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Protein kinase Cepsilon is required for macrophage activation and defense against bacterial infection.

Castrillo A, Pennington DJ, Otto F, Parker PJ, Owen MJ, Boscá L - J. Exp. Med. (2001)

Bottom Line: Macrophages from PKCepsilon(-/-) animals demonstrated a severely attenuated response to lipopolysaccharide (LPS) and interferon (IFN)gamma, characterized by a dramatic reduction in the generation of NO, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta.This study provides direct evidence that PKCepsilon is critically involved at an early stage of LPS-mediated signaling in activated macrophages.Furthermore, we demonstrate that in the absence of PKCepsilon, host defense against bacterial infection is severely compromised, resulting in an increased incidence of mortality.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Cientificas-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain.

ABSTRACT
To assess directly the role of protein kinase C (PKC)epsilon in the immune system, we generated mice that carried a homozygous disruption of the PKCepsilon locus. PKCepsilon(-/-) animals appeared normal and were generally healthy, although female mice frequently developed a bacterial infection of the uterus. Macrophages from PKCepsilon(-/-) animals demonstrated a severely attenuated response to lipopolysaccharide (LPS) and interferon (IFN)gamma, characterized by a dramatic reduction in the generation of NO, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta. Further analysis revealed that LPS-stimulated macrophages from PKCepsilon(-/-) mice were deficient in the induction of nitric oxide synthase (NOS)-2, demonstrating a decrease in the activation of IkappaB kinase, a reduction in IkappaB degradation, and a decrease in nuclear factor (NF)kappaB nuclear translocation. After intravenous administration of Gram-negative or Gram-positive bacteria, PKCepsilon(-/-) mice demonstrated a significantly decreased period of survival. This study provides direct evidence that PKCepsilon is critically involved at an early stage of LPS-mediated signaling in activated macrophages. Furthermore, we demonstrate that in the absence of PKCepsilon, host defense against bacterial infection is severely compromised, resulting in an increased incidence of mortality.

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The release of proinflammatory cytokines and prostaglandin E2 are impaired in PKCε−/− mice. (A–C) Thioglycollate-elicited peritoneal macrophages were activated for the indicated times with 200 ng/ml of LPS and 20 U/ml of IFNγ. The amount of TNF-α, IL-1β, and PGE2 in the culture medium was then determined. (D–E) Bar graphs demonstrating the serum levels of TNF-α (1 h after injection) and IL-1β (4 h after injection) after the intraperitoneal injection of either 5 mg/kg or 40 mg/kg of LPS. (F) Apoptosis of thioglycollate-elicited peritoneal macrophages 24 h after activation by LPS (200 ng/ml), LPS plus IFNγ (200 ng/ml and 20 U/ml, respectively), or LPS, IFNγ, and 15dPGJ2 (2 μM). Results show the mean ± SEM of three experiments. The * and ** denote P < 0.05 and P < 0.01, respectively, for experimental vs. wild-type animals, for the parameters indicated.
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fig4: The release of proinflammatory cytokines and prostaglandin E2 are impaired in PKCε−/− mice. (A–C) Thioglycollate-elicited peritoneal macrophages were activated for the indicated times with 200 ng/ml of LPS and 20 U/ml of IFNγ. The amount of TNF-α, IL-1β, and PGE2 in the culture medium was then determined. (D–E) Bar graphs demonstrating the serum levels of TNF-α (1 h after injection) and IL-1β (4 h after injection) after the intraperitoneal injection of either 5 mg/kg or 40 mg/kg of LPS. (F) Apoptosis of thioglycollate-elicited peritoneal macrophages 24 h after activation by LPS (200 ng/ml), LPS plus IFNγ (200 ng/ml and 20 U/ml, respectively), or LPS, IFNγ, and 15dPGJ2 (2 μM). Results show the mean ± SEM of three experiments. The * and ** denote P < 0.05 and P < 0.01, respectively, for experimental vs. wild-type animals, for the parameters indicated.

Mentions: In addition to the synthesis of NO, activated macrophages are known to release a wide range of cytokines and other gene products that promote an inflammatory response and help to initiate adaptive immunity. The production of TNFα, IL-1β, and prostaglandin E2 (PGE2) were therefore analyzed after in vitro LPS and IFNγ stimulation of thioglycollate-elicited macrophages. Fig. 4, A–C , clearly demonstrates that macrophages from PKCε−/− mice generated severely attenuated levels of TNFα, IL-1β, and PGE2 compared with macrophages from wild-type animals. The release of these proinflammatory mediators was also assessed in vivo after the intraperitoneal administration of LPS after sensitization with D-galactosamine (17). Serum levels of TNFα and IL-1β were both significantly reduced in PKCε−/− mice when compared with the levels observed in littermate controls (Fig. 4, D and E).


