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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 specifically enhances LPS-mediated neutrophil activation and requires a direct interaction with LPS for its effect.A: PMNs were primed for 30 min at 37°C with Gal 3 (0.2 µM) or E. coli LPS (1 µg/mL) and then stimulated with LPS (1 µg/mL) or Gal 3 (0.2 µM), respectively. Alternatively, Gal 3 (1 µM) and LPS (5 µg/mL) were preincubated together for 30 min at 37°C and then diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) to be used to stimulate PMNs (n = 2). B: Effect of increasing LPS and Gal 3 concentrations in the pre-incubation mixture. Increasing concentrations of Gal 3 (0.2 µM to 25 µM) and LPS (1 µg/mL to 125 µg/mL) were preincubated together for 30 min at 37°C. They were then diluted in HBSS2+ to the final Gal 3 − 0.2 µM and LPS – 1 µg/ml concentrations and added to PMN for a 45-min incubation at 37°C. C: Time-response curve of LPS and Gal 3 preincubation. Gal 3 (7 µM) and LPS (35 µg/mL) were preincubated together for 0–30 min, as indicated, at 37°C and then diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) to be used to stimulate PMNs for a 45-min incubation at 37°C (representative from 2 independent experiments). D: PMNs were stimulated with LPS (1 µg/ml), fMLP (10−6 M), TNF-α (10 ng/ml) or IL-8 (25 ng/ml), which had all been preincubated without (black) or with Gal 3 (0.4 µM, grey) for 30 min at 37°C. Neutrophil CD11b expression was measured as in figure 1 (n = 5). E: Gal 3 effect on neutrophil activation by LPS from various strains of bacteria. Gal 3 (1 µM) was preincubated with 10 µg/ml LPS from E. coli, K. pneumonia or S. minnesota R7 for 30 min at 37°C, in presence or absence of 10 mM lactose (lac). Then, each mixture was diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) concentrations and used to stimulate PMNs (2×106/mL) for 45 min at 37°C, and analyzed for CD11b expression as above. Results are expressed as mean fluorescence intensity (MFI) (n = 3). * p<0.05, **p<0.01, ***p<0.001.
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pone-0026004-g003: Gal 3 specifically enhances LPS-mediated neutrophil activation and requires a direct interaction with LPS for its effect.A: PMNs were primed for 30 min at 37°C with Gal 3 (0.2 µM) or E. coli LPS (1 µg/mL) and then stimulated with LPS (1 µg/mL) or Gal 3 (0.2 µM), respectively. Alternatively, Gal 3 (1 µM) and LPS (5 µg/mL) were preincubated together for 30 min at 37°C and then diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) to be used to stimulate PMNs (n = 2). B: Effect of increasing LPS and Gal 3 concentrations in the pre-incubation mixture. Increasing concentrations of Gal 3 (0.2 µM to 25 µM) and LPS (1 µg/mL to 125 µg/mL) were preincubated together for 30 min at 37°C. They were then diluted in HBSS2+ to the final Gal 3 − 0.2 µM and LPS – 1 µg/ml concentrations and added to PMN for a 45-min incubation at 37°C. C: Time-response curve of LPS and Gal 3 preincubation. Gal 3 (7 µM) and LPS (35 µg/mL) were preincubated together for 0–30 min, as indicated, at 37°C and then diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) to be used to stimulate PMNs for a 45-min incubation at 37°C (representative from 2 independent experiments). D: PMNs were stimulated with LPS (1 µg/ml), fMLP (10−6 M), TNF-α (10 ng/ml) or IL-8 (25 ng/ml), which had all been preincubated without (black) or with Gal 3 (0.4 µM, grey) for 30 min at 37°C. Neutrophil CD11b expression was measured as in figure 1 (n = 5). E: Gal 3 effect on neutrophil activation by LPS from various strains of bacteria. Gal 3 (1 µM) was preincubated with 10 µg/ml LPS from E. coli, K. pneumonia or S. minnesota R7 for 30 min at 37°C, in presence or absence of 10 mM lactose (lac). Then, each mixture was diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) concentrations and used to stimulate PMNs (2×106/mL) for 45 min at 37°C, and analyzed for CD11b expression as above. Results are expressed as mean fluorescence intensity (MFI) (n = 3). * p<0.05, **p<0.01, ***p<0.001.

