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LPS-induced galectin-3 oligomerization results in enhancement of neutrophil activation.

Fermino ML, Polli CD, Toledo KA, Liu FT, Hsu DK, Roque-Barreira MC, Pereira-da-Silva G, Bernardes ES, Halbwachs-Mecarelli L - PLoS ONE (2011)

Bottom Line: Although it is well established that Gal 3 can interact with LPS, the pathophysiological importance of LPS/Gal 3 interactions is not fully understood.This result emphasizes the importance of an N-terminus-mediated Gal 3 oligomerization induced by its interaction with LPS.Finally we demonstrated that Balb/C mice were more susceptible to LPS-mediated shock when LPS was pretreated with Gal 3.

View Article: PubMed Central - PubMed

Affiliation: Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

ABSTRACT
Galectin-3 (Gal 3) is a glycan-binding protein that can be secreted by activated macrophages and mast cells at inflammation sites and plays an important role in inflammatory diseases caused by Bacteria and their products, such as lipopolysaccharides (LPS). Although it is well established that Gal 3 can interact with LPS, the pathophysiological importance of LPS/Gal 3 interactions is not fully understood. Data presented herein demonstrate for the first time that the interaction of Gal 3, either via its carbohydrate binding C-terminal domain or via its N-terminal part, with LPS from different bacterial strains, enhances the LPS-mediated neutrophil activation in vitro. Gal 3 allowed low LPS concentrations (1 µg/mL without serum, 1 ng/mL with serum) to upregulate CD11b expression and reactive oxygen species (ROS) generation on human neutrophils in vitro and drastically enhanced the binding efficiency of LPS to the neutrophil surface. These effects required LPS preincubation with Gal 3, before neutrophil stimulation and involved specific Gal 3/LPS interaction. A C-terminal Gal-3 fragment, which retains the lectin domain but lacks the N-terminal part, was still able to bind both to Escherichia coli LPS and to neutrophils, but had lost the ability to enhance neutrophil response to LPS. This result emphasizes the importance of an N-terminus-mediated Gal 3 oligomerization induced by its interaction with LPS. Finally we demonstrated that Balb/C mice were more susceptible to LPS-mediated shock when LPS was pretreated with Gal 3. Altogether, these results suggest that multimeric interactions between Gal 3 oligomers and LPS potentiate its pro-inflammatory effects on neutrophils.

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LPS induces oligomerization of Gal 3.A: A 96-well microplate was coated with 1 µg of recombinant Gal 3 per well. After blocking with gelatin 3%, 0.2 µM biotin-labeled Gal 3 ([b]Gal 3) and the indicated concentrations of unlabeled Gal 3 were added to each well in the presence (solid squares) or absence (open squares) of 5 µg/mL E. coli LPS. The signal was generated by neutravidin-HRP using OPD as a substrate. B and C: PMNs (2×106/mL) were incubated for 30 min at 4°C with increasing concentrations of truncated Galectin-3 (Gal 3C), lacking the N-terminal part (B), or increasing concentration of full-length Galectin-3 (Gal 3) (C). After washing, a specific polyclonal goat anti-Gal 3C (B) or goat anti-Gal 3 (C) and an FITC-labeled secondary anti-goat IgG antibody were used to detect Gal 3C on neutrophils by flow cytometry (n = 2). D: PMNs (10×106/mL) were treated for 20 min at 37°C with FITC-LPS (12.5 µg/mL) preincubated with Gal 3 (8 µM) or with Gal 3C (8 µM). The binding of FITC-LPS was evaluated by flow cytometry as in figure 4. E: PMNs (2×106/mL) were treated for 45 min at 37°C with the indicated concentrations of full-length Gal 3 (Gal 3) or Gal 3C, preincubated or not with E. coli LPS (1 µg/mL), and analyzed for CD11b expression by flow cytometry. ***p<0.001.
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pone-0026004-g005: LPS induces oligomerization of Gal 3.A: A 96-well microplate was coated with 1 µg of recombinant Gal 3 per well. After blocking with gelatin 3%, 0.2 µM biotin-labeled Gal 3 ([b]Gal 3) and the indicated concentrations of unlabeled Gal 3 were added to each well in the presence (solid squares) or absence (open squares) of 5 µg/mL E. coli LPS. The signal was generated by neutravidin-HRP using OPD as a substrate. B and C: PMNs (2×106/mL) were incubated for 30 min at 4°C with increasing concentrations of truncated Galectin-3 (Gal 3C), lacking the N-terminal part (B), or increasing concentration of full-length Galectin-3 (Gal 3) (C). After washing, a specific polyclonal goat anti-Gal 3C (B) or goat anti-Gal 3 (C) and an FITC-labeled secondary anti-goat IgG antibody were used to detect Gal 3C on neutrophils by flow cytometry (n = 2). D: PMNs (10×106/mL) were treated for 20 min at 37°C with FITC-LPS (12.5 µg/mL) preincubated with Gal 3 (8 µM) or with Gal 3C (8 µM). The binding of FITC-LPS was evaluated by flow cytometry as in figure 4. E: PMNs (2×106/mL) were treated for 45 min at 37°C with the indicated concentrations of full-length Gal 3 (Gal 3) or Gal 3C, preincubated or not with E. coli LPS (1 µg/mL), and analyzed for CD11b expression by flow cytometry. ***p<0.001.

