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CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species.

Yeo YA, Martínez Gómez JM, Croxford JL, Gasser S, Ling EA, Schwarz H - J Neuroinflammation (2012)

Bottom Line: CD137L signaling is also important for microglia activation in vivo, since CD137L-deficient mice exhibited profoundly less microglia activation during experimental autoimmune encephalomyelitis (EAE) which is a well-established murine model for neuroinflammation and human multiple sclerosis (MS).In vitro co-culture confirmed that CD137L-activated microglia induces apoptosis in oligodendrocytes, and identified reactive oxygen species as the mechanism of apoptosis induction.These data demonstrate activating effects of CD137L signaling to microglia, and show for the first time that the CD137 receptor/ligand system may be a mediator of neuroinflammatory and neurodegenerative disease, by activating microglia which in turn kill oligodendrocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive #14-02T, Singapore, 117599, Singapore.

ABSTRACT
CD137 (4-1BB, TNFRSF9), a member of the tumor necrosis factor (TNF) receptor family, is a potent T cell co-stimulatory molecule. CD137 ligand (CD137L) is expressed by antigen presenting cells (APC) as a transmembrane protein and transmits activating signals into APC. In this study we investigated the effects of CD137L signaling in microglia, the resident APC in the central nervous system. In vitro, the murine microglia cell lines BV-2 and N9, as well as primary murine microglia responded with activation as evidenced by adherence and secretion of proinflammatory cytokines, MMP-9, and soluble intercellular adhesion molecule (ICAM). CD137L signaling is also important for microglia activation in vivo, since CD137L-deficient mice exhibited profoundly less microglia activation during experimental autoimmune encephalomyelitis (EAE) which is a well-established murine model for neuroinflammation and human multiple sclerosis (MS). Also CD137 is expressed in the CNS of mice during EAE. Activated microglia has been reported to mediate the destruction of axonal myelin sheaths and cause the death of oligodendrocytes, the main pathogenic mechanisms in EAE and MS. Corresponding to the lower microglia activation there were also fewer apoptotic oligodendrocytes in the CNS of CD137L-deficient mice. In vitro co-culture confirmed that CD137L-activated microglia induces apoptosis in oligodendrocytes, and identified reactive oxygen species as the mechanism of apoptosis induction. These data demonstrate activating effects of CD137L signaling to microglia, and show for the first time that the CD137 receptor/ligand system may be a mediator of neuroinflammatory and neurodegenerative disease, by activating microglia which in turn kill oligodendrocytes.

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CD137L signaling activates microgliain vitro. Microglia cells were grown on plates that had been coated with PBS (grey bars) or 10 μg/mL of Fc control protein (white bars) or 10 μg/mL of CD137-Fc protein (black bars). (A) Attachment and morphological changes of primary microglia were documented by photography (40× magnification) 1 h after plating. Scale bar: 20 μm. (B) Attachment and morphological changes of BV-2 and N9 cells and primary microglia were documented by photography (63×) 24 h after plating. Cells exposed to immobilized CD137-Fc protein developed long spiky projections* and became amoeboid cells^ with shortened protrusions#. Scale bar: 20 μm. (C) The concentrations of cytokines in supernatants of primary microglia were determined by ELISA at indicated time points. Depicted are means ± standard deviations of triplicate measurements. (D) The phagocytic capacity of the cells was determined by adding FITC-labeled latex beads for 1 h before analysis by flow cytometry. Control: Autofluorescence of the cells. Numbers above the histograms state the percentages of positive cells and mean fluorescence intensities (MFI). These experiments were repeated three times with similar results. * P <0.05; ** P <0.01.
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Figure 2: CD137L signaling activates microgliain vitro. Microglia cells were grown on plates that had been coated with PBS (grey bars) or 10 μg/mL of Fc control protein (white bars) or 10 μg/mL of CD137-Fc protein (black bars). (A) Attachment and morphological changes of primary microglia were documented by photography (40× magnification) 1 h after plating. Scale bar: 20 μm. (B) Attachment and morphological changes of BV-2 and N9 cells and primary microglia were documented by photography (63×) 24 h after plating. Cells exposed to immobilized CD137-Fc protein developed long spiky projections* and became amoeboid cells^ with shortened protrusions#. Scale bar: 20 μm. (C) The concentrations of cytokines in supernatants of primary microglia were determined by ELISA at indicated time points. Depicted are means ± standard deviations of triplicate measurements. (D) The phagocytic capacity of the cells was determined by adding FITC-labeled latex beads for 1 h before analysis by flow cytometry. Control: Autofluorescence of the cells. Numbers above the histograms state the percentages of positive cells and mean fluorescence intensities (MFI). These experiments were repeated three times with similar results. * P <0.05; ** P <0.01.

Mentions: The activation of microglia by CD137L signaling was reflected by morphological changes such as increased adherence and cell spreading. The attachment of primary microglia on tissue culture plates was already visible after 1 h of seeding in the presence of CD137-Fc but not under the PBS and Fc control conditions (Figure 2A). Morphological changes of BV-2, N9, and primary microglia were evident a day after CD137L signaling (Figure 2B).


CD137 ligand activated microglia induces oligodendrocyte apoptosis via reactive oxygen species.