Mentions: We initially concluded, as others [26], [27], that the effect of Gal 3 on neutrophils required cell priming by LPS. However, surprisingly, the enhancing effect of Gal 3 on LPS-mediated neutrophil activation was not observed when neutrophils were primed with 1 µg/mL of LPS before the addition of Gal 3 (0.2 µM) or vice-versa (Figure 3A). Indeed, LPS- or Gal 3-primed cells did not differ significantly from cells treated with Gal 3 or LPS alone (Figure 3A). Importantly, Gal 3 effects on neutrophil activation required a prior interaction of Gal 3 with LPS, since only the pre-incubated Gal 3/LPS was able to significantly increase neutrophil activation (Figure 3A). In fact, we observed a dose-dependent up-regulation of CD11b expression with increasing concentrations of LPS/Gal 3 during the preincubation step, although the final concentration in contact with neutrophils was kept constant (0.2 µM Gal 3, 1 µg/ml LPS) (Figure 3B). Moreover, a time-response curve for LPS/Gal 3 preincubation revealed that the ability of Gal 3 to interact with LPS and to induce CD11b up-regulation of expression was very rapid in onset, reaching a plateau after 10 min of preincubation (Figure 3C). These results further emphasize the importance of a direct interaction of Gal 3 with LPS. The ability of Gal 3 to enhance neutrophil activation was specifically observed with LPS and not upon pre-incubation with other stimuli such as fMLP, TNF-α or IL-8 (Figure 3D).


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 specifically enhances LPS-mediated neutrophil activation and requires a direct interaction with LPS for its effect.A: PMNs were primed for 30 min at 37°C with Gal 3 (0.2 µM) or E. coli LPS (1 µg/mL) and then stimulated with LPS (1 µg/mL) or Gal 3 (0.2 µM), respectively. Alternatively, Gal 3 (1 µM) and LPS (5 µg/mL) were preincubated together for 30 min at 37°C and then diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) to be used to stimulate PMNs (n = 2). B: Effect of increasing LPS and Gal 3 concentrations in the pre-incubation mixture. Increasing concentrations of Gal 3 (0.2 µM to 25 µM) and LPS (1 µg/mL to 125 µg/mL) were preincubated together for 30 min at 37°C. They were then diluted in HBSS2+ to the final Gal 3 − 0.2 µM and LPS – 1 µg/ml concentrations and added to PMN for a 45-min incubation at 37°C. C: Time-response curve of LPS and Gal 3 preincubation. Gal 3 (7 µM) and LPS (35 µg/mL) were preincubated together for 0–30 min, as indicated, at 37°C and then diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) to be used to stimulate PMNs for a 45-min incubation at 37°C (representative from 2 independent experiments). D: PMNs were stimulated with LPS (1 µg/ml), fMLP (10−6 M), TNF-α (10 ng/ml) or IL-8 (25 ng/ml), which had all been preincubated without (black) or with Gal 3 (0.4 µM, grey) for 30 min at 37°C. Neutrophil CD11b expression was measured as in figure 1 (n = 5). E: Gal 3 effect on neutrophil activation by LPS from various strains of bacteria. Gal 3 (1 µM) was preincubated with 10 µg/ml LPS from E. coli, K. pneumonia or S. minnesota R7 for 30 min at 37°C, in presence or absence of 10 mM lactose (lac). Then, each mixture was diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) concentrations and used to stimulate PMNs (2×106/mL) for 45 min at 37°C, and analyzed for CD11b expression as above. Results are expressed as mean fluorescence intensity (MFI) (n = 3). * p<0.05, **p<0.01, ***p<0.001.
