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Flow cytometric characterization of brain dendritic cell subsets after murine stroke.

Pösel C, Uri A, Schulz I, Boltze J, Weise G, Wagner DC - Exp Transl Stroke Med (2014)

Bottom Line: Sterile inflammation is a substantial element of post-stroke pathophysiology with the determination of autoimmunity versus tolerance being one of its most important aspects.The latter could be differentiated into the classical migratory DC subpopulations (CD11b+ and CD103+), but no CD4 or CD8+ DC were found.Finally, stroke caused a significant increase of CD11b/CD103 double-positive DC in the affected brain hemisphere.

View Article: PubMed Central - PubMed

Affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

ABSTRACT

Background: Sterile inflammation is a substantial element of post-stroke pathophysiology with the determination of autoimmunity versus tolerance being one of its most important aspects. It is believed that this determination is initiated relatively early after stroke onset by clearing macrophages and migratory dendritic cells (DC). However, the phenotypic differentiation of macrophages and DC is intricate particularly in the disease context. Here, we utilized a set of surface markers used in mucosal immunity research to investigate the involvement of macrophages and DC subpopulations in post-stroke inflammation in mice.

Findings: Photothrombotic stroke induced a significant increase of lineage (CD3, B220, Ly6G and CD49b) negative CD11b+ cells in the brain primarily consisting of F4/80+ macrophages and, to a lesser extent, F4/80-/CD11c-/CD11b+ monocytes and F4/80-/CD11c+ DC. The latter could be differentiated into the classical migratory DC subpopulations (CD11b+ and CD103+), but no CD4 or CD8+ DC were found. Finally, stroke caused a significant increase of CD11b/CD103 double-positive DC in the affected brain hemisphere.

Conclusions: The surface marker combination used in this study allowed a phenotypic differentiation of macrophages and DC subpopulations after stroke, thus providing an important prerequisite to study post-stroke immunity and tolerance.

No MeSH data available.


Related in: MedlinePlus

Differentiation of mononuclear phagocytes after photothrombotic stroke (PT) in mice. (A), in both experiments, PT consistently caused a significant increase of CD45high + leukocytes in the ipsilateral (ipsi) hemisphere. (B), subset analysis revealed a strong increase of F4/80+ macrophages which could be further categorized by CD11c and MHCII expression (C; experiment 2, n = 3). (D), monocytes (Mo) and Lin-/CD11c+ cDC also significantly contribute to the inflammatory response to stroke. *p < 0.05, ** p < 0.01, ***p < 0.001 by one way ANOVA and Bonferronis’ post-hoc test. Contra, contralateral.
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Fig2: Differentiation of mononuclear phagocytes after photothrombotic stroke (PT) in mice. (A), in both experiments, PT consistently caused a significant increase of CD45high + leukocytes in the ipsilateral (ipsi) hemisphere. (B), subset analysis revealed a strong increase of F4/80+ macrophages which could be further categorized by CD11c and MHCII expression (C; experiment 2, n = 3). (D), monocytes (Mo) and Lin-/CD11c+ cDC also significantly contribute to the inflammatory response to stroke. *p < 0.05, ** p < 0.01, ***p < 0.001 by one way ANOVA and Bonferronis’ post-hoc test. Contra, contralateral.

Mentions: Brain infiltrating cells were defined as CD45high+ cells that could be unambiguously differentiated from CD45 intermediate (int) microglia (Figure 1, cell gate). Macrophages were delineated as CD11b+/F4/80+ cells. The remaining cells were then distinguished by a lineage (Lin) combination (CD3, B220, Ly6G, CD49b) and CD11c. Lin-/CD11c-/CD11b+ cells were categorized as monocytes, Lin-/CD11c+ as conventional DC (cDC) and Lin+ (B220+)/CD11c+ cells as plasmacytoid DC (pDC; Figure 1, non-macrophage gate).Flow cytometric quantification revealed a significant increase of CD45+ cells in the ischemic hemisphere at day 6 after stroke (naive: 115.2 ± 27.5 × 10E3 cells and PT contralateral: 107.8 ± 26.1 x 10E3 cells versus PT ipsilateral: 183.2 ± 17.1 × 10E3 cells; p < 0.01). This difference was primarily caused by CD45high leukocytes (Figure 2A) and, to a lesser degree, by CD45int microglia (naive: 103.3 ± 28.9 × 10E3 cells and PT contralateral: 85.3 ± 20.6 × 10E3 cells versus PT ipsilateral: 124.5 ± 16.7 × 10E3 cells; p > 0.05). Compared to naive controls, experimental stroke induced a 40 fold increase of F4/80+ macrophages within the ipsilateral hemisphere (Figure 2B). The majority of these cells did not co-express CD11c or major histocompatibility complex (MHC)-II, but we also observed CD11c- or MHCII-single positive and CD11c/MHCII-double positive macrophages (Figure 2C).We found a comparable increase of monocytes and Lin-/CD11c+ cDC (Figure 2D) within the ischemic hemisphere. Finally, stroke induced a slight, but statistically significant increase of pDC (naive: 135 ± 104 cells and PT contralateral: 186 ± 105 cells versus PT ipsilateral: 524 ± 215 cells; p < 0.05)Figure 2


Flow cytometric characterization of brain dendritic cell subsets after murine stroke.

