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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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Macrophages from PKCε−/− mice fail to fully induce NOS-2 expression after LPS activation. (A) Western blot for NOS-2 levels in thioglycollate-elicited peritoneal macrophages after 18 or 24 h activation with LPS (200 ng/ml) and IFNγ (20 U/ml). (B) Western blot showing NOS-2 expression in peritoneal macrophages 24 h after an intraperitoneal in vivo challenge with LPS. (C) NOS-2 mRNA levels in macrophages after 4 and 6 h activation with LPS and IFNγ. Loading was normalized by using a probe for 18S ribosomal RNA as a control.
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fig3: Macrophages from PKCε−/− mice fail to fully induce NOS-2 expression after LPS activation. (A) Western blot for NOS-2 levels in thioglycollate-elicited peritoneal macrophages after 18 or 24 h activation with LPS (200 ng/ml) and IFNγ (20 U/ml). (B) Western blot showing NOS-2 expression in peritoneal macrophages 24 h after an intraperitoneal in vivo challenge with LPS. (C) NOS-2 mRNA levels in macrophages after 4 and 6 h activation with LPS and IFNγ. Loading was normalized by using a probe for 18S ribosomal RNA as a control.

Mentions: As macrophages are known to generate NO via the upregulation of the expression of NOS-2, the protein level of NOS-2 was assessed in vitro after the activation of thioglycollate-elicited macrophages by LPS and IFNγ. Fig. 3 A demonstrates that cultured macrophages from PKCε−/− mice displayed a severely reduced level of NOS-2 protein expression after both 18 and 24 h of stimulation when compared with wild-type cells. To verify this decrease in LPS-induced NOS-2 expression, LPS was administered in vivo via an intraperitoneal injection. After 24 h, macrophages were isolated from the peritoneal cavity and again assayed for NOS-2 protein expression. As shown in Fig. 3 B, the extent of induction of NOS-2 protein in response to LPS is severely attenuated in macrophages from PKCε-deficient animals. As NOS-2 activity is mainly controlled at the level of transcription (23), the in vitro expression of NOS-2 RNA in response to LPS plus IFNγ challenge was determined. Fig. 3 C demonstrates that after such a challenge NOS-2 RNA levels were significantly decreased in macrophages from PKCε−/− mice when compared with controls. These data suggest that the reduction in LPS-induced NO generation in macrophages from PKCε−/− mice is a result of the failure to upregulate the expression of both NOS-2 RNA and protein.


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)

Macrophages from PKCε−/− mice fail to fully induce NOS-2 expression after LPS activation. (A) Western blot for NOS-2 levels in thioglycollate-elicited peritoneal macrophages after 18 or 24 h activation with LPS (200 ng/ml) and IFNγ (20 U/ml). (B) Western blot showing NOS-2 expression in peritoneal macrophages 24 h after an intraperitoneal in vivo challenge with LPS. (C) NOS-2 mRNA levels in macrophages after 4 and 6 h activation with LPS and IFNγ. Loading was normalized by using a probe for 18S ribosomal RNA as a control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195971&req=5

fig3: Macrophages from PKCε−/− mice fail to fully induce NOS-2 expression after LPS activation. (A) Western blot for NOS-2 levels in thioglycollate-elicited peritoneal macrophages after 18 or 24 h activation with LPS (200 ng/ml) and IFNγ (20 U/ml). (B) Western blot showing NOS-2 expression in peritoneal macrophages 24 h after an intraperitoneal in vivo challenge with LPS. (C) NOS-2 mRNA levels in macrophages after 4 and 6 h activation with LPS and IFNγ. Loading was normalized by using a probe for 18S ribosomal RNA as a control.
Mentions: As macrophages are known to generate NO via the upregulation of the expression of NOS-2, the protein level of NOS-2 was assessed in vitro after the activation of thioglycollate-elicited macrophages by LPS and IFNγ. Fig. 3 A demonstrates that cultured macrophages from PKCε−/− mice displayed a severely reduced level of NOS-2 protein expression after both 18 and 24 h of stimulation when compared with wild-type cells. To verify this decrease in LPS-induced NOS-2 expression, LPS was administered in vivo via an intraperitoneal injection. After 24 h, macrophages were isolated from the peritoneal cavity and again assayed for NOS-2 protein expression. As shown in Fig. 3 B, the extent of induction of NOS-2 protein in response to LPS is severely attenuated in macrophages from PKCε-deficient animals. As NOS-2 activity is mainly controlled at the level of transcription (23), the in vitro expression of NOS-2 RNA in response to LPS plus IFNγ challenge was determined. Fig. 3 C demonstrates that after such a challenge NOS-2 RNA levels were significantly decreased in macrophages from PKCε−/− mice when compared with controls. These data suggest that the reduction in LPS-induced NO generation in macrophages from PKCε−/− mice is a result of the failure to upregulate the expression of both NOS-2 RNA and protein.

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