Limits...
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.

Show MeSH

Related in: MedlinePlus

Macrophages from PKCε−/− mice generate less NO in response to LPS challenge. (A) Graphical representation of the percentage of thioglycollate-elicited peritoneal macrophages that expressed cell-surface CD11b and CD14. (B) Graph showing the release of nitrite into the culture medium after the activation of thioglycollate-elicited peritoneal macrophages with increasing concentrations of LPS for 24 h. (C) Graphical view of nitrate release into the culture medium after the activation of thioglycollate-elicited peritoneal macrophages with a fixed concentration of both LPS (200 ng/ml) and IFNγ (20 U/ml) for increasing periods of time. Results show the mean ± SEM of three experiments. The * denotes P < 0.05 for experimental vs. wild-type animals for the parameters indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195971&req=5

fig2: Macrophages from PKCε−/− mice generate less NO in response to LPS challenge. (A) Graphical representation of the percentage of thioglycollate-elicited peritoneal macrophages that expressed cell-surface CD11b and CD14. (B) Graph showing the release of nitrite into the culture medium after the activation of thioglycollate-elicited peritoneal macrophages with increasing concentrations of LPS for 24 h. (C) Graphical view of nitrate release into the culture medium after the activation of thioglycollate-elicited peritoneal macrophages with a fixed concentration of both LPS (200 ng/ml) and IFNγ (20 U/ml) for increasing periods of time. Results show the mean ± SEM of three experiments. The * denotes P < 0.05 for experimental vs. wild-type animals for the parameters indicated.

Mentions: As PKCε−/− mice were succumbing to E. coli–related bacterial infection, it was hypothesized that their response to Gram-negative bacteria was somehow compromised. To test this, macrophages were elicited via intraperitoneal thioglycollate injection and their response to in vitro administration of LPS was assessed measuring nitrite generation as a functional end point. The number of macrophages obtained from PKCε−/− mice were comparable to littermate controls, and the percentage of cells expressing cell-surface levels of both CD14, an integral component of the LPS receptor, and CD11b was similar (Fig. 2 A). At lower doses of LPS, below 200 ng/ml, macrophages from PKCε−/− mice generated significantly lower amounts of NO than macrophages from control animals (Fig. 2 B). However, at substantially higher concentrations of LPS, above 500 ng/ml, a comparable production of NO was detected from both groups. Macrophages can be activated to a greater degree if IFNγ is allowed to act synergistically with LPS. Using a set concentration of both LPS and IFNγ, the generation of NO over a 36-h period was also demonstrated to be significantly reduced in macrophages from PKCε−/− mice when compared with macrophages from wild-type controls (Fig. 2 C). It should be noted that IFNγ treatment alone can result in the generation of NO from macrophages, although this response is lower and delayed with respect to the response observed with LPS (16). In PKCε−/− animals the generation of NO after in vitro IFNγ treatment of elicited macrophages was also reduced in comparison to wild-type littermates (data not shown). The basis of this reduced response to IFNγ in the PKCε−/− mice is presently unclear. However, studies are underway to assess the extent of this defect and to characterize the cellular components involved.


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 generate less NO in response to LPS challenge. (A) Graphical representation of the percentage of thioglycollate-elicited peritoneal macrophages that expressed cell-surface CD11b and CD14. (B) Graph showing the release of nitrite into the culture medium after the activation of thioglycollate-elicited peritoneal macrophages with increasing concentrations of LPS for 24 h. (C) Graphical view of nitrate release into the culture medium after the activation of thioglycollate-elicited peritoneal macrophages with a fixed concentration of both LPS (200 ng/ml) and IFNγ (20 U/ml) for increasing periods of time. Results show the mean ± SEM of three experiments. The * denotes P < 0.05 for experimental vs. wild-type animals for the parameters indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195971&req=5

fig2: Macrophages from PKCε−/− mice generate less NO in response to LPS challenge. (A) Graphical representation of the percentage of thioglycollate-elicited peritoneal macrophages that expressed cell-surface CD11b and CD14. (B) Graph showing the release of nitrite into the culture medium after the activation of thioglycollate-elicited peritoneal macrophages with increasing concentrations of LPS for 24 h. (C) Graphical view of nitrate release into the culture medium after the activation of thioglycollate-elicited peritoneal macrophages with a fixed concentration of both LPS (200 ng/ml) and IFNγ (20 U/ml) for increasing periods of time. Results show the mean ± SEM of three experiments. The * denotes P < 0.05 for experimental vs. wild-type animals for the parameters indicated.
Mentions: As PKCε−/− mice were succumbing to E. coli–related bacterial infection, it was hypothesized that their response to Gram-negative bacteria was somehow compromised. To test this, macrophages were elicited via intraperitoneal thioglycollate injection and their response to in vitro administration of LPS was assessed measuring nitrite generation as a functional end point. The number of macrophages obtained from PKCε−/− mice were comparable to littermate controls, and the percentage of cells expressing cell-surface levels of both CD14, an integral component of the LPS receptor, and CD11b was similar (Fig. 2 A). At lower doses of LPS, below 200 ng/ml, macrophages from PKCε−/− mice generated significantly lower amounts of NO than macrophages from control animals (Fig. 2 B). However, at substantially higher concentrations of LPS, above 500 ng/ml, a comparable production of NO was detected from both groups. Macrophages can be activated to a greater degree if IFNγ is allowed to act synergistically with LPS. Using a set concentration of both LPS and IFNγ, the generation of NO over a 36-h period was also demonstrated to be significantly reduced in macrophages from PKCε−/− mice when compared with macrophages from wild-type controls (Fig. 2 C). It should be noted that IFNγ treatment alone can result in the generation of NO from macrophages, although this response is lower and delayed with respect to the response observed with LPS (16). In PKCε−/− animals the generation of NO after in vitro IFNγ treatment of elicited macrophages was also reduced in comparison to wild-type littermates (data not shown). The basis of this reduced response to IFNγ in the PKCε−/− mice is presently unclear. However, studies are underway to assess the extent of this defect and to characterize the cellular components involved.

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