Mentions: Since the binding of FITC-LPS to neutrophils is more pronounced as the concentration of Gal 3 reaches a threshold concentration (2 µM, Figure 4C), we next tested the ability of LPS to trigger galectin-3 self-association. Biotin-labeled Gal 3, without or with LPS, was allowed to interact with a galectin 3-coated plate, in the presence of increasing amounts of unlabeled Gal 3. As shown in figure 5A, the unlabeled Gal 3 did not compete with the biotin-galectin 3, since the amount of biotin-galectin 3 binding was not decreased, but on the contrary slightly enhanced in the presence of a 10 time excess of unlabeled Gal 3. This suggests the occurrence of Gal 3 oligomerization, which was enhanced in the presence of LPS (Figure 5A).


LPS-induced galectin-3 oligomerization results in enhancement of neutrophil activation.

Fermino ML, Polli CD, Toledo KA, Liu FT, Hsu DK, Roque-Barreira MC, Pereira-da-Silva G, Bernardes ES, Halbwachs-Mecarelli L - PLoS ONE (2011)

LPS induces oligomerization of Gal 3.A: A 96-well microplate was coated with 1 µg of recombinant Gal 3 per well. After blocking with gelatin 3%, 0.2 µM biotin-labeled Gal 3 ([b]Gal 3) and the indicated concentrations of unlabeled Gal 3 were added to each well in the presence (solid squares) or absence (open squares) of 5 µg/mL E. coli LPS. The signal was generated by neutravidin-HRP using OPD as a substrate. B and C: PMNs (2×106/mL) were incubated for 30 min at 4°C with increasing concentrations of truncated Galectin-3 (Gal 3C), lacking the N-terminal part (B), or increasing concentration of full-length Galectin-3 (Gal 3) (C). After washing, a specific polyclonal goat anti-Gal 3C (B) or goat anti-Gal 3 (C) and an FITC-labeled secondary anti-goat IgG antibody were used to detect Gal 3C on neutrophils by flow cytometry (n = 2). D: PMNs (10×106/mL) were treated for 20 min at 37°C with FITC-LPS (12.5 µg/mL) preincubated with Gal 3 (8 µM) or with Gal 3C (8 µM). The binding of FITC-LPS was evaluated by flow cytometry as in figure 4. E: PMNs (2×106/mL) were treated for 45 min at 37°C with the indicated concentrations of full-length Gal 3 (Gal 3) or Gal 3C, preincubated or not with E. coli LPS (1 µg/mL), and analyzed for CD11b expression by flow cytometry. ***p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198732&req=5

