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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

Galectin-2 binds CD14 and mimics LPS.(A) Human monocytes were incubated with biotinylated rh-gal-2 or—rh-gal-1. Binding of galectins (expressed as MFI) was analyzed by flow cytometry in the presence or absence of neutralizing anti-human CD14 antibody (100ug/ml). (B) IFN-β gene expression was measured in human monocytes by real-time PCR after treatment with TLR4 ligand LPS (10 ng/ml), TLR2 ligand PGN (1 μg/ml), rh-gal-2 (10 μg/ml), or combinations for three hours. Expression levels were compared of untreated vs. h-gal-2, LPS, and PGN. LPS vs. LPS + h-gal-2, and PGN vs. PGN + h-gal-2. Gene expression is expressed relative to untreated sample (set at 1). *P <0.05, **P < 0.01. Results in panel A and B are presented as mean ± SEM from at least 3 independent experiments. (C) Whole blood from WT, TLR4-/-, or CD14-/- mice was stimulated with rm-gal-2,or LPS in the absence or presence of polymyxin B (PMB). TNF-α protein levels were measured in supernatants and compared between wt and CD14- or TLR4-deficient cells stimulated as indicated. Within the mouse strains the effect of PMB was evaluated. *P < 0.05, **P < 0.01, ***P < 0.001, wt vs. knockout. '''P < 0.001, LPS vs. LPS + PMB. (D) Human monocytes were incubated with rh-gal-2 (10 μg/ml) or LPS (as indicated) for three hours in the presence or absence of neutralizing anti-human CD14 antibody (20 μg/ml).IFN-β gene expression was determined by real-time PCR. Results represent the mean expression ± SD of one experiment measured in triplicate. ***P < 0.001, ****P < 0.0001
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pone.0124347.g002: Galectin-2 binds CD14 and mimics LPS.(A) Human monocytes were incubated with biotinylated rh-gal-2 or—rh-gal-1. Binding of galectins (expressed as MFI) was analyzed by flow cytometry in the presence or absence of neutralizing anti-human CD14 antibody (100ug/ml). (B) IFN-β gene expression was measured in human monocytes by real-time PCR after treatment with TLR4 ligand LPS (10 ng/ml), TLR2 ligand PGN (1 μg/ml), rh-gal-2 (10 μg/ml), or combinations for three hours. Expression levels were compared of untreated vs. h-gal-2, LPS, and PGN. LPS vs. LPS + h-gal-2, and PGN vs. PGN + h-gal-2. Gene expression is expressed relative to untreated sample (set at 1). *P <0.05, **P < 0.01. Results in panel A and B are presented as mean ± SEM from at least 3 independent experiments. (C) Whole blood from WT, TLR4-/-, or CD14-/- mice was stimulated with rm-gal-2,or LPS in the absence or presence of polymyxin B (PMB). TNF-α protein levels were measured in supernatants and compared between wt and CD14- or TLR4-deficient cells stimulated as indicated. Within the mouse strains the effect of PMB was evaluated. *P < 0.05, **P < 0.01, ***P < 0.001, wt vs. knockout. '''P < 0.001, LPS vs. LPS + PMB. (D) Human monocytes were incubated with rh-gal-2 (10 μg/ml) or LPS (as indicated) for three hours in the presence or absence of neutralizing anti-human CD14 antibody (20 μg/ml).IFN-β gene expression was determined by real-time PCR. Results represent the mean expression ± SD of one experiment measured in triplicate. ***P < 0.001, ****P < 0.0001

Mentions: Because of the association of galectin-2-monocyte binding with CD14 expression levels, we examined whether galectin-2 is a ligand for CD14.CD14 antibodies that neutralize activation of the CD14-associated TLR2 and TLR4 receptors [23,24] caused a 50% reduction in binding of human galectin-2 to human monocytes, whereas binding of human galectin-1 was not affected by these antibodies(Fig 2A).Next, we examined whether binding of galectin-2 to CD14 leads to TLR activation and the possible role of the two CD14-associated TLRs in activation. We analyzed whether galectin-2 is able to induce IFN-β gene expression. IFN-β activity is induced by the TLR4 ligand LPS, but not by the TLR2 ligand PGN (Fig 2B).[25]The observation that galectin-2 was able to induce IFN-β expression, comparable to LPS and irrespective of TLR2 stimulation, suggests that galectin-2 activates TLR4.To further explore this possibility, we stimulated mouse whole blood cells deficient for TLR4 or CD14 with m-galectin-2. Induction of TNF-α protein production was determined by ELISA, showing that both LPS and m-galectin-2 required CD14 and TLR4 for signaling (Fig 2C). The LPS response, but not the galectin-2-induced response was inhibited by polymyxin B. These results indicate that galectin-2 acts independent of LPS, and requires both CD14 and TLR4 to mediate its effects. Also in human monocytes, galectin-2 required CD14 for IFN-β induction. Neutralizing CD14 antibodies indeed inhibited the induction of IFN-β expression by approximately 50% (Fig 2D).


