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Galectin-2 induces a proinflammatory, anti-arteriogenic phenotype in monocytes and macrophages.

Yıldırım C, Vogel DY, Hollander MR, Baggen JM, Fontijn RD, Nieuwenhuis S, Haverkamp A, de Vries MR, Quax PH, Garcia-Vallejo JJ, van der Laan AM, Dijkstra CD, van der Pouw Kraan TC, van Royen N, Horrevoets AJ - PLoS ONE (2015)

Bottom Line: The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown.Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire.This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1).

View Article: PubMed Central - PubMed

Affiliation: Dept of Molecular Cell Biology and Immunology, VU University Medical Centre, Amsterdam, the Netherlands.

ABSTRACT
Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.

No MeSH data available.


Related in: MedlinePlus

Binding analysis of recombinant human galectin-2 to monocytes and macrophages.Cells were incubated with 10 μg/ml biotinylated recombinant human galectin-2 (rh-gal-2). (A) The effect of lactose (20mM) and thiodigalactoside (20mM) on rh-gal-2 binding to monocytes. (B) Binding (expressed as MFI) of human galectin-2 to human monocyte subsets i.e. non-classical (gate R1; CD14+CD16+), intermediate (gate R2; CD14++/CD16+), and classical (gate R3; CD14++CD16-). Results are presented as mean ± SEM from at least 3 independent experiments. * P < 0.05, **P < 0.01, ***P < 0.001, untreated vs. h-gal-2. (C) Correlation of rh-gal-2 binding with CD14 expression. (D) Binding of rh-gal-2 to human monocyte-derived M0 macrophages and mouse macrophages (RAW264.7). Representative histograms from three independent experiments are depicted in all three panels.
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pone.0124347.g001: Binding analysis of recombinant human galectin-2 to monocytes and macrophages.Cells were incubated with 10 μg/ml biotinylated recombinant human galectin-2 (rh-gal-2). (A) The effect of lactose (20mM) and thiodigalactoside (20mM) on rh-gal-2 binding to monocytes. (B) Binding (expressed as MFI) of human galectin-2 to human monocyte subsets i.e. non-classical (gate R1; CD14+CD16+), intermediate (gate R2; CD14++/CD16+), and classical (gate R3; CD14++CD16-). Results are presented as mean ± SEM from at least 3 independent experiments. * P < 0.05, **P < 0.01, ***P < 0.001, untreated vs. h-gal-2. (C) Correlation of rh-gal-2 binding with CD14 expression. (D) Binding of rh-gal-2 to human monocyte-derived M0 macrophages and mouse macrophages (RAW264.7). Representative histograms from three independent experiments are depicted in all three panels.

Mentions: We evaluated the binding of human and mouse biotinylatedgalectin-2 to the surface of human monocytes by flow cytometry. Human monocytes bound both human and mouse galectin-2 with a high affinity (apparent Kd of 1.1 μg/ml (77 nM) and 2.5 ug/ml, respectively, S2 Fig). Both galectins bound in a concentration dependent manner with near maximal binding at10 μg/ml and an apparent Bmax of 93.6 and80.7 for human and mouse galectin-2, respectively. By contrast, a much lower binding was observed for human galectin-1 at equal fluorescent labeling (apparent Kd 0.2 μg/ml, Bmax of 28.9). Next, the carbohydrate-binding specificity of human galectin-2 was determined by adding either lactose or thiodigalactoside together with human galectin-2 to human monocytes. These disaccharides did not inhibit binding of human galectin-2 (Fig 1A), suggesting that the binding is carbohydrate-independent. Interestingly, different human monocyte subsets (classical CD14++CD16-; intermediate CD14++CD16+; and non-classical CD14+CD16+monocytes) showed differential binding of human galectin-2 (Fig 1B). CD14low monocytes (non-classical) showed a lower binding of human galectin-2 than CD14high monocytes (classical and intermediate), reaching significance for the comparison with intermediate monocytes (Fig 1B). These results suggest that binding is associated with expression levels of CD14. Additionally, binding of human galectin-2 to different types of macrophages was investigated. As shown in Fig 1C, human monocyte-derived macrophages (M0) and mouse macrophages (RAW264.7 cells) bound human galectin-2.Human monocyte-derived dendritic cells (DCs), and human T-cells did not bind human galectin-2 (S3 Fig).


Galectin-2 induces a proinflammatory, anti-arteriogenic phenotype in monocytes and macrophages.

Yıldırım C, Vogel DY, Hollander MR, Baggen JM, Fontijn RD, Nieuwenhuis S, Haverkamp A, de Vries MR, Quax PH, Garcia-Vallejo JJ, van der Laan AM, Dijkstra CD, van der Pouw Kraan TC, van Royen N, Horrevoets AJ - PLoS ONE (2015)

