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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 enhances LPS binding to the neutrophil surface.A and B: PMNs (2×106/mL) were stimulated for 45 min with Gal 3 (0.4 µM) and E. coli LPS (1 µg/ml), preincubated as in figure 3. TLR4 (A) and CD14 (B) expressions were determined by flow cytometry. The results are expressed as mean fluorescence intensity (MFI) (n = 3). C: FITC-labeled E. coli LPS (12.5 µg/mL) was preincubated with increasing molar concentrations of Gal 3 (from 0.25 to 8 µM) and then added to PMNs (10×106/mL) for additional 20 min incubation at 37°C with (n = 3) D. Gal 3 was treated with 12.5 mM lactose or sucrose for 15 min before being incubated with FITC-LPS (12.5 µg/mL) and added to PMNs (10×106/mL). The binding of FITC-LPS was determined by flow cytometry and expressed as mean fluorescence intensity (MFI).
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pone-0026004-g004: Gal 3 enhances LPS binding to the neutrophil surface.A and B: PMNs (2×106/mL) were stimulated for 45 min with Gal 3 (0.4 µM) and E. coli LPS (1 µg/ml), preincubated as in figure 3. TLR4 (A) and CD14 (B) expressions were determined by flow cytometry. The results are expressed as mean fluorescence intensity (MFI) (n = 3). C: FITC-labeled E. coli LPS (12.5 µg/mL) was preincubated with increasing molar concentrations of Gal 3 (from 0.25 to 8 µM) and then added to PMNs (10×106/mL) for additional 20 min incubation at 37°C with (n = 3) D. Gal 3 was treated with 12.5 mM lactose or sucrose for 15 min before being incubated with FITC-LPS (12.5 µg/mL) and added to PMNs (10×106/mL). The binding of FITC-LPS was determined by flow cytometry and expressed as mean fluorescence intensity (MFI).

Mentions: To test whether the Gal 3-mediated increase in LPS binding might be caused by an up-regulation of LPS receptors on neutrophils, we analyzed the expression of CD14 and TLR4. As shown in figure 4, there was no significant change in TLR4 (A) and CD14 (B) expressions after neutrophil stimulation with the Gal 3/LPS complex. We then hypothesized that Gal 3 enhances the effects of LPS by increasing its binding to neutrophil surface. We thus preincubated FITC-labeled E. coli LPS with increasing concentrations of Gal 3 for 30 min before adding it to neutrophils. One should point out that low concentrations of FITC-LPS (1 µg/mL) preincubated with 0.4 µM of Gal 3, in the absence of serum, did not result in a level of fluorescence intensity detected by flow cytometry. Therefore, we increased proportionally the doses of FITC-LPS and Gal 3 and found that the dose of 12.5 µg/mL LPS allowed the detection of cell-bound LPS. Preincubation of this amount of LPS with increasing concentrations of Gal 3 showed a significant increase of LPS binding to neutrophils for a Gal 3/LPS proportion (4 µM Gal 3 per 10 µg LPS) similar to that required for neutrophil activation. As expected, previous treatment of Gal 3 with lactose (12.5 mM) specifically inhibited the Gal 3-induced binding of LPS to neutrophils (Figure 4D). Gal 3/LPS interactions thus strikingly enhance the binding of LPS to the cell surface.


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 enhances LPS binding to the neutrophil surface.A and B: PMNs (2×106/mL) were stimulated for 45 min with Gal 3 (0.4 µM) and E. coli LPS (1 µg/ml), preincubated as in figure 3. TLR4 (A) and CD14 (B) expressions were determined by flow cytometry. The results are expressed as mean fluorescence intensity (MFI) (n = 3). C: FITC-labeled E. coli LPS (12.5 µg/mL) was preincubated with increasing molar concentrations of Gal 3 (from 0.25 to 8 µM) and then added to PMNs (10×106/mL) for additional 20 min incubation at 37°C with (n = 3) D. Gal 3 was treated with 12.5 mM lactose or sucrose for 15 min before being incubated with FITC-LPS (12.5 µg/mL) and added to PMNs (10×106/mL). The binding of FITC-LPS was determined by flow cytometry and expressed as mean fluorescence intensity (MFI).
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Related In: Results  -  Collection

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pone-0026004-g004: Gal 3 enhances LPS binding to the neutrophil surface.A and B: PMNs (2×106/mL) were stimulated for 45 min with Gal 3 (0.4 µM) and E. coli LPS (1 µg/ml), preincubated as in figure 3. TLR4 (A) and CD14 (B) expressions were determined by flow cytometry. The results are expressed as mean fluorescence intensity (MFI) (n = 3). C: FITC-labeled E. coli LPS (12.5 µg/mL) was preincubated with increasing molar concentrations of Gal 3 (from 0.25 to 8 µM) and then added to PMNs (10×106/mL) for additional 20 min incubation at 37°C with (n = 3) D. Gal 3 was treated with 12.5 mM lactose or sucrose for 15 min before being incubated with FITC-LPS (12.5 µg/mL) and added to PMNs (10×106/mL). The binding of FITC-LPS was determined by flow cytometry and expressed as mean fluorescence intensity (MFI).
Mentions: To test whether the Gal 3-mediated increase in LPS binding might be caused by an up-regulation of LPS receptors on neutrophils, we analyzed the expression of CD14 and TLR4. As shown in figure 4, there was no significant change in TLR4 (A) and CD14 (B) expressions after neutrophil stimulation with the Gal 3/LPS complex. We then hypothesized that Gal 3 enhances the effects of LPS by increasing its binding to neutrophil surface. We thus preincubated FITC-labeled E. coli LPS with increasing concentrations of Gal 3 for 30 min before adding it to neutrophils. One should point out that low concentrations of FITC-LPS (1 µg/mL) preincubated with 0.4 µM of Gal 3, in the absence of serum, did not result in a level of fluorescence intensity detected by flow cytometry. Therefore, we increased proportionally the doses of FITC-LPS and Gal 3 and found that the dose of 12.5 µg/mL LPS allowed the detection of cell-bound LPS. Preincubation of this amount of LPS with increasing concentrations of Gal 3 showed a significant increase of LPS binding to neutrophils for a Gal 3/LPS proportion (4 µM Gal 3 per 10 µg LPS) similar to that required for neutrophil activation. As expected, previous treatment of Gal 3 with lactose (12.5 mM) specifically inhibited the Gal 3-induced binding of LPS to neutrophils (Figure 4D). Gal 3/LPS interactions thus strikingly enhance the binding of LPS to the cell surface.

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