Yeo YA, Martínez Gómez JM, Croxford JL, Gasser S, Ling EA, Schwarz H - J Neuroinflammation (2012)

CD137L signaling activates microgliain vitro. Microglia cells were grown on plates that had been coated with PBS (grey bars) or 10 μg/mL of Fc control protein (white bars) or 10 μg/mL of CD137-Fc protein (black bars). (A) Attachment and morphological changes of primary microglia were documented by photography (40× magnification) 1 h after plating. Scale bar: 20 μm. (B) Attachment and morphological changes of BV-2 and N9 cells and primary microglia were documented by photography (63×) 24 h after plating. Cells exposed to immobilized CD137-Fc protein developed long spiky projections* and became amoeboid cells^ with shortened protrusions#. Scale bar: 20 μm. (C) The concentrations of cytokines in supernatants of primary microglia were determined by ELISA at indicated time points. Depicted are means ± standard deviations of triplicate measurements. (D) The phagocytic capacity of the cells was determined by adding FITC-labeled latex beads for 1 h before analysis by flow cytometry. Control: Autofluorescence of the cells. Numbers above the histograms state the percentages of positive cells and mean fluorescence intensities (MFI). These experiments were repeated three times with similar results. * P <0.05; ** P <0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: CD137L signaling activates microgliain vitro. Microglia cells were grown on plates that had been coated with PBS (grey bars) or 10 μg/mL of Fc control protein (white bars) or 10 μg/mL of CD137-Fc protein (black bars). (A) Attachment and morphological changes of primary microglia were documented by photography (40× magnification) 1 h after plating. Scale bar: 20 μm. (B) Attachment and morphological changes of BV-2 and N9 cells and primary microglia were documented by photography (63×) 24 h after plating. Cells exposed to immobilized CD137-Fc protein developed long spiky projections* and became amoeboid cells^ with shortened protrusions#. Scale bar: 20 μm. (C) The concentrations of cytokines in supernatants of primary microglia were determined by ELISA at indicated time points. Depicted are means ± standard deviations of triplicate measurements. (D) The phagocytic capacity of the cells was determined by adding FITC-labeled latex beads for 1 h before analysis by flow cytometry. Control: Autofluorescence of the cells. Numbers above the histograms state the percentages of positive cells and mean fluorescence intensities (MFI). These experiments were repeated three times with similar results. * P <0.05; ** P <0.01.
Mentions: The activation of microglia by CD137L signaling was reflected by morphological changes such as increased adherence and cell spreading. The attachment of primary microglia on tissue culture plates was already visible after 1 h of seeding in the presence of CD137-Fc but not under the PBS and Fc control conditions (Figure 2A). Morphological changes of BV-2, N9, and primary microglia were evident a day after CD137L signaling (Figure 2B).

Bottom Line: CD137L signaling is also important for microglia activation in vivo, since CD137L-deficient mice exhibited profoundly less microglia activation during experimental autoimmune encephalomyelitis (EAE) which is a well-established murine model for neuroinflammation and human multiple sclerosis (MS).In vitro co-culture confirmed that CD137L-activated microglia induces apoptosis in oligodendrocytes, and identified reactive oxygen species as the mechanism of apoptosis induction.These data demonstrate activating effects of CD137L signaling to microglia, and show for the first time that the CD137 receptor/ligand system may be a mediator of neuroinflammatory and neurodegenerative disease, by activating microglia which in turn kill oligodendrocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Centre for Translational Medicine, 14 Medical Drive #14-02T, Singapore, 117599, Singapore.

ABSTRACT
CD137 (4-1BB, TNFRSF9), a member of the tumor necrosis factor (TNF) receptor family, is a potent T cell co-stimulatory molecule. CD137 ligand (CD137L) is expressed by antigen presenting cells (APC) as a transmembrane protein and transmits activating signals into APC. In this study we investigated the effects of CD137L signaling in microglia, the resident APC in the central nervous system. In vitro, the murine microglia cell lines BV-2 and N9, as well as primary murine microglia responded with activation as evidenced by adherence and secretion of proinflammatory cytokines, MMP-9, and soluble intercellular adhesion molecule (ICAM). CD137L signaling is also important for microglia activation in vivo, since CD137L-deficient mice exhibited profoundly less microglia activation during experimental autoimmune encephalomyelitis (EAE) which is a well-established murine model for neuroinflammation and human multiple sclerosis (MS). Also CD137 is expressed in the CNS of mice during EAE. Activated microglia has been reported to mediate the destruction of axonal myelin sheaths and cause the death of oligodendrocytes, the main pathogenic mechanisms in EAE and MS. Corresponding to the lower microglia activation there were also fewer apoptotic oligodendrocytes in the CNS of CD137L-deficient mice. In vitro co-culture confirmed that CD137L-activated microglia induces apoptosis in oligodendrocytes, and identified reactive oxygen species as the mechanism of apoptosis induction. These data demonstrate activating effects of CD137L signaling to microglia, and show for the first time that the CD137 receptor/ligand system may be a mediator of neuroinflammatory and neurodegenerative disease, by activating microglia which in turn kill oligodendrocytes.

Show MeSH
Related in: MedlinePlus