Binding analysis of recombinant human galectin-2 to monocytes and macrophages.Cells were incubated with 10 μg/ml biotinylated recombinant human galectin-2 (rh-gal-2). (A) The effect of lactose (20mM) and thiodigalactoside (20mM) on rh-gal-2 binding to monocytes. (B) Binding (expressed as MFI) of human galectin-2 to human monocyte subsets i.e. non-classical (gate R1; CD14+CD16+), intermediate (gate R2; CD14++/CD16+), and classical (gate R3; CD14++CD16-). Results are presented as mean ± SEM from at least 3 independent experiments. * P < 0.05, **P < 0.01, ***P < 0.001, untreated vs. h-gal-2. (C) Correlation of rh-gal-2 binding with CD14 expression. (D) Binding of rh-gal-2 to human monocyte-derived M0 macrophages and mouse macrophages (RAW264.7). Representative histograms from three independent experiments are depicted in all three panels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401781&req=5

pone.0124347.g001: Binding analysis of recombinant human galectin-2 to monocytes and macrophages.Cells were incubated with 10 μg/ml biotinylated recombinant human galectin-2 (rh-gal-2). (A) The effect of lactose (20mM) and thiodigalactoside (20mM) on rh-gal-2 binding to monocytes. (B) Binding (expressed as MFI) of human galectin-2 to human monocyte subsets i.e. non-classical (gate R1; CD14+CD16+), intermediate (gate R2; CD14++/CD16+), and classical (gate R3; CD14++CD16-). Results are presented as mean ± SEM from at least 3 independent experiments. * P < 0.05, **P < 0.01, ***P < 0.001, untreated vs. h-gal-2. (C) Correlation of rh-gal-2 binding with CD14 expression. (D) Binding of rh-gal-2 to human monocyte-derived M0 macrophages and mouse macrophages (RAW264.7). Representative histograms from three independent experiments are depicted in all three panels.
Mentions: We evaluated the binding of human and mouse biotinylatedgalectin-2 to the surface of human monocytes by flow cytometry. Human monocytes bound both human and mouse galectin-2 with a high affinity (apparent Kd of 1.1 μg/ml (77 nM) and 2.5 ug/ml, respectively, S2 Fig). Both galectins bound in a concentration dependent manner with near maximal binding at10 μg/ml and an apparent Bmax of 93.6 and80.7 for human and mouse galectin-2, respectively. By contrast, a much lower binding was observed for human galectin-1 at equal fluorescent labeling (apparent Kd 0.2 μg/ml, Bmax of 28.9). Next, the carbohydrate-binding specificity of human galectin-2 was determined by adding either lactose or thiodigalactoside together with human galectin-2 to human monocytes. These disaccharides did not inhibit binding of human galectin-2 (Fig 1A), suggesting that the binding is carbohydrate-independent. Interestingly, different human monocyte subsets (classical CD14++CD16-; intermediate CD14++CD16+; and non-classical CD14+CD16+monocytes) showed differential binding of human galectin-2 (Fig 1B). CD14low monocytes (non-classical) showed a lower binding of human galectin-2 than CD14high monocytes (classical and intermediate), reaching significance for the comparison with intermediate monocytes (Fig 1B). These results suggest that binding is associated with expression levels of CD14. Additionally, binding of human galectin-2 to different types of macrophages was investigated. As shown in Fig 1C, human monocyte-derived macrophages (M0) and mouse macrophages (RAW264.7 cells) bound human galectin-2.Human monocyte-derived dendritic cells (DCs), and human T-cells did not bind human galectin-2 (S3 Fig).

Bottom Line: The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown.Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire.This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1).

View Article: PubMed Central - PubMed

Affiliation: Dept of Molecular Cell Biology and Immunology, VU University Medical Centre, Amsterdam, the Netherlands.

ABSTRACT
Galectin-2 is a monocyte-expressed carbohydrate-binding lectin, for which increased expression is genetically determined and associated with decreased collateral arteriogenesis in obstructive coronary artery disease patients. The inhibiting effect of galectin-2 on arteriogenesis was confirmed in vivo, but the mechanism is largely unknown. In this study we aimed to explore the effects of galectin-2 on monocyte/macrophage phenotype in vitro and vivo, and to identify the receptor by which galectin-2 exerts these effects. We now show that the binding of galectin-2 to different circulating human monocyte subsets is dependent on monocyte surface expression levels of CD14. The high affinity binding is blocked by an anti-CD14 antibody but not by carbohydrates, indicating a specific protein-protein interaction. Galectin-2 binding to human monocytes modulated their transcriptome by inducing proinflammatory cytokines and inhibiting pro-arteriogenic factors, while attenuating monocyte migration. Using specific knock-out mice, we show that galectin-2 acts through the CD14/toll-like receptor (TLR)-4 pathway. Furthermore, galectin-2 skews human macrophages to a M1-like proinflammatory phenotype, characterized by a reduced motility and expression of an anti-arteriogenic cytokine/growth factor repertoire. This is accompanied by a switch in surface protein expression to CD40-high and CD206-low (M1). In a murine model we show that galectin-2 administration, known to attenuate arteriogenesis, leads to increased numbers of CD40-positive (M1) and reduced numbers of CD206-positive (M2) macrophages surrounding actively remodeling collateral arteries. In conclusion galectin-2 is the first endogenous CD14/TLR4 ligand that induces a proinflammatory, non-arteriogenic phenotype in monocytes/macrophages. Interference with CD14-Galectin-2 interaction may provide a new intervention strategy to stimulate growth of collateral arteries in genetically compromised cardiovascular patients.

No MeSH data available.


Related in: MedlinePlus