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The immune reaction against allogeneic necrotic cells is reduced in Annexin A5 knock out mice whose macrophages display an anti-inflammatory phenotype.

Frey B, Munoz LE, Pausch F, Sieber R, Franz S, Brachvogel B, Poschl E, Schneider H, Rödel F, Sauer R, Fietkau R, Herrmann M, Gaipl US - J. Cell. Mol. Med. (2008)

Bottom Line: We found that Anx A5 KO mice have a strongly reduced allogeneic cellular immune reaction against primary as well as secondary necrotic cells.The tumour size of an allogeneic tumour regressed faster when endogenous Anx A5 was present.These data demonstrate that endogenous Anx A5 influences the phagocytosis of necrotic cells, modulates the immune response towards allogeneic cells and acts as an inflammatory protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen-Nürnberg, Germany.

ABSTRACT
Proteins of the annexin family bind to phospholipids in a Ca(2+) dependent manner. The exposure of phosphatidylserine (PS) by apoptotic as well as necrotic cells is one major eat-me-signal for macrophages. Annexin A5 (Anx A5) preferentially binds to PS. The availability of Anx A5 knock out (KO) mice allowed us to investigate for the first time if endogenous Anx A5 modulates the immune response towards allogeneic cells. Furthermore, the effect of Anx A5 gene deletion on the phagocytic process as well as on the inflammatory reaction of macrophages was explored. We found that Anx A5 KO mice have a strongly reduced allogeneic cellular immune reaction against primary as well as secondary necrotic cells. In vivo phagocytosis experiments revealed that macrophages of Anx A5 KO mice displayed an increased uptake of necrotic cells. Additionally, an increased secretion of the anti-inflammatory cytokine IL-10 of isolated macrophages of Anx A5 KO mice after contact with necrotic cells was observed. Furthermore, the promoter activity of the Anx A5 gene was enhanced after stimulation of macrophages. The tumour size of an allogeneic tumour regressed faster when endogenous Anx A5 was present. These data demonstrate that endogenous Anx A5 influences the phagocytosis of necrotic cells, modulates the immune response towards allogeneic cells and acts as an inflammatory protein.

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Anx A5 promoter activity of activated macrophages. Macrophages were obtainedfrom the peritoneal lavage of Anx A5 KO mice. Following, the macrophages werecultured for 24 hrs in medium without (w/o) or with LPS. The peritonealmacrophages were stained with 5-bromo-4-chloro-3-indoxyl beta-D-galactoside(X-Gal) for b-galactosidase activity (A), which represents the promoteractivity of the Anx A5 gene. The X-Gal staining of macrophages (deep blackareas) clearly demonstrates the expression of Anx A5 within the macrophagepopulation (arrows). The b-galactosidase activity was also quantified bystaining the Anx A5 knock out macrophages with fluorescein di-β-D-galactopyranoside (FDG) reagent. F4/80 positive macrophages displayed a4.3-fold higher ß-Gal activity after stimulation with LPS. One out ofthree representative set of experiments is shown. KO: knock out; WT: wild type;LPS: lipopolysac-charide
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fig04: Anx A5 promoter activity of activated macrophages. Macrophages were obtainedfrom the peritoneal lavage of Anx A5 KO mice. Following, the macrophages werecultured for 24 hrs in medium without (w/o) or with LPS. The peritonealmacrophages were stained with 5-bromo-4-chloro-3-indoxyl beta-D-galactoside(X-Gal) for b-galactosidase activity (A), which represents the promoteractivity of the Anx A5 gene. The X-Gal staining of macrophages (deep blackareas) clearly demonstrates the expression of Anx A5 within the macrophagepopulation (arrows). The b-galactosidase activity was also quantified bystaining the Anx A5 knock out macrophages with fluorescein di-β-D-galactopyranoside (FDG) reagent. F4/80 positive macrophages displayed a4.3-fold higher ß-Gal activity after stimulation with LPS. One out ofthree representative set of experiments is shown. KO: knock out; WT: wild type;LPS: lipopolysac-charide

Mentions: Fluorescein di-β-D-galactopyranoside (FDG; Molecular Probes, Invitrogen) canbe used to detect the transient expression of b-galactosidase constructs. Peritonealmacrophages were stained with F4/80-PE or CD11b-PE and additional with FDG asfollows: 5 × 105 cells of the F4/80 or CD11b stained cells werere-suspended in 10 μl PBS/FCS. The FDG stock solution was diluted to 2 mM inH2O. 10 μl of this FDG dilution was added to 10 μl of thecell suspension. After vortexing, the cells were incubated for 75 seconds at37°C. Immediately afterwards, 125 μl of ice cold PBS/FCS was added andthe suspension incubated for 2.0 hrs on ice in the dark. FDG positive macrophagescould then easily be quantified by flow cytometry (see Fig. 4).


