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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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Schematic view of putative mechanisms responsible for galectin-3 enhancement of LPS-induced neutrophil activation.Monomeric Galectin-3 interacts with LPS glycans from LPS aggregates via its C-terminal domain. LPS/Gal 3 interactions induce Gal 3 oligomerization, which promotes the dissolution of LPS aggregates, stabilizes LPS monomers and enhances the LPS interaction with surface receptors, leading to increased neutrophil activation. CD14 only is shown as the initial LPS ligand on neutrophils, because it is highly glycosylated and could, potentially, interact directly with Gal 3. Moreover, being GPI-anchored, CD14 would be most easily clustered on the cell surface. However, this implies a secondary clustering of TLR/MD2, which would transmit or modulate signals into the cell.
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pone-0026004-g008: Schematic view of putative mechanisms responsible for galectin-3 enhancement of LPS-induced neutrophil activation.Monomeric Galectin-3 interacts with LPS glycans from LPS aggregates via its C-terminal domain. LPS/Gal 3 interactions induce Gal 3 oligomerization, which promotes the dissolution of LPS aggregates, stabilizes LPS monomers and enhances the LPS interaction with surface receptors, leading to increased neutrophil activation. CD14 only is shown as the initial LPS ligand on neutrophils, because it is highly glycosylated and could, potentially, interact directly with Gal 3. Moreover, being GPI-anchored, CD14 would be most easily clustered on the cell surface. However, this implies a secondary clustering of TLR/MD2, which would transmit or modulate signals into the cell.

Mentions: Our results raise the question as to how galectin-3 oligomerization could lower the activation threshold of neutrophils to LPS. One possibility is that each galectin-3 oligomer, binding several LPS molecules, cross-links LPS receptors such as CD14 and TLR4 on the cell surface, thereby increasing their cell activating efficiency, as schematized in figure 8. Moreover, simultaneous interaction of galectin-3 carbohydrate recognition domain with glycans on LPS receptors such as CD14 [31], [40], [41] could further enhance the binding of LPS to the cell surface. Another hypothesis, which does not exclude the former one, is that galectin 3 dissociates LPS aggregates, as has been observed with LPS-binding protein (LBP). Indeed, LPS, in aqueous suspension, forms aggregates with low cell activating efficiency, which are disrupted by LPS-binding protein (LBP) [42]. This accelerates LPS binding to CD14 and enhances the resulting TLR-4-mediated cell signaling. We observed that the fluorescence of FITC-LPS was enhanced upon incubation with Galectin 3 (Figure 6), which indicates that galectin-3 somehow interferes with LPS aggregate structure. However, at this point we are not able to assert that Gal 3 promotes LPS disaggregation in a way similar to LBP or that it just interferes with the space between FITC molecules, thus promoting dequenching [30]. Gal 3 putative dissociation of LPS aggregates (Figure 8) could account for the ability of low concentrations of LPS to stimulate neutrophils, when pre-incubated with Gal 3, even in the absence of serum LBP. An intriguing observation is galectin-3 enhancement of cell activation induced by Salmonella minesota LPS, which only binds to galectin-3 via its N-terminal part. This indicates that galectin-3 oligomerization may be triggered by the engagement of the N-terminal part with ligands, an observation not described so far, opening a new avenue in the study of galectin-3 functions.


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)

Schematic view of putative mechanisms responsible for galectin-3 enhancement of LPS-induced neutrophil activation.Monomeric Galectin-3 interacts with LPS glycans from LPS aggregates via its C-terminal domain. LPS/Gal 3 interactions induce Gal 3 oligomerization, which promotes the dissolution of LPS aggregates, stabilizes LPS monomers and enhances the LPS interaction with surface receptors, leading to increased neutrophil activation. CD14 only is shown as the initial LPS ligand on neutrophils, because it is highly glycosylated and could, potentially, interact directly with Gal 3. Moreover, being GPI-anchored, CD14 would be most easily clustered on the cell surface. However, this implies a secondary clustering of TLR/MD2, which would transmit or modulate signals into the cell.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198732&req=5

pone-0026004-g008: Schematic view of putative mechanisms responsible for galectin-3 enhancement of LPS-induced neutrophil activation.Monomeric Galectin-3 interacts with LPS glycans from LPS aggregates via its C-terminal domain. LPS/Gal 3 interactions induce Gal 3 oligomerization, which promotes the dissolution of LPS aggregates, stabilizes LPS monomers and enhances the LPS interaction with surface receptors, leading to increased neutrophil activation. CD14 only is shown as the initial LPS ligand on neutrophils, because it is highly glycosylated and could, potentially, interact directly with Gal 3. Moreover, being GPI-anchored, CD14 would be most easily clustered on the cell surface. However, this implies a secondary clustering of TLR/MD2, which would transmit or modulate signals into the cell.
Mentions: Our results raise the question as to how galectin-3 oligomerization could lower the activation threshold of neutrophils to LPS. One possibility is that each galectin-3 oligomer, binding several LPS molecules, cross-links LPS receptors such as CD14 and TLR4 on the cell surface, thereby increasing their cell activating efficiency, as schematized in figure 8. Moreover, simultaneous interaction of galectin-3 carbohydrate recognition domain with glycans on LPS receptors such as CD14 [31], [40], [41] could further enhance the binding of LPS to the cell surface. Another hypothesis, which does not exclude the former one, is that galectin 3 dissociates LPS aggregates, as has been observed with LPS-binding protein (LBP). Indeed, LPS, in aqueous suspension, forms aggregates with low cell activating efficiency, which are disrupted by LPS-binding protein (LBP) [42]. This accelerates LPS binding to CD14 and enhances the resulting TLR-4-mediated cell signaling. We observed that the fluorescence of FITC-LPS was enhanced upon incubation with Galectin 3 (Figure 6), which indicates that galectin-3 somehow interferes with LPS aggregate structure. However, at this point we are not able to assert that Gal 3 promotes LPS disaggregation in a way similar to LBP or that it just interferes with the space between FITC molecules, thus promoting dequenching [30]. Gal 3 putative dissociation of LPS aggregates (Figure 8) could account for the ability of low concentrations of LPS to stimulate neutrophils, when pre-incubated with Gal 3, even in the absence of serum LBP. An intriguing observation is galectin-3 enhancement of cell activation induced by Salmonella minesota LPS, which only binds to galectin-3 via its N-terminal part. This indicates that galectin-3 oligomerization may be triggered by the engagement of the N-terminal part with ligands, an observation not described so far, opening a new avenue in the study of galectin-3 functions.

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