng/mL TNFα were used, * p<0.04 vs each individual cytokine at 24 h. ** p<0.001 vs each individual cytokine at 48 h. & p<0.001 vs control. Mean±SEM of 6 different experiments. C) Dose-response in HPMC. Mean±SEM of 4 different experiments * p<0.05 vs control. D) Quantification of apoptosis in HOMC by flow cytometry. The combination TNFα/IFNγ for 48 h induces apoptosis. *p<0.005 vs. control. Mean±SEM of 4 different experiments.
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pone-0006634-g001: Cooperation of cytokines induces apoptosis in mesothelial cells.A) Representative flow cytometry of DNA content diagrams. Note the increase in the hypodiploid, apoptotic population in HPMC cultured for 48 h in presence of TNFα/IFNγ. B) Quantification of apoptosis in HPMC by flow cytometry. The combination of TNFα/IFNγ induces apoptosis. When not specified, 300 U/mL IFNγ and 250 ng/mL TNFα were used, * p<0.04 vs each individual cytokine at 24 h. ** p<0.001 vs each individual cytokine at 48 h. & p<0.001 vs control. Mean±SEM of 6 different experiments. C) Dose-response in HPMC. Mean±SEM of 4 different experiments * p<0.05 vs control. D) Quantification of apoptosis in HOMC by flow cytometry. The combination TNFα/IFNγ for 48 h induces apoptosis. *p<0.005 vs. control. Mean±SEM of 4 different experiments.

Mentions: Acute demesothelization occurs during experimental and human peritonitis, and is associated with an increased rate of mesothelial cell apoptosis [3]–[6]. During peritoneal inflammation numerous cytokines are released locally. Among them we find IFNγ and TNFα [33], [34]. Although TNFα is, by itself, a potentially lethal cytokine [19], we did not observe a lethal effect of TNFα in human peritoneal mesothelial cells (HPMC) cultured from the effluent of stable PD patients (Fig. 1. A, B). IFNγ increased TNFα lethality (Fig. 1.A, B) [6]. The combination of cytokines induced HPMC apoptosis in a time- and dose-dependent manner (Fig. 1.B, C). By contrast, cytokines had no effect on the percentage of cells on the S or M phase of the cell cycle, as assessed by flow cytometry of DNA content (range 9 to 10% for the different conditions). The combination of IFNγ and TNFα also induced apoptosis in human omental mesothelial cells (HOMC) cultured from the omentum of non-uremic patients (Fig. 1.D).


A nanoconjugate Apaf-1 inhibitor protects mesothelial cells from cytokine-induced injury.

Santamaría B, Benito-Martin A, Ucero AC, Aroeira LS, Reyero A, Vicent MJ, Orzáez M, Celdrán A, Esteban J, Selgas R, Ruíz-Ortega M, Cabrera ML, Egido J, Pérez-Payá E, Ortiz A - PLoS ONE (2009)

Cooperation of cytokines induces apoptosis in mesothelial cells.A) Representative flow cytometry of DNA content diagrams. Note the increase in the hypodiploid, apoptotic population in HPMC cultured for 48 h in presence of TNFα/IFNγ. B) Quantification of apoptosis in HPMC by flow cytometry. The combination of TNFα/IFNγ induces apoptosis. When not specified, 300 U/mL IFNγ and 250 ng/mL TNFα were used, * p<0.04 vs each individual cytokine at 24 h. ** p<0.001 vs each individual cytokine at 48 h. & p<0.001 vs control. Mean±SEM of 6 different experiments. C) Dose-response in HPMC. Mean±SEM of 4 different experiments * p<0.05 vs control. D) Quantification of apoptosis in HOMC by flow cytometry. The combination TNFα/IFNγ for 48 h induces apoptosis. *p<0.005 vs. control. Mean±SEM of 4 different experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2722088&req=5

pone-0006634-g001: Cooperation of cytokines induces apoptosis in mesothelial cells.A) Representative flow cytometry of DNA content diagrams. Note the increase in the hypodiploid, apoptotic population in HPMC cultured for 48 h in presence of TNFα/IFNγ. B) Quantification of apoptosis in HPMC by flow cytometry. The combination of TNFα/IFNγ induces apoptosis. When not specified, 300 U/mL IFNγ and 250 ng/mL TNFα were used, * p<0.04 vs each individual cytokine at 24 h. ** p<0.001 vs each individual cytokine at 48 h. & p<0.001 vs control. Mean±SEM of 6 different experiments. C) Dose-response in HPMC. Mean±SEM of 4 different experiments * p<0.05 vs control. D) Quantification of apoptosis in HOMC by flow cytometry. The combination TNFα/IFNγ for 48 h induces apoptosis. *p<0.005 vs. control. Mean±SEM of 4 different experiments.
Mentions: Acute demesothelization occurs during experimental and human peritonitis, and is associated with an increased rate of mesothelial cell apoptosis [3]–[6]. During peritoneal inflammation numerous cytokines are released locally. Among them we find IFNγ and TNFα [33], [34]. Although TNFα is, by itself, a potentially lethal cytokine [19], we did not observe a lethal effect of TNFα in human peritoneal mesothelial cells (HPMC) cultured from the effluent of stable PD patients (Fig. 1. A, B). IFNγ increased TNFα lethality (Fig. 1.A, B) [6]. The combination of cytokines induced HPMC apoptosis in a time- and dose-dependent manner (Fig. 1.B, C). By contrast, cytokines had no effect on the percentage of cells on the S or M phase of the cell cycle, as assessed by flow cytometry of DNA content (range 9 to 10% for the different conditions). The combination of IFNγ and TNFα also induced apoptosis in human omental mesothelial cells (HOMC) cultured from the omentum of non-uremic patients (Fig. 1.D).

Bottom Line: Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells.Specific caspase-8 and caspase-3 inhibitors were similarly effective.Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration.

View Article: PubMed Central - PubMed

Affiliation: Dialysis Unit, Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Instituto Reina Sofía de Investigación Nefrológica, Madrid, Spain.

ABSTRACT

Background: Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization.

Methodology: Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma. Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice.

Conclusion: Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.

Show MeSH
Related in: MedlinePlus