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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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Gal 3/LPS interaction enhances the fluorescence of FITC-LPS.FITC-labeled E. coli LPS (10 µg/ml) was incubated with 4 µM Gal 3 or 2% serum for 2 hours, and then applied to a gel filtration column (Superdex TM 75 HR 10/30 column). FITC-LPS alone or Gal 3 alone were also analyzed. 0.5 ml fractions of FITC-LPS ± Gal 3 or ±2% serum were collected and further analyzed on a fluorescence microplate reader. The excitation and emission wavelengths were at 475 nm and 520 nm, respectively. Data represents the fluorescence of the main peak of FITC-LPS recovered in the void volume. ** p<0.01, ***p<0.001.
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pone-0026004-g006: Gal 3/LPS interaction enhances the fluorescence of FITC-LPS.FITC-labeled E. coli LPS (10 µg/ml) was incubated with 4 µM Gal 3 or 2% serum for 2 hours, and then applied to a gel filtration column (Superdex TM 75 HR 10/30 column). FITC-LPS alone or Gal 3 alone were also analyzed. 0.5 ml fractions of FITC-LPS ± Gal 3 or ±2% serum were collected and further analyzed on a fluorescence microplate reader. The excitation and emission wavelengths were at 475 nm and 520 nm, respectively. Data represents the fluorescence of the main peak of FITC-LPS recovered in the void volume. ** p<0.01, ***p<0.001.

Mentions: The fact that Gal 3/LPS interaction induces greater-than 20-fold increase in FITC-LPS binding to neutrophils but only a three-fold increase in LPS-mediated CD11b up-regulation raised the possibility that galectin-3 might interfere with the intramolecular self-quenching of fluorescein. It has long been known that the fluorescence of FITC-LPS aggregates is self quenched but should increase when the aggregates dissociate [29] or the spacing between FITC-LPS molecules within aggregates increase [30], because of dequenching. Therefore, FITC-LPS, preincubated or not with Gal 3 or with serum LPB, was analyzed by gel filtration followed by fluorescence detection. Figure 6 represents the analysis of the main peak of FITC-LPS recovered in the void volume and clearly demonstrates that the presence of Gal 3 enhances three times the FITC-LPS fluorescence. Similar results were obtained when FITC-LPS was preincubated with serum LBP, which it is known to promote LPS disaggregation [31]. This result suggests that Gal 3/LPS interaction is able to modify the LPS aggregate structure, which may account, at least in part, for its potentiating effect on LPS-induced neutrophil activation.


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)

Gal 3/LPS interaction enhances the fluorescence of FITC-LPS.FITC-labeled E. coli LPS (10 µg/ml) was incubated with 4 µM Gal 3 or 2% serum for 2 hours, and then applied to a gel filtration column (Superdex TM 75 HR 10/30 column). FITC-LPS alone or Gal 3 alone were also analyzed. 0.5 ml fractions of FITC-LPS ± Gal 3 or ±2% serum were collected and further analyzed on a fluorescence microplate reader. The excitation and emission wavelengths were at 475 nm and 520 nm, respectively. Data represents the fluorescence of the main peak of FITC-LPS recovered in the void volume. ** p<0.01, ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026004-g006: Gal 3/LPS interaction enhances the fluorescence of FITC-LPS.FITC-labeled E. coli LPS (10 µg/ml) was incubated with 4 µM Gal 3 or 2% serum for 2 hours, and then applied to a gel filtration column (Superdex TM 75 HR 10/30 column). FITC-LPS alone or Gal 3 alone were also analyzed. 0.5 ml fractions of FITC-LPS ± Gal 3 or ±2% serum were collected and further analyzed on a fluorescence microplate reader. The excitation and emission wavelengths were at 475 nm and 520 nm, respectively. Data represents the fluorescence of the main peak of FITC-LPS recovered in the void volume. ** p<0.01, ***p<0.001.
Mentions: The fact that Gal 3/LPS interaction induces greater-than 20-fold increase in FITC-LPS binding to neutrophils but only a three-fold increase in LPS-mediated CD11b up-regulation raised the possibility that galectin-3 might interfere with the intramolecular self-quenching of fluorescein. It has long been known that the fluorescence of FITC-LPS aggregates is self quenched but should increase when the aggregates dissociate [29] or the spacing between FITC-LPS molecules within aggregates increase [30], because of dequenching. Therefore, FITC-LPS, preincubated or not with Gal 3 or with serum LPB, was analyzed by gel filtration followed by fluorescence detection. Figure 6 represents the analysis of the main peak of FITC-LPS recovered in the void volume and clearly demonstrates that the presence of Gal 3 enhances three times the FITC-LPS fluorescence. Similar results were obtained when FITC-LPS was preincubated with serum LBP, which it is known to promote LPS disaggregation [31]. This result suggests that Gal 3/LPS interaction is able to modify the LPS aggregate structure, which may account, at least in part, for its potentiating effect on LPS-induced neutrophil activation.

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