LPS-Induced Galectin-3 Oligomerization Results in Enhancement of Neutrophil Activation

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.

Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

“…Each cell pellet was then resuspended in 1 ml of HBHA buffer, and binding to an optimal concentration of rLILRA3-APtag-His (30 nM) was performed as described above. To determine whether glycosylation contrib-utes for ligand binding, U937 cells were preincubated with 0.1-0.2 M ␤-lactose or control sucrose or NaCl (Sigma) for 1 h at 37°C followed by a brief wash with cold HBHA buffer and binding to 30 nM rLILRA3-APtag-His or rAPtag-His control performed as described (14,15).…”

Galectin-3 Plays an Important Role in Innate Immunity to Gastric Infection by Helicobacter pylori

“…Of note, the phagocytosis-promoting functions of Gal3 appear to operate mostly through intracellular mechanisms, with Gal3 being localized in phagocytic cups and phagosomes of macrophages containing phagocytosed erythrocytes (18) or in bacterium-containing phagosomes of Mycobacterium-infected macrophages (17). Furthermore, extracellular Gal3 promotes neutrophil functions such as survival, phagocytosis, and extravasation (19)(20)(21). Gal3 also binds to LPS from various bacterial species and may serve as a negative regulator of LPS-induced inflammation, protecting hosts from endotoxin shock (22).…”

Detection of Galectin-3 Interaction with Commensal Bacteria

“…Pathogenic interaction with gal-3 may result in suppressed or exaggerated states of endotoxic shock (12,19) or increased adhesion to host tissues (20). These studies highlight the ability of pathogens to capitalize on the presence of gal-3 to augment their capacity to colonize and survive within the host environment.…”

“…Gal-3 was first demonstrated to associate with the lipopolysaccharide (LPS) of Klebsiella pneumoniae through the binding of ␤-galactoside glycans in the outer core. In addition, interactions with Escherichia coli (LPS) (12), Pseudomonas aeruginosa (outer core of LPS) (13), Neisseria gonorrhoeae (lipooligosaccharides) (14), and Helicobacter pylori (O antigen of LPS) (15) have been described. In a recent paper, Fermino et al (12) suggested that gal-3 may have a broader specificity than LPS.…”

“…In addition, interactions with Escherichia coli (LPS) (12), Pseudomonas aeruginosa (outer core of LPS) (13), Neisseria gonorrhoeae (lipooligosaccharides) (14), and Helicobacter pylori (O antigen of LPS) (15) have been described. In a recent paper, Fermino et al (12) suggested that gal-3 may have a broader specificity than LPS. Indeed, gal-3 has been shown to bind mycolic acid, the major constituent of the Mycobacterium tuberculosis cell envelope (16).…”