The expression of ABO(H) blood group antigens causes deletion of cells that generate self anti-blood group antibodies, but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against these pathogens, given such limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectins-4 and -8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing E. coli, while failing to alter viability of other E. coli strains or other gram-negative or gram-positive organisms both in vitro and in vivo. Killing by both galectins-4 and -8 resides within their C-terminal domains, occurs rapidly and independently of complement, and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that display blood group self-antigens on their surface.

We report that human galectin-1 (dGal-1), a small dimeric ␤-galactoside-binding protein, induces phosphatidylserine ( It is believed that the turnover of neutrophils and other leukocytes in tissues involves programmed cell death (apoptosis) and then phagocytosis by tissue macrophages (1-4). However, the factors regulating turnover of leukocytes are unclear. Although Fas and Fas ligand (FasL) 1 induce apoptosis of mature, circulating neutrophils in vitro (5-7), mice deficient in FasL (gld) or Fas (lpr) have essentially normal numbers of circulating mature granulocytes (8). FasL/Fas-mediated apoptosis is not essential in regulating the clearance of neutrophils during inflammation (9). Fas and FasL may promote, rather than decrease, inflammatory responses in vivo (10 -12). In transgenic mice expressing bcl-2 in mature neutrophils, apoptosis of circulating cells is inhibited, but neutrophil homeostasis is unaltered, and macrophage-mediated phagocytosis of neutrophils is normal (13). Phagocytosis is required for resolution of the inflammatory process and leukocyte homeostasis in vivo (14 -16). These results suggest that factors not yet defined may regulate leukocyte turnover in tissues. Such observations led us to explore whether the basement membrane and extracellular matrix might harbor other proteins capable of binding to leukocytes and inducing their apoptosis or phagocytic recognition. A candidate protein is the ␤-galactoside-binding protein termed galectin-1 (dGal-1), which binds to most leukocytes. dGal-1 is a widely expressed dimeric protein (subunit ϳ14.6 kDa), which is a member of the galectin family of lectins (17)(18)(19). It is secreted by many cell types, including human endothelial cells (20,21), and is found in the basement membrane and extracellular matrices around capillary walls (22,23). dGal-1 has been reported to have various biological activities, including effects on neurite outgrowth (24, 25), growth inhibition of non-neural cells (26 -28), cell growth stimulation (29,30), and apoptosis of immature thymocytes (31, 32), and to activate human T cells and T cell lines (33,34).To explore the biological activity of dGal-1 toward leukocytes, we prepared a recombinant form of dimeric human dGal-1 and a mutated, monomeric form of galectin-1 (mGal-1). We explored the interactions of these lectins with HL-60 cells, MOLT-4 cells, and both resting and activated human neutrophils. Our results show that dGal-1, but not mGal-1, rapidly enhances surface staining with Annexin V (phosphatidylserine (PS) exposure) in desialylated HL-60 cells, desialylated MOLT-4 cells, and activated, but not resting, human neutrophils. The exposure of PS is often associated with apoptosis * This work was supported by National Institutes of Health Grants AI48075 (to R. D. C.) and HL34363 and RR15577 (to R. P. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate ...

Galectin-1 (Gal-1) and galectin-3 (Gal-3) exhibit profound but unique immunomodulatory activities in animals but their molecular mechanisms are incompletely understood. Early studies suggested that Gal-1 inhibits leukocyte function by inducing apoptotic cell death and removal, but recent studies show that some galectins induce exposure of the common death signal phosphatidylserine (PS) independently of apoptosis. In this study, we report that Gal-3, but not Gal-1, induces both PS exposure and apoptosis in primary activated human T cells, whereas both Gal-1 and Gal-3 induce PS exposure in neutrophils in the absence of cell death. Gal-1 and Gal-3 bind differently to the surfaces of T cells and only Gal-3 mobilizes intracellular Ca2+ in these cells, although Gal-1 and Gal-3 bind their respective T cell ligands with similar affinities. Although Gal-1 does not alter T cell viability, it induces IL-10 production and attenuates IFN-γ production in activated T cells, suggesting a mechanism for Gal-1-mediated immunosuppression in vivo. These studies demonstrate that Gal-1 and Gal-3 induce differential responses in T cells and neutrophils, and identify the first factor, Gal-3, capable of inducing PS exposure with or without accompanying apoptosis in different leukocytes, thus providing a possible mechanism for galectin-mediated immunomodulation in vivo.

IntroductionThe physiologic causes of leukocyte turnover in homeostasis and during disease conditions are not well understood. It is believed that leukocytes are partly eliminated by programmed cell death or apoptosis 1-4 through phagocytosis by macrophages, dendritic cells, or neighboring cells. 5,6 The efficient removal of dying cells is important in homeostasis, since it limits accumulation of cellular debris that could be potentially immunogenic or toxic. [7][8][9] However, the role of apoptosis in removing large numbers of cells in inflammation and during the resolution phase remains uncertain. Although human neutrophils undergo apoptosis spontaneously when cultured in vitro, the role of apoptosis in regulating neutrophil turnover in vivo is unclear. 6,[10][11][12] Apoptosis impairs cellular functions and might impair proinflammatory functions of neutrophils. 13 However, excessive neutrophil influx with loss of membrane integrity during late apoptotic events could contribute to neutrophil-mediated injury of surrounding viable parenchymal tissue. 14 Exuberant apoptosis may therefore be proinflammatory instead of anti-inflammatory, [15][16][17] presumably due to the release of cellular contents prior to phagocytic removal. In this regard, nonapoptotic neutrophils can be cleared by phagocytosis in vivo. 18 Finally, factors known to induce or to block apoptosis, such as ligation of Fas/FasL and expression of bcl-2, respectively, do not alter neutrophil turnover in mouse models. [19][20][21][22] These studies suggest that unidentified factors may be involved in the phagocytic removal of viable, rather than apoptotic, cells.The removal of apoptotic cells occurs partly through tethering to phagocytic cells due to receptor-ligand interactions involving recognition of phosphatidylserine (PS) exposed on the surfaces of apoptotic cells. 23 Surface PS is recognized by a defined PS-