Biochimica et Biophysica Acta (BBA) - General Subjects
Neutrophil activation induced by the lectin KM+ involves binding to CXCR2
Introduction
Neutrophils play a crucial role in inflammation and their accumulation within injured tissue is a hallmark of the acute inflammatory response. The movement of neutrophils from the circulation to the sites of injury is initiated and directed by numerous exogenous and endogenous attractants that bind and activate seven-transmembrane G-protein-coupled receptors (GPCR) [1], [2], [3], [4]. Neutrophils stimulated by GPCR-binding attractants such as chemokines, C5a and N-formyl peptide, rapidly change their morphology from rounded cells covered with microvilli to elongated cells with ruffles. The activated cells also have pseudopodia that form broad lamellepodia that are extended anteriorly in the direction of the increasing concentration of attractant, whereas a contractile uropod is formed posteriorly. The result is a morphologically polarized cell that is efficient at vectorial migration [5], [6], [7], [8].
Lectins are included among the multiple attractants that stimulate the neutrophil migration. Lectins are ubiquitous proteins that exhibit specific and reversible carbohydrate-binding activities [9]. Since lectins have a great versatility and diversity in binding to carbohydrates, they function as cell recognition mediators in a wide range of biological systems [10]. Studies have shown that many plant lectins can induce an inflammatory response characterized by dose-dependent plasma exudation and neutrophil migration in rats or mice [11], [12], [13], [14], [15], [16], [17], [18], [19].
Indeed, lectin–carbohydrate interactions may trigger many neutrophil functions. Lectins with different carbohydrate specificities such as concanavalin A (mannose-specific), wheat germ agglutinin (WGA-specific for N-acetylglucosamine and sialic acid), selectins (specific for sialic acid containing structures, especially sialyl-Lewisx), and some members of the galectin family (lactose-specific) have been shown to induce neutrophil responses [20], [21], [22], [23], [24], [25]. Selectins and their counter-receptors were extensively studied as mediators of the initial tethering and rolling of neutrophils along the endothelium of the vessel wall [26] and the function of these molecules was thought to be restricted to regulating of cell–cell adhesive interactions. More recent studies demonstrate their role in intracellular signaling and leukocyte activation. Binding of selectin molecules on neutrophils initiates transmembrane signals that modulate specific functional roles, such as adhesion-dependent leukocyte functions, β2-integrin activation, O2− production. Protein kinase-dependent pathway plays a key role in selectin-dependent leukocyte activation, which is illustrated by the fact that binding of L-selectin to sulfatides on the surface of neutrophils induces tyrosine phosphorylation of a variety of cellular proteins including MAP kinase, and PLCγ [27]. Other studies have reported that some cytotoxic functions also are initiated by lectins such as WGA [28], galectin-3 [24], and galectin-1 [25]. For members 1 and 3 of the galectin family, their increased binding to the neutrophil surface is associated with an enhancement of neutrophil response, which involves a mechanism dependent on granule mobilization [24], [25]. Recently, it had been shown that galectin-3 increases the phagocytic activity of neutrophils, modulates neutrophil degranulation and influences neutrophil survival, probably through a mechanism involving the stimulation of p38 MAPK [29]. A plant lectin, Sambucus nigra agglutinin, is also reported as able to induce neutrophil degranulation, through a mechanism that apparently involves signaling systems dependent on G proteins and lipid metabolism [30].
In a previous study, we have demonstrated that a structurally well defined d-mannose-specific lectin from Artocarpus integrifolia seeds, KM+, also known as artocarpin, is able to induce neutrophil migration both in vivo and in vitro. Its carbohydrate recognition domain is directly responsible for the effect of inducing neutrophil migration, [12] which occurs by haptotaxis and is mediated by simultaneous interaction of the tetrameric lectin with their glycoligands on the extracellular matrix (ECM) and on the neutrophil surface [12], [31]. We have recently identified laminin as being the ligand for KM+ on the ECM and have demonstrated that this interaction potentiates the KM+-induced neutrophil migration. The interaction of KM+ with laminin promotes formation of a substrate-bound gradient of KM+, which is necessary for the haptotatic movement of neutrophils that transmigrate through the vascular wall [32]. Although the tissue ligand for KM+ has been identified, its ligand on the neutrophil surface remains unknown.
In the present work, we demonstrate that KM+ interacts with glycans associated with a G-protein coupled receptor (GPCR), which corresponds to CXCR2. The responses triggered by the interaction of KM+ with its neutrophil ligand are comparable to those observed following the binding of well-known neutrophil attractants, indicating that the induction of neutrophil migration should be considered as one of the multiple activities attributed to lectins.
Section snippets
Reagents
Biobond, cacodylate, Embed 812, fluormount-G, glutaraldehyde, osmium tetroxide, thiocarbohydrizide, and uranyl acetate were purchased from Electron Microscopy Sciences, Fort Washington, PA, USA. fMLP, paraformaldehyde, RPMI medium, and saponin were obtained from Sigma Chemical Company, St. Louis, MO, USA. Formaldehyde was purchased from Merck, Darmstadt, Germany, pertussis toxin from List Biological Laboratories, Campbell, CA, USA, recombinant human IL-8 from R&D Systems Inc., Minneapolis, MN,
KM+ binding to glycoligands on the neutrophil surface
To study the interaction of KM+ with glycoligands on the surface of human neutrophils, cells were incubated with the biotinylated lectin and analyzed by flow cytometry and fluorescence microscopy. Inhibition of KM+ binding to the cell surface by specific sugars was also assayed. By flow cytometry, 96% of the neutrophils incubated with biotinylated KM+ were labeled. In the presence of d-mannose, but not of d-galactose, KM+ binding to neutrophils was abolished, as only 1% of the cells was labeled
Discussion
In the present study, we have expanded our characterization of the neutrophil migration inducing activity of the lectin KM+. We have demonstrated that this activity is inhibited by exposure to pertussis toxin, suggesting that KM+ acts through GPCR. In addition, we have shown that inhibition of CXCR2 activity also inhibits neutrophil migration induced by KM+, suggesting that it is due, at least in part, to KM+ binding to CXCR2.
We have previously shown that KM+ can mimic some mammalian lectins
Acknowledgments
We thank Mrs. Sandra M.O. Thomaz, Mrs. Imaculada C. Bragheto, and Mrs. Patricia Vendruscolo for technical assistance, and Mrs. Erica Vensdruscolo for assistance in preparing the manuscript. We are indebted to Mrs. Marcia S.Z. Graeff, Mrs. Maria Tereza P. Maglia, and Mr. Jose Augusto Maulin for the expert assistance with confocal and electron microscopy. This study was supported by FAPESP (00/09333-2), CNPq (350418/00-4) and FAEPA grants. G.P.S. received a PhD research fellowship from FAPESP
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