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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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LPS-mediated signaling events are disrupted in macrophages from PKCε−/− mice. (A and B) Thioglycollate-elicited peritoneal macrophages were activated with 200 ng/ml of LPS, and at the indicated times cytosolic and nuclear protein extracts were prepared. The levels of phosphorylated and total p42 ERK and p44 ERK (A), and p38 MAP kinase (B) were determined by Western blot. (C) The effect of p42 and p44 ERK and p38 MAP kinase on the expression of NOS-2 was determined measuring the release of nitrite to the culture medium (24 h) in cells incubated with 50 μM PD 98059, an inhibitor of ERK, and 20 μM of SB203580 and SB20190, inhibitors of p38 MAP kinase. Results show the mean ± SEM of three experiments. The * denotes P < 0.05 with respect to the LPS (200 ng/ml) and IFNγ (20 U/ml) responses in control vs. experimental animals.
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fig6: LPS-mediated signaling events are disrupted in macrophages from PKCε−/− mice. (A and B) Thioglycollate-elicited peritoneal macrophages were activated with 200 ng/ml of LPS, and at the indicated times cytosolic and nuclear protein extracts were prepared. The levels of phosphorylated and total p42 ERK and p44 ERK (A), and p38 MAP kinase (B) were determined by Western blot. (C) The effect of p42 and p44 ERK and p38 MAP kinase on the expression of NOS-2 was determined measuring the release of nitrite to the culture medium (24 h) in cells incubated with 50 μM PD 98059, an inhibitor of ERK, and 20 μM of SB203580 and SB20190, inhibitors of p38 MAP kinase. Results show the mean ± SEM of three experiments. The * denotes P < 0.05 with respect to the LPS (200 ng/ml) and IFNγ (20 U/ml) responses in control vs. experimental animals.

Mentions: Previous work has implicated several signaling pathways in the upregulation of expression of genes such as NOS-2, TNFα, and IL-1β after the LPS stimulation of macrophages. Two such pathways are those involving the ERK1/2 MAP kinases and the family of p38 MAP kinases. The activation of these kinases was therefore assessed by detection of the phosphorylated forms of p44ERK, p42ERK, and p38 in LPS-stimulated macrophage cytosolic extracts. The amount of phosphorylated p44, p42, and p38 is clearly reduced in extracts from LPS-stimulated macrophages from PKCε−/− mice, particularly at early time points after stimulation (Fig. 6, A and B) . The relevance of this reduction in LPS-induced p44/p42ERK and p38 activation with respect to NO synthesis was evaluated. Whereas PD 98059, an inhibitor of ERK, was unable to modify NO synthesis in response to LPS in macrophages from wild-type animals, SB 203580 and SB 202190, two inhibitors of p38 MAPK, significantly inhibited NO synthesis in activated macrophages from PKCε+/+ mice (Fig. 6 C), confirming previous data (26). However, neither SB 203580 nor SB 202190 affected the already lowered synthesis of NO in PKCε−/− mice (Fig. 6 C). These results suggest that although both the ERK and p38 MAP kinase signaling cascades are attenuated in PKCε−/− mice in response to LPS, only the p38 MAP kinase pathway is directly involved in NO production in a PKCε-dependent manner.


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)

LPS-mediated signaling events are disrupted in macrophages from PKCε−/− mice. (A and B) Thioglycollate-elicited peritoneal macrophages were activated with 200 ng/ml of LPS, and at the indicated times cytosolic and nuclear protein extracts were prepared. The levels of phosphorylated and total p42 ERK and p44 ERK (A), and p38 MAP kinase (B) were determined by Western blot. (C) The effect of p42 and p44 ERK and p38 MAP kinase on the expression of NOS-2 was determined measuring the release of nitrite to the culture medium (24 h) in cells incubated with 50 μM PD 98059, an inhibitor of ERK, and 20 μM of SB203580 and SB20190, inhibitors of p38 MAP kinase. Results show the mean ± SEM of three experiments. The * denotes P < 0.05 with respect to the LPS (200 ng/ml) and IFNγ (20 U/ml) responses in control vs. experimental animals.
© Copyright Policy
Related In: Results  -  Collection

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fig6: LPS-mediated signaling events are disrupted in macrophages from PKCε−/− mice. (A and B) Thioglycollate-elicited peritoneal macrophages were activated with 200 ng/ml of LPS, and at the indicated times cytosolic and nuclear protein extracts were prepared. The levels of phosphorylated and total p42 ERK and p44 ERK (A), and p38 MAP kinase (B) were determined by Western blot. (C) The effect of p42 and p44 ERK and p38 MAP kinase on the expression of NOS-2 was determined measuring the release of nitrite to the culture medium (24 h) in cells incubated with 50 μM PD 98059, an inhibitor of ERK, and 20 μM of SB203580 and SB20190, inhibitors of p38 MAP kinase. Results show the mean ± SEM of three experiments. The * denotes P < 0.05 with respect to the LPS (200 ng/ml) and IFNγ (20 U/ml) responses in control vs. experimental animals.
Mentions: Previous work has implicated several signaling pathways in the upregulation of expression of genes such as NOS-2, TNFα, and IL-1β after the LPS stimulation of macrophages. Two such pathways are those involving the ERK1/2 MAP kinases and the family of p38 MAP kinases. The activation of these kinases was therefore assessed by detection of the phosphorylated forms of p44ERK, p42ERK, and p38 in LPS-stimulated macrophage cytosolic extracts. The amount of phosphorylated p44, p42, and p38 is clearly reduced in extracts from LPS-stimulated macrophages from PKCε−/− mice, particularly at early time points after stimulation (Fig. 6, A and B) . The relevance of this reduction in LPS-induced p44/p42ERK and p38 activation with respect to NO synthesis was evaluated. Whereas PD 98059, an inhibitor of ERK, was unable to modify NO synthesis in response to LPS in macrophages from wild-type animals, SB 203580 and SB 202190, two inhibitors of p38 MAPK, significantly inhibited NO synthesis in activated macrophages from PKCε+/+ mice (Fig. 6 C), confirming previous data (26). However, neither SB 203580 nor SB 202190 affected the already lowered synthesis of NO in PKCε−/− mice (Fig. 6 C). These results suggest that although both the ERK and p38 MAP kinase signaling cascades are attenuated in PKCε−/− mice in response to LPS, only the p38 MAP kinase pathway is directly involved in NO production in a PKCε-dependent manner.

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