Protein kinase Cepsilon is required for macrophage activation and defense against bacterial infection.

Castrillo A, Pennington DJ, Otto F, Parker PJ, Owen MJ, Boscá L - J. Exp. Med. (2001)

The release of proinflammatory cytokines and prostaglandin E2 are impaired in PKCε−/− mice. (A–C) Thioglycollate-elicited peritoneal macrophages were activated for the indicated times with 200 ng/ml of LPS and 20 U/ml of IFNγ. The amount of TNF-α, IL-1β, and PGE2 in the culture medium was then determined. (D–E) Bar graphs demonstrating the serum levels of TNF-α (1 h after injection) and IL-1β (4 h after injection) after the intraperitoneal injection of either 5 mg/kg or 40 mg/kg of LPS. (F) Apoptosis of thioglycollate-elicited peritoneal macrophages 24 h after activation by LPS (200 ng/ml), LPS plus IFNγ (200 ng/ml and 20 U/ml, respectively), or LPS, IFNγ, and 15dPGJ2 (2 μM). Results show the mean ± SEM of three experiments. The * and ** denote P < 0.05 and P < 0.01, respectively, for experimental vs. wild-type animals, for the parameters indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195971&req=5

fig4: The release of proinflammatory cytokines and prostaglandin E2 are impaired in PKCε−/− mice. (A–C) Thioglycollate-elicited peritoneal macrophages were activated for the indicated times with 200 ng/ml of LPS and 20 U/ml of IFNγ. The amount of TNF-α, IL-1β, and PGE2 in the culture medium was then determined. (D–E) Bar graphs demonstrating the serum levels of TNF-α (1 h after injection) and IL-1β (4 h after injection) after the intraperitoneal injection of either 5 mg/kg or 40 mg/kg of LPS. (F) Apoptosis of thioglycollate-elicited peritoneal macrophages 24 h after activation by LPS (200 ng/ml), LPS plus IFNγ (200 ng/ml and 20 U/ml, respectively), or LPS, IFNγ, and 15dPGJ2 (2 μM). Results show the mean ± SEM of three experiments. The * and ** denote P < 0.05 and P < 0.01, respectively, for experimental vs. wild-type animals, for the parameters indicated.
Mentions: In addition to the synthesis of NO, activated macrophages are known to release a wide range of cytokines and other gene products that promote an inflammatory response and help to initiate adaptive immunity. The production of TNFα, IL-1β, and prostaglandin E2 (PGE2) were therefore analyzed after in vitro LPS and IFNγ stimulation of thioglycollate-elicited macrophages. Fig. 4, A–C , clearly demonstrates that macrophages from PKCε−/− mice generated severely attenuated levels of TNFα, IL-1β, and PGE2 compared with macrophages from wild-type animals. The release of these proinflammatory mediators was also assessed in vivo after the intraperitoneal administration of LPS after sensitization with D-galactosamine (17). Serum levels of TNFα and IL-1β were both significantly reduced in PKCε−/− mice when compared with the levels observed in littermate controls (Fig. 4, D and E).

Bottom Line: Macrophages from PKCepsilon(-/-) animals demonstrated a severely attenuated response to lipopolysaccharide (LPS) and interferon (IFN)gamma, characterized by a dramatic reduction in the generation of NO, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta.This study provides direct evidence that PKCepsilon is critically involved at an early stage of LPS-mediated signaling in activated macrophages.Furthermore, we demonstrate that in the absence of PKCepsilon, host defense against bacterial infection is severely compromised, resulting in an increased incidence of mortality.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Cientificas-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain.

ABSTRACT
To assess directly the role of protein kinase C (PKC)epsilon in the immune system, we generated mice that carried a homozygous disruption of the PKCepsilon locus. PKCepsilon(-/-) animals appeared normal and were generally healthy, although female mice frequently developed a bacterial infection of the uterus. Macrophages from PKCepsilon(-/-) animals demonstrated a severely attenuated response to lipopolysaccharide (LPS) and interferon (IFN)gamma, characterized by a dramatic reduction in the generation of NO, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta. Further analysis revealed that LPS-stimulated macrophages from PKCepsilon(-/-) mice were deficient in the induction of nitric oxide synthase (NOS)-2, demonstrating a decrease in the activation of IkappaB kinase, a reduction in IkappaB degradation, and a decrease in nuclear factor (NF)kappaB nuclear translocation. After intravenous administration of Gram-negative or Gram-positive bacteria, PKCepsilon(-/-) mice demonstrated a significantly decreased period of survival. This study provides direct evidence that PKCepsilon is critically involved at an early stage of LPS-mediated signaling in activated macrophages. Furthermore, we demonstrate that in the absence of PKCepsilon, host defense against bacterial infection is severely compromised, resulting in an increased incidence of mortality.

Show MeSH
Related in: MedlinePlus