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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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Uptake by macrophages of necrotic cells in presence or absence of endogenousAnx A5. Macrophages in the peritoneum of the mice had contact withCFSE-labelled heat-induced (30 min., 56°C) primary necrotic WEHI 231cells for 0.75 (A, B) or 3.5 hrs (C, D). Following, a peritoneal lavage wasperformed and the phagocytosis by macrophages, which were identified with F4/80staining, of the necrotic prey was determined. In (A) and (C), representativedot plots of the analyses of the phagocytosis by flow cytometry are shown.Macrophages of Anx A5 KO mice showed a significantly enhanced uptake of primarynecrotic cells after 0.75 hr (B) as well as after 3.5 hrs (D). Note: Thephagocytic ability of peritoneal macrophages of Anx A5 KO mice to take upmultiple necrotic cells per phagocyte (high positive macrophages) was highlysignificantly enhanced when compared with macrophages of WT animals (D, rightgraph). KO: knock out; WT: wild type; Mph: macrophages; Gates R3,R2:%CFSE positive Mph; Gate R7:% CFSE high positive Mph; One outof two representative set of experiments is shown. *P< 0.05; **P < 0.01
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fig02: Uptake by macrophages of necrotic cells in presence or absence of endogenousAnx A5. Macrophages in the peritoneum of the mice had contact withCFSE-labelled heat-induced (30 min., 56°C) primary necrotic WEHI 231cells for 0.75 (A, B) or 3.5 hrs (C, D). Following, a peritoneal lavage wasperformed and the phagocytosis by macrophages, which were identified with F4/80staining, of the necrotic prey was determined. In (A) and (C), representativedot plots of the analyses of the phagocytosis by flow cytometry are shown.Macrophages of Anx A5 KO mice showed a significantly enhanced uptake of primarynecrotic cells after 0.75 hr (B) as well as after 3.5 hrs (D). Note: Thephagocytic ability of peritoneal macrophages of Anx A5 KO mice to take upmultiple necrotic cells per phagocyte (high positive macrophages) was highlysignificantly enhanced when compared with macrophages of WT animals (D, rightgraph). KO: knock out; WT: wild type; Mph: macrophages; Gates R3,R2:%CFSE positive Mph; Gate R7:% CFSE high positive Mph; One outof two representative set of experiments is shown. *P< 0.05; **P < 0.01

Mentions: Animals were injected i.p. with CFSE-labelled heat-induced primary necrotic cellsallowing resident macrophages to interact with the dead cells for up to 3.5 hrs. Themice were killed after 0.75 hr or 3.5 hrs and the uptake of the fluorescent necroticcells in F4/80 positive macrophages, present in the peritoneal lavage, was monitoredby flow cytometry. F4/80 positive peritoneal macrophages of Anx A5 KO micephagozytosed significantly more primary necrotic cells in comparison to macrophagesof WT mice (Fig. 2). This was to be observedat 0.75 hr (Fig. 2A and B) as well as at 3.5 hrs (Fig.2C and D) after injection of thenecrotic cells. The uptake of multiple necrotic cells per phagocyte (high positivemacrophages) was highly significantly enhanced in Anx A5 KO mice when compared withAnx A5 WT animals (Fig. 2D, right graph).Similar effects were observed by using mechanical stress-induced primary necroticcells for the phagocytosis assays (suppl. 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)

Uptake by macrophages of necrotic cells in presence or absence of endogenousAnx A5. Macrophages in the peritoneum of the mice had contact withCFSE-labelled heat-induced (30 min., 56°C) primary necrotic WEHI 231cells for 0.75 (A, B) or 3.5 hrs (C, D). Following, a peritoneal lavage wasperformed and the phagocytosis by macrophages, which were identified with F4/80staining, of the necrotic prey was determined. In (A) and (C), representativedot plots of the analyses of the phagocytosis by flow cytometry are shown.Macrophages of Anx A5 KO mice showed a significantly enhanced uptake of primarynecrotic cells after 0.75 hr (B) as well as after 3.5 hrs (D). Note: Thephagocytic ability of peritoneal macrophages of Anx A5 KO mice to take upmultiple necrotic cells per phagocyte (high positive macrophages) was highlysignificantly enhanced when compared with macrophages of WT animals (D, rightgraph). KO: knock out; WT: wild type; Mph: macrophages; Gates R3,R2:%CFSE positive Mph; Gate R7:% CFSE high positive Mph; One outof two representative set of experiments is shown. *P< 0.05; **P < 0.01
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fig02: Uptake by macrophages of necrotic cells in presence or absence of endogenousAnx A5. Macrophages in the peritoneum of the mice had contact withCFSE-labelled heat-induced (30 min., 56°C) primary necrotic WEHI 231cells for 0.75 (A, B) or 3.5 hrs (C, D). Following, a peritoneal lavage wasperformed and the phagocytosis by macrophages, which were identified with F4/80staining, of the necrotic prey was determined. In (A) and (C), representativedot plots of the analyses of the phagocytosis by flow cytometry are shown.Macrophages of Anx A5 KO mice showed a significantly enhanced uptake of primarynecrotic cells after 0.75 hr (B) as well as after 3.5 hrs (D). Note: Thephagocytic ability of peritoneal macrophages of Anx A5 KO mice to take upmultiple necrotic cells per phagocyte (high positive macrophages) was highlysignificantly enhanced when compared with macrophages of WT animals (D, rightgraph). KO: knock out; WT: wild type; Mph: macrophages; Gates R3,R2:%CFSE positive Mph; Gate R7:% CFSE high positive Mph; One outof two representative set of experiments is shown. *P< 0.05; **P < 0.01
Mentions: Animals were injected i.p. with CFSE-labelled heat-induced primary necrotic cellsallowing resident macrophages to interact with the dead cells for up to 3.5 hrs. Themice were killed after 0.75 hr or 3.5 hrs and the uptake of the fluorescent necroticcells in F4/80 positive macrophages, present in the peritoneal lavage, was monitoredby flow cytometry. F4/80 positive peritoneal macrophages of Anx A5 KO micephagozytosed significantly more primary necrotic cells in comparison to macrophagesof WT mice (Fig. 2). This was to be observedat 0.75 hr (Fig. 2A and B) as well as at 3.5 hrs (Fig.2C and D) after injection of thenecrotic cells. The uptake of multiple necrotic cells per phagocyte (high positivemacrophages) was highly significantly enhanced in Anx A5 KO mice when compared withAnx A5 WT animals (Fig. 2D, right graph).Similar effects were observed by using mechanical stress-induced primary necroticcells for the phagocytosis assays (suppl. 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