pone-0026004-g005: LPS induces oligomerization of Gal 3.A: A 96-well microplate was coated with 1 µg of recombinant Gal 3 per well. After blocking with gelatin 3%, 0.2 µM biotin-labeled Gal 3 ([b]Gal 3) and the indicated concentrations of unlabeled Gal 3 were added to each well in the presence (solid squares) or absence (open squares) of 5 µg/mL E. coli LPS. The signal was generated by neutravidin-HRP using OPD as a substrate. B and C: PMNs (2×106/mL) were incubated for 30 min at 4°C with increasing concentrations of truncated Galectin-3 (Gal 3C), lacking the N-terminal part (B), or increasing concentration of full-length Galectin-3 (Gal 3) (C). After washing, a specific polyclonal goat anti-Gal 3C (B) or goat anti-Gal 3 (C) and an FITC-labeled secondary anti-goat IgG antibody were used to detect Gal 3C on neutrophils by flow cytometry (n = 2). D: PMNs (10×106/mL) were treated for 20 min at 37°C with FITC-LPS (12.5 µg/mL) preincubated with Gal 3 (8 µM) or with Gal 3C (8 µM). The binding of FITC-LPS was evaluated by flow cytometry as in figure 4. E: PMNs (2×106/mL) were treated for 45 min at 37°C with the indicated concentrations of full-length Gal 3 (Gal 3) or Gal 3C, preincubated or not with E. coli LPS (1 µg/mL), and analyzed for CD11b expression by flow cytometry. ***p<0.001.
Mentions: Since the binding of FITC-LPS to neutrophils is more pronounced as the concentration of Gal 3 reaches a threshold concentration (2 µM, Figure 4C), we next tested the ability of LPS to trigger galectin-3 self-association. Biotin-labeled Gal 3, without or with LPS, was allowed to interact with a galectin 3-coated plate, in the presence of increasing amounts of unlabeled Gal 3. As shown in figure 5A, the unlabeled Gal 3 did not compete with the biotin-galectin 3, since the amount of biotin-galectin 3 binding was not decreased, but on the contrary slightly enhanced in the presence of a 10 time excess of unlabeled Gal 3. This suggests the occurrence of Gal 3 oligomerization, which was enhanced in the presence of LPS (Figure 5A).

Bottom Line: Although it is well established that Gal 3 can interact with LPS, the pathophysiological importance of LPS/Gal 3 interactions is not fully understood.This result emphasizes the importance of an N-terminus-mediated Gal 3 oligomerization induced by its interaction with LPS.Finally we demonstrated that Balb/C mice were more susceptible to LPS-mediated shock when LPS was pretreated with Gal 3.

View Article: PubMed Central - PubMed

Affiliation: Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

ABSTRACT
Galectin-3 (Gal 3) is a glycan-binding protein that can be secreted by activated macrophages and mast cells at inflammation sites and plays an important role in inflammatory diseases caused by Bacteria and their products, such as lipopolysaccharides (LPS). Although it is well established that Gal 3 can interact with LPS, the pathophysiological importance of LPS/Gal 3 interactions is not fully understood. Data presented herein demonstrate for the first time that the interaction of Gal 3, either via its carbohydrate binding C-terminal domain or via its N-terminal part, with LPS from different bacterial strains, enhances the LPS-mediated neutrophil activation in vitro. Gal 3 allowed low LPS concentrations (1 µg/mL without serum, 1 ng/mL with serum) to upregulate CD11b expression and reactive oxygen species (ROS) generation on human neutrophils in vitro and drastically enhanced the binding efficiency of LPS to the neutrophil surface. These effects required LPS preincubation with Gal 3, before neutrophil stimulation and involved specific Gal 3/LPS interaction. A C-terminal Gal-3 fragment, which retains the lectin domain but lacks the N-terminal part, was still able to bind both to Escherichia coli LPS and to neutrophils, but had lost the ability to enhance neutrophil response to LPS. This result emphasizes the importance of an N-terminus-mediated Gal 3 oligomerization induced by its interaction with LPS. Finally we demonstrated that Balb/C mice were more susceptible to LPS-mediated shock when LPS was pretreated with Gal 3. Altogether, these results suggest that multimeric interactions between Gal 3 oligomers and LPS potentiate its pro-inflammatory effects on neutrophils.

Show MeSH
Related in: MedlinePlus