The immune reaction against allogeneic necrotic cells is reduced in Annexin A5 knock out mice whose macrophages display an anti-inflammatory phenotype.

Frey B, Munoz LE, Pausch F, Sieber R, Franz S, Brachvogel B, Poschl E, Schneider H, Rödel F, Sauer R, Fietkau R, Herrmann M, Gaipl US - J. Cell. Mol. Med. (2008)

Anx A5 promoter activity of activated macrophages. Macrophages were obtainedfrom the peritoneal lavage of Anx A5 KO mice. Following, the macrophages werecultured for 24 hrs in medium without (w/o) or with LPS. The peritonealmacrophages were stained with 5-bromo-4-chloro-3-indoxyl beta-D-galactoside(X-Gal) for b-galactosidase activity (A), which represents the promoteractivity of the Anx A5 gene. The X-Gal staining of macrophages (deep blackareas) clearly demonstrates the expression of Anx A5 within the macrophagepopulation (arrows). The b-galactosidase activity was also quantified bystaining the Anx A5 knock out macrophages with fluorescein di-β-D-galactopyranoside (FDG) reagent. F4/80 positive macrophages displayed a4.3-fold higher ß-Gal activity after stimulation with LPS. One out ofthree representative set of experiments is shown. KO: knock out; WT: wild type;LPS: lipopolysac-charide
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496152&req=5

fig04: Anx A5 promoter activity of activated macrophages. Macrophages were obtainedfrom the peritoneal lavage of Anx A5 KO mice. Following, the macrophages werecultured for 24 hrs in medium without (w/o) or with LPS. The peritonealmacrophages were stained with 5-bromo-4-chloro-3-indoxyl beta-D-galactoside(X-Gal) for b-galactosidase activity (A), which represents the promoteractivity of the Anx A5 gene. The X-Gal staining of macrophages (deep blackareas) clearly demonstrates the expression of Anx A5 within the macrophagepopulation (arrows). The b-galactosidase activity was also quantified bystaining the Anx A5 knock out macrophages with fluorescein di-β-D-galactopyranoside (FDG) reagent. F4/80 positive macrophages displayed a4.3-fold higher ß-Gal activity after stimulation with LPS. One out ofthree representative set of experiments is shown. KO: knock out; WT: wild type;LPS: lipopolysac-charide
Mentions: Fluorescein di-β-D-galactopyranoside (FDG; Molecular Probes, Invitrogen) canbe used to detect the transient expression of b-galactosidase constructs. Peritonealmacrophages were stained with F4/80-PE or CD11b-PE and additional with FDG asfollows: 5 × 105 cells of the F4/80 or CD11b stained cells werere-suspended in 10 μl PBS/FCS. The FDG stock solution was diluted to 2 mM inH2O. 10 μl of this FDG dilution was added to 10 μl of thecell suspension. After vortexing, the cells were incubated for 75 seconds at37°C. Immediately afterwards, 125 μl of ice cold PBS/FCS was added andthe suspension incubated for 2.0 hrs on ice in the dark. FDG positive macrophagescould then easily be quantified by flow cytometry (see Fig. 4).

Bottom Line: We found that Anx A5 KO mice have a strongly reduced allogeneic cellular immune reaction against primary as well as secondary necrotic cells.The tumour size of an allogeneic tumour regressed faster when endogenous Anx A5 was present.These data demonstrate that endogenous Anx A5 influences the phagocytosis of necrotic cells, modulates the immune response towards allogeneic cells and acts as an inflammatory protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen-Nürnberg, Germany.

ABSTRACT
Proteins of the annexin family bind to phospholipids in a Ca(2+) dependent manner. The exposure of phosphatidylserine (PS) by apoptotic as well as necrotic cells is one major eat-me-signal for macrophages. Annexin A5 (Anx A5) preferentially binds to PS. The availability of Anx A5 knock out (KO) mice allowed us to investigate for the first time if endogenous Anx A5 modulates the immune response towards allogeneic cells. Furthermore, the effect of Anx A5 gene deletion on the phagocytic process as well as on the inflammatory reaction of macrophages was explored. We found that Anx A5 KO mice have a strongly reduced allogeneic cellular immune reaction against primary as well as secondary necrotic cells. In vivo phagocytosis experiments revealed that macrophages of Anx A5 KO mice displayed an increased uptake of necrotic cells. Additionally, an increased secretion of the anti-inflammatory cytokine IL-10 of isolated macrophages of Anx A5 KO mice after contact with necrotic cells was observed. Furthermore, the promoter activity of the Anx A5 gene was enhanced after stimulation of macrophages. The tumour size of an allogeneic tumour regressed faster when endogenous Anx A5 was present. These data demonstrate that endogenous Anx A5 influences the phagocytosis of necrotic cells, modulates the immune response towards allogeneic cells and acts as an inflammatory protein.

Show MeSH
Related in: MedlinePlus