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)

Galectin-2 binds CD14 and mimics LPS.(A) Human monocytes were incubated with biotinylated rh-gal-2 or—rh-gal-1. Binding of galectins (expressed as MFI) was analyzed by flow cytometry in the presence or absence of neutralizing anti-human CD14 antibody (100ug/ml). (B) IFN-β gene expression was measured in human monocytes by real-time PCR after treatment with TLR4 ligand LPS (10 ng/ml), TLR2 ligand PGN (1 μg/ml), rh-gal-2 (10 μg/ml), or combinations for three hours. Expression levels were compared of untreated vs. h-gal-2, LPS, and PGN. LPS vs. LPS + h-gal-2, and PGN vs. PGN + h-gal-2. Gene expression is expressed relative to untreated sample (set at 1). *P <0.05, **P < 0.01. Results in panel A and B are presented as mean ± SEM from at least 3 independent experiments. (C) Whole blood from WT, TLR4-/-, or CD14-/- mice was stimulated with rm-gal-2,or LPS in the absence or presence of polymyxin B (PMB). TNF-α protein levels were measured in supernatants and compared between wt and CD14- or TLR4-deficient cells stimulated as indicated. Within the mouse strains the effect of PMB was evaluated. *P < 0.05, **P < 0.01, ***P < 0.001, wt vs. knockout. '''P < 0.001, LPS vs. LPS + PMB. (D) Human monocytes were incubated with rh-gal-2 (10 μg/ml) or LPS (as indicated) for three hours in the presence or absence of neutralizing anti-human CD14 antibody (20 μg/ml).IFN-β gene expression was determined by real-time PCR. Results represent the mean expression ± SD of one experiment measured in triplicate. ***P < 0.001, ****P < 0.0001
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401781&req=5

pone.0124347.g002: Galectin-2 binds CD14 and mimics LPS.(A) Human monocytes were incubated with biotinylated rh-gal-2 or—rh-gal-1. Binding of galectins (expressed as MFI) was analyzed by flow cytometry in the presence or absence of neutralizing anti-human CD14 antibody (100ug/ml). (B) IFN-β gene expression was measured in human monocytes by real-time PCR after treatment with TLR4 ligand LPS (10 ng/ml), TLR2 ligand PGN (1 μg/ml), rh-gal-2 (10 μg/ml), or combinations for three hours. Expression levels were compared of untreated vs. h-gal-2, LPS, and PGN. LPS vs. LPS + h-gal-2, and PGN vs. PGN + h-gal-2. Gene expression is expressed relative to untreated sample (set at 1). *P <0.05, **P < 0.01. Results in panel A and B are presented as mean ± SEM from at least 3 independent experiments. (C) Whole blood from WT, TLR4-/-, or CD14-/- mice was stimulated with rm-gal-2,or LPS in the absence or presence of polymyxin B (PMB). TNF-α protein levels were measured in supernatants and compared between wt and CD14- or TLR4-deficient cells stimulated as indicated. Within the mouse strains the effect of PMB was evaluated. *P < 0.05, **P < 0.01, ***P < 0.001, wt vs. knockout. '''P < 0.001, LPS vs. LPS + PMB. (D) Human monocytes were incubated with rh-gal-2 (10 μg/ml) or LPS (as indicated) for three hours in the presence or absence of neutralizing anti-human CD14 antibody (20 μg/ml).IFN-β gene expression was determined by real-time PCR. Results represent the mean expression ± SD of one experiment measured in triplicate. ***P < 0.001, ****P < 0.0001
Mentions: Because of the association of galectin-2-monocyte binding with CD14 expression levels, we examined whether galectin-2 is a ligand for CD14.CD14 antibodies that neutralize activation of the CD14-associated TLR2 and TLR4 receptors [23,24] caused a 50% reduction in binding of human galectin-2 to human monocytes, whereas binding of human galectin-1 was not affected by these antibodies(Fig 2A).Next, we examined whether binding of galectin-2 to CD14 leads to TLR activation and the possible role of the two CD14-associated TLRs in activation. We analyzed whether galectin-2 is able to induce IFN-β gene expression. IFN-β activity is induced by the TLR4 ligand LPS, but not by the TLR2 ligand PGN (Fig 2B).[25]The observation that galectin-2 was able to induce IFN-β expression, comparable to LPS and irrespective of TLR2 stimulation, suggests that galectin-2 activates TLR4.To further explore this possibility, we stimulated mouse whole blood cells deficient for TLR4 or CD14 with m-galectin-2. Induction of TNF-α protein production was determined by ELISA, showing that both LPS and m-galectin-2 required CD14 and TLR4 for signaling (Fig 2C). The LPS response, but not the galectin-2-induced response was inhibited by polymyxin B. These results indicate that galectin-2 acts independent of LPS, and requires both CD14 and TLR4 to mediate its effects. Also in human monocytes, galectin-2 required CD14 for IFN-β induction. Neutralizing CD14 antibodies indeed inhibited the induction of IFN-β expression by approximately 50% (Fig 2D).

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