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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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Contaminating LPS interferes with the ability of Gal 3 to activate neutrophils.Freshly isolated neutrophils (2.0×106/ml) were incubated for 45 min at 37°C in HBSS2+ in the presence of: A: Recombinant Gal 3 preparation (0.2 µM), which had been depleted of endotoxin (detoxi) or not (extract), by absorption on detoxigel (n = 3). B: 0.2 µM of extract or endotoxin-depleted recombinant Gal 3, which had been preincubated for 30 min at 37°C with polymyxin B (PMX, 40 mg/ml) (n = 3). C: Increasing molar concentrations of endotoxin-depleted Gal 3 (n = 3). After staining with a specific anti-CD11b mAb, cells were analyzed by flow cytometry. D: Non-stimulated PMN (2.0×106/mL) were incubated with FITC-labeled Gal 3 (0.2 µM) for 30 min at 4°C, in the presence or absence of 20 mM sucrose or lactose. The binding of FITC-Gal 3 to neutrophils was analyzed by flow cytometry (n = 3). Results are expressed as mean fluorescence intensity (MFI). * p<0.05; **p<0.01; ***p<0.001.
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pone-0026004-g001: Contaminating LPS interferes with the ability of Gal 3 to activate neutrophils.Freshly isolated neutrophils (2.0×106/ml) were incubated for 45 min at 37°C in HBSS2+ in the presence of: A: Recombinant Gal 3 preparation (0.2 µM), which had been depleted of endotoxin (detoxi) or not (extract), by absorption on detoxigel (n = 3). B: 0.2 µM of extract or endotoxin-depleted recombinant Gal 3, which had been preincubated for 30 min at 37°C with polymyxin B (PMX, 40 mg/ml) (n = 3). C: Increasing molar concentrations of endotoxin-depleted Gal 3 (n = 3). After staining with a specific anti-CD11b mAb, cells were analyzed by flow cytometry. D: Non-stimulated PMN (2.0×106/mL) were incubated with FITC-labeled Gal 3 (0.2 µM) for 30 min at 4°C, in the presence or absence of 20 mM sucrose or lactose. The binding of FITC-Gal 3 to neutrophils was analyzed by flow cytometry (n = 3). Results are expressed as mean fluorescence intensity (MFI). * p<0.05; **p<0.01; ***p<0.001.

Mentions: We first analyzed the effects of recombinant Gal 3, isolated from transfected E. coli as described [22], without (Gal 3 extract) and with (detoxi) LPS elimination on detoxi-gel and incubated with freshly isolated unprimed neutrophils. As shown in figure 1A, the incubation with 0.2 µM Gal 3 extract resulted in neutrophil activation, as measured by the increase of CD11b expression. This effect was not observed with the same molar concentration of endotoxin-free detoxigel-treated Gal 3 (detoxi-Gal 3). LPS contamination of the Gal 3 extract preparation, as measured by Limulus Amebocyte Lysate test, was less than 400 nanograms of LPS per mg of Gal 3 (data not shown). We then pretreated the Gal 3 extract preparation with polymyxin B (PMX), to block the effect of contaminating LPS. As shown in figure 1B, PMX prevented CD11b up-regulation promoted by Gal 3 extract preparation or by LPS. We then analyzed the effect of increasing doses of endotoxin-free detoxigel-treated Gal 3 on unprimed neutrophils. As shown in figure 1C, detoxi-Gal 3 concentrations up to 0.4 µM had no significant effect on neutrophil activation state. One should point out, however, that high molar concentrations (≥0.8 µM) resulted in a significant up-regulation of CD11b membrane expression which, in this case, was PMX-insensitive.


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)

Contaminating LPS interferes with the ability of Gal 3 to activate neutrophils.Freshly isolated neutrophils (2.0×106/ml) were incubated for 45 min at 37°C in HBSS2+ in the presence of: A: Recombinant Gal 3 preparation (0.2 µM), which had been depleted of endotoxin (detoxi) or not (extract), by absorption on detoxigel (n = 3). B: 0.2 µM of extract or endotoxin-depleted recombinant Gal 3, which had been preincubated for 30 min at 37°C with polymyxin B (PMX, 40 mg/ml) (n = 3). C: Increasing molar concentrations of endotoxin-depleted Gal 3 (n = 3). After staining with a specific anti-CD11b mAb, cells were analyzed by flow cytometry. D: Non-stimulated PMN (2.0×106/mL) were incubated with FITC-labeled Gal 3 (0.2 µM) for 30 min at 4°C, in the presence or absence of 20 mM sucrose or lactose. The binding of FITC-Gal 3 to neutrophils was analyzed by flow cytometry (n = 3). Results are expressed as mean fluorescence intensity (MFI). * p<0.05; **p<0.01; ***p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198732&req=5

pone-0026004-g001: Contaminating LPS interferes with the ability of Gal 3 to activate neutrophils.Freshly isolated neutrophils (2.0×106/ml) were incubated for 45 min at 37°C in HBSS2+ in the presence of: A: Recombinant Gal 3 preparation (0.2 µM), which had been depleted of endotoxin (detoxi) or not (extract), by absorption on detoxigel (n = 3). B: 0.2 µM of extract or endotoxin-depleted recombinant Gal 3, which had been preincubated for 30 min at 37°C with polymyxin B (PMX, 40 mg/ml) (n = 3). C: Increasing molar concentrations of endotoxin-depleted Gal 3 (n = 3). After staining with a specific anti-CD11b mAb, cells were analyzed by flow cytometry. D: Non-stimulated PMN (2.0×106/mL) were incubated with FITC-labeled Gal 3 (0.2 µM) for 30 min at 4°C, in the presence or absence of 20 mM sucrose or lactose. The binding of FITC-Gal 3 to neutrophils was analyzed by flow cytometry (n = 3). Results are expressed as mean fluorescence intensity (MFI). * p<0.05; **p<0.01; ***p<0.001.
Mentions: We first analyzed the effects of recombinant Gal 3, isolated from transfected E. coli as described [22], without (Gal 3 extract) and with (detoxi) LPS elimination on detoxi-gel and incubated with freshly isolated unprimed neutrophils. As shown in figure 1A, the incubation with 0.2 µM Gal 3 extract resulted in neutrophil activation, as measured by the increase of CD11b expression. This effect was not observed with the same molar concentration of endotoxin-free detoxigel-treated Gal 3 (detoxi-Gal 3). LPS contamination of the Gal 3 extract preparation, as measured by Limulus Amebocyte Lysate test, was less than 400 nanograms of LPS per mg of Gal 3 (data not shown). We then pretreated the Gal 3 extract preparation with polymyxin B (PMX), to block the effect of contaminating LPS. As shown in figure 1B, PMX prevented CD11b up-regulation promoted by Gal 3 extract preparation or by LPS. We then analyzed the effect of increasing doses of endotoxin-free detoxigel-treated Gal 3 on unprimed neutrophils. As shown in figure 1C, detoxi-Gal 3 concentrations up to 0.4 µM had no significant effect on neutrophil activation state. One should point out, however, that high molar concentrations (≥0.8 µM) resulted in a significant up-regulation of CD11b membrane expression which, in this case, was PMX-insensitive.

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