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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 increases the ability of LPS to activate neutrophils and induces oxidative responses.PMNs (2.0×106/ml) were incubated for 45 min at 37°C with E. coli LPS (1 µg/mL) and Gal 3 (0.4 µM), which had been preincubated separately or together for 30 min at 37°C; after labeling with a specific anti-CD11b mAb, cells were analyzed by flow cytometry. A: Representative histrogram of neutrophil CD11b expression. B: Bar graph representation of data obtained from 5 experiments (mean ± SD). C: PMNs were incubated at 37°C for 30 min with DCFHDA and either Gal 3 (5 µg/ml), LPS (1 µg/ml) or a mixture of Gal 3 and LPS pre-incubated for 20 min at 37°C. Then, PMN were further activated or not with fMLP 10−6 M for 7 minutes. The reaction was stopped by the addition of ice-cold PBS-azide 0.1% and the oxydation-dependent fluorescence of intracellular DCFHDA was measured as described in Methods. The results are given as DCFHDA index, i.e. the mean fluorescence intensity MFI of each sample/MFI of resting PMN (n = 3). D: Increasing concentrations of LPS (0.1 to 1000 ng/ml), in 0.2% autologous serum, were preincubated or not with Gal 3 (0.4 µM) and then added to PMNs. CD11b expression was measured as above after a 45-min incubation and expressed as mean MFI ± SD (n = 3). *p<0.05, ** p<0.01.
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pone-0026004-g002: Gal 3 increases the ability of LPS to activate neutrophils and induces oxidative responses.PMNs (2.0×106/ml) were incubated for 45 min at 37°C with E. coli LPS (1 µg/mL) and Gal 3 (0.4 µM), which had been preincubated separately or together for 30 min at 37°C; after labeling with a specific anti-CD11b mAb, cells were analyzed by flow cytometry. A: Representative histrogram of neutrophil CD11b expression. B: Bar graph representation of data obtained from 5 experiments (mean ± SD). C: PMNs were incubated at 37°C for 30 min with DCFHDA and either Gal 3 (5 µg/ml), LPS (1 µg/ml) or a mixture of Gal 3 and LPS pre-incubated for 20 min at 37°C. Then, PMN were further activated or not with fMLP 10−6 M for 7 minutes. The reaction was stopped by the addition of ice-cold PBS-azide 0.1% and the oxydation-dependent fluorescence of intracellular DCFHDA was measured as described in Methods. The results are given as DCFHDA index, i.e. the mean fluorescence intensity MFI of each sample/MFI of resting PMN (n = 3). D: Increasing concentrations of LPS (0.1 to 1000 ng/ml), in 0.2% autologous serum, were preincubated or not with Gal 3 (0.4 µM) and then added to PMNs. CD11b expression was measured as above after a 45-min incubation and expressed as mean MFI ± SD (n = 3). *p<0.05, ** p<0.01.

Mentions: The effects of Gal 3 on LPS-mediated neutrophil activation were then investigated. LPS is known to lose most of its neutrophil activating efficiency in the absence of serum LPS-binding protein (LBP) and soluble CD14 [24] and concentrations as high as 10 µg/ml of LPS are required to activate neutrophils in the absence of serum (data not shown). When unprimed human neutrophils were incubated with suboptimal E. coli LPS concentration (1 µg/ml) or with 0.4 µM detoxi-Gal 3, separately, these stimuli induced little or no neutrophil activation (Figure 2A). By contrast, a two- to three-fold increase of CD11b expression was observed when neutrophils were treated with the same amount of LPS that had been preincubated with Gal 3 (Figure 2A, 2B). We also tested whether Gal 3 was able to enhance neutrophil oxidative responses to LPS. LPS is not an efficient trigger of the oxidative burst but it is known to prime PMN for an enhanced oxidative response to fMLP [25]. Therefore, we first primed PMN with either Gal 3, LPS or a pre-incubated mixture of Gal 3 and LPS, in the presence of the fluorescent probe DCFHDA, and then further activated PMN with fMLP. The results show that LPS preincubation with Gal 3 increases the oxidant response of PMN to LPS itself (DCFHDA indexes of 2.77±0,2 and 2±0.09 with LPS/Gal3 and LPS alone, respectively) (Figure 2C). Moreover, it potentiates LPS priming effect: PMN preincubation with the LPS/Gal3 mixture lead to up to 3 fold the oxidative response to fMLP, as compared to the 1.8 fold increase observed after priming by LPS alone (Figure 2C).


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 increases the ability of LPS to activate neutrophils and induces oxidative responses.PMNs (2.0×106/ml) were incubated for 45 min at 37°C with E. coli LPS (1 µg/mL) and Gal 3 (0.4 µM), which had been preincubated separately or together for 30 min at 37°C; after labeling with a specific anti-CD11b mAb, cells were analyzed by flow cytometry. A: Representative histrogram of neutrophil CD11b expression. B: Bar graph representation of data obtained from 5 experiments (mean ± SD). C: PMNs were incubated at 37°C for 30 min with DCFHDA and either Gal 3 (5 µg/ml), LPS (1 µg/ml) or a mixture of Gal 3 and LPS pre-incubated for 20 min at 37°C. Then, PMN were further activated or not with fMLP 10−6 M for 7 minutes. The reaction was stopped by the addition of ice-cold PBS-azide 0.1% and the oxydation-dependent fluorescence of intracellular DCFHDA was measured as described in Methods. The results are given as DCFHDA index, i.e. the mean fluorescence intensity MFI of each sample/MFI of resting PMN (n = 3). D: Increasing concentrations of LPS (0.1 to 1000 ng/ml), in 0.2% autologous serum, were preincubated or not with Gal 3 (0.4 µM) and then added to PMNs. CD11b expression was measured as above after a 45-min incubation and expressed as mean MFI ± SD (n = 3). *p<0.05, ** p<0.01.
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Related In: Results  -  Collection

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pone-0026004-g002: Gal 3 increases the ability of LPS to activate neutrophils and induces oxidative responses.PMNs (2.0×106/ml) were incubated for 45 min at 37°C with E. coli LPS (1 µg/mL) and Gal 3 (0.4 µM), which had been preincubated separately or together for 30 min at 37°C; after labeling with a specific anti-CD11b mAb, cells were analyzed by flow cytometry. A: Representative histrogram of neutrophil CD11b expression. B: Bar graph representation of data obtained from 5 experiments (mean ± SD). C: PMNs were incubated at 37°C for 30 min with DCFHDA and either Gal 3 (5 µg/ml), LPS (1 µg/ml) or a mixture of Gal 3 and LPS pre-incubated for 20 min at 37°C. Then, PMN were further activated or not with fMLP 10−6 M for 7 minutes. The reaction was stopped by the addition of ice-cold PBS-azide 0.1% and the oxydation-dependent fluorescence of intracellular DCFHDA was measured as described in Methods. The results are given as DCFHDA index, i.e. the mean fluorescence intensity MFI of each sample/MFI of resting PMN (n = 3). D: Increasing concentrations of LPS (0.1 to 1000 ng/ml), in 0.2% autologous serum, were preincubated or not with Gal 3 (0.4 µM) and then added to PMNs. CD11b expression was measured as above after a 45-min incubation and expressed as mean MFI ± SD (n = 3). *p<0.05, ** p<0.01.
Mentions: The effects of Gal 3 on LPS-mediated neutrophil activation were then investigated. LPS is known to lose most of its neutrophil activating efficiency in the absence of serum LPS-binding protein (LBP) and soluble CD14 [24] and concentrations as high as 10 µg/ml of LPS are required to activate neutrophils in the absence of serum (data not shown). When unprimed human neutrophils were incubated with suboptimal E. coli LPS concentration (1 µg/ml) or with 0.4 µM detoxi-Gal 3, separately, these stimuli induced little or no neutrophil activation (Figure 2A). By contrast, a two- to three-fold increase of CD11b expression was observed when neutrophils were treated with the same amount of LPS that had been preincubated with Gal 3 (Figure 2A, 2B). We also tested whether Gal 3 was able to enhance neutrophil oxidative responses to LPS. LPS is not an efficient trigger of the oxidative burst but it is known to prime PMN for an enhanced oxidative response to fMLP [25]. Therefore, we first primed PMN with either Gal 3, LPS or a pre-incubated mixture of Gal 3 and LPS, in the presence of the fluorescent probe DCFHDA, and then further activated PMN with fMLP. The results show that LPS preincubation with Gal 3 increases the oxidant response of PMN to LPS itself (DCFHDA indexes of 2.77±0,2 and 2±0.09 with LPS/Gal3 and LPS alone, respectively) (Figure 2C). Moreover, it potentiates LPS priming effect: PMN preincubation with the LPS/Gal3 mixture lead to up to 3 fold the oxidative response to fMLP, as compared to the 1.8 fold increase observed after priming by LPS alone (Figure 2C).

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