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pone-0026004-g003: Gal 3 specifically enhances LPS-mediated neutrophil activation and requires a direct interaction with LPS for its effect.A: PMNs were primed for 30 min at 37°C with Gal 3 (0.2 µM) or E. coli LPS (1 µg/mL) and then stimulated with LPS (1 µg/mL) or Gal 3 (0.2 µM), respectively. Alternatively, Gal 3 (1 µM) and LPS (5 µg/mL) were preincubated together for 30 min at 37°C and then diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) to be used to stimulate PMNs (n = 2). B: Effect of increasing LPS and Gal 3 concentrations in the pre-incubation mixture. Increasing concentrations of Gal 3 (0.2 µM to 25 µM) and LPS (1 µg/mL to 125 µg/mL) were preincubated together for 30 min at 37°C. They were then diluted in HBSS2+ to the final Gal 3 − 0.2 µM and LPS – 1 µg/ml concentrations and added to PMN for a 45-min incubation at 37°C. C: Time-response curve of LPS and Gal 3 preincubation. Gal 3 (7 µM) and LPS (35 µg/mL) were preincubated together for 0–30 min, as indicated, at 37°C and then diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) to be used to stimulate PMNs for a 45-min incubation at 37°C (representative from 2 independent experiments). D: PMNs were stimulated with LPS (1 µg/ml), fMLP (10−6 M), TNF-α (10 ng/ml) or IL-8 (25 ng/ml), which had all been preincubated without (black) or with Gal 3 (0.4 µM, grey) for 30 min at 37°C. Neutrophil CD11b expression was measured as in figure 1 (n = 5). E: Gal 3 effect on neutrophil activation by LPS from various strains of bacteria. Gal 3 (1 µM) was preincubated with 10 µg/ml LPS from E. coli, K. pneumonia or S. minnesota R7 for 30 min at 37°C, in presence or absence of 10 mM lactose (lac). Then, each mixture was diluted to 0.2 µM (Gal 3) and 1 µg/mL (LPS) concentrations and used to stimulate PMNs (2×106/mL) for 45 min at 37°C, and analyzed for CD11b expression as above. Results are expressed as mean fluorescence intensity (MFI) (n = 3). * p<0.05, **p<0.01, ***p<0.001.
Mentions: We initially concluded, as others [26], [27], that the effect of Gal 3 on neutrophils required cell priming by LPS. However, surprisingly, the enhancing effect of Gal 3 on LPS-mediated neutrophil activation was not observed when neutrophils were primed with 1 µg/mL of LPS before the addition of Gal 3 (0.2 µM) or vice-versa (Figure 3A). Indeed, LPS- or Gal 3-primed cells did not differ significantly from cells treated with Gal 3 or LPS alone (Figure 3A). Importantly, Gal 3 effects on neutrophil activation required a prior interaction of Gal 3 with LPS, since only the pre-incubated Gal 3/LPS was able to significantly increase neutrophil activation (Figure 3A). In fact, we observed a dose-dependent up-regulation of CD11b expression with increasing concentrations of LPS/Gal 3 during the preincubation step, although the final concentration in contact with neutrophils was kept constant (0.2 µM Gal 3, 1 µg/ml LPS) (Figure 3B). Moreover, a time-response curve for LPS/Gal 3 preincubation revealed that the ability of Gal 3 to interact with LPS and to induce CD11b up-regulation of expression was very rapid in onset, reaching a plateau after 10 min of preincubation (Figure 3C). These results further emphasize the importance of a direct interaction of Gal 3 with LPS. The ability of Gal 3 to enhance neutrophil activation was specifically observed with LPS and not upon pre-incubation with other stimuli such as fMLP, TNF-α or IL-8 (Figure 3D).

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