Pösel C, Uri A, Schulz I, Boltze J, Weise G, Wagner DC - Exp Transl Stroke Med (2014)

Differentiation of mononuclear phagocytes after photothrombotic stroke (PT) in mice. (A), in both experiments, PT consistently caused a significant increase of CD45high + leukocytes in the ipsilateral (ipsi) hemisphere. (B), subset analysis revealed a strong increase of F4/80+ macrophages which could be further categorized by CD11c and MHCII expression (C; experiment 2, n = 3). (D), monocytes (Mo) and Lin-/CD11c+ cDC also significantly contribute to the inflammatory response to stroke. *p < 0.05, ** p < 0.01, ***p < 0.001 by one way ANOVA and Bonferronis’ post-hoc test. Contra, contralateral.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Differentiation of mononuclear phagocytes after photothrombotic stroke (PT) in mice. (A), in both experiments, PT consistently caused a significant increase of CD45high + leukocytes in the ipsilateral (ipsi) hemisphere. (B), subset analysis revealed a strong increase of F4/80+ macrophages which could be further categorized by CD11c and MHCII expression (C; experiment 2, n = 3). (D), monocytes (Mo) and Lin-/CD11c+ cDC also significantly contribute to the inflammatory response to stroke. *p < 0.05, ** p < 0.01, ***p < 0.001 by one way ANOVA and Bonferronis’ post-hoc test. Contra, contralateral.
Mentions: Brain infiltrating cells were defined as CD45high+ cells that could be unambiguously differentiated from CD45 intermediate (int) microglia (Figure 1, cell gate). Macrophages were delineated as CD11b+/F4/80+ cells. The remaining cells were then distinguished by a lineage (Lin) combination (CD3, B220, Ly6G, CD49b) and CD11c. Lin-/CD11c-/CD11b+ cells were categorized as monocytes, Lin-/CD11c+ as conventional DC (cDC) and Lin+ (B220+)/CD11c+ cells as plasmacytoid DC (pDC; Figure 1, non-macrophage gate).Flow cytometric quantification revealed a significant increase of CD45+ cells in the ischemic hemisphere at day 6 after stroke (naive: 115.2 ± 27.5 × 10E3 cells and PT contralateral: 107.8 ± 26.1 x 10E3 cells versus PT ipsilateral: 183.2 ± 17.1 × 10E3 cells; p < 0.01). This difference was primarily caused by CD45high leukocytes (Figure 2A) and, to a lesser degree, by CD45int microglia (naive: 103.3 ± 28.9 × 10E3 cells and PT contralateral: 85.3 ± 20.6 × 10E3 cells versus PT ipsilateral: 124.5 ± 16.7 × 10E3 cells; p > 0.05). Compared to naive controls, experimental stroke induced a 40 fold increase of F4/80+ macrophages within the ipsilateral hemisphere (Figure 2B). The majority of these cells did not co-express CD11c or major histocompatibility complex (MHC)-II, but we also observed CD11c- or MHCII-single positive and CD11c/MHCII-double positive macrophages (Figure 2C).We found a comparable increase of monocytes and Lin-/CD11c+ cDC (Figure 2D) within the ischemic hemisphere. Finally, stroke induced a slight, but statistically significant increase of pDC (naive: 135 ± 104 cells and PT contralateral: 186 ± 105 cells versus PT ipsilateral: 524 ± 215 cells; p < 0.05)Figure 2

Bottom Line: Sterile inflammation is a substantial element of post-stroke pathophysiology with the determination of autoimmunity versus tolerance being one of its most important aspects.The latter could be differentiated into the classical migratory DC subpopulations (CD11b+ and CD103+), but no CD4 or CD8+ DC were found.Finally, stroke caused a significant increase of CD11b/CD103 double-positive DC in the affected brain hemisphere.

View Article: PubMed Central - PubMed

Affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

ABSTRACT

Background: Sterile inflammation is a substantial element of post-stroke pathophysiology with the determination of autoimmunity versus tolerance being one of its most important aspects. It is believed that this determination is initiated relatively early after stroke onset by clearing macrophages and migratory dendritic cells (DC). However, the phenotypic differentiation of macrophages and DC is intricate particularly in the disease context. Here, we utilized a set of surface markers used in mucosal immunity research to investigate the involvement of macrophages and DC subpopulations in post-stroke inflammation in mice.

Findings: Photothrombotic stroke induced a significant increase of lineage (CD3, B220, Ly6G and CD49b) negative CD11b+ cells in the brain primarily consisting of F4/80+ macrophages and, to a lesser extent, F4/80-/CD11c-/CD11b+ monocytes and F4/80-/CD11c+ DC. The latter could be differentiated into the classical migratory DC subpopulations (CD11b+ and CD103+), but no CD4 or CD8+ DC were found. Finally, stroke caused a significant increase of CD11b/CD103 double-positive DC in the affected brain hemisphere.

Conclusions: The surface marker combination used in this study allowed a phenotypic differentiation of macrophages and DC subpopulations after stroke, thus providing an important prerequisite to study post-stroke immunity and tolerance.

No MeSH data available.


Related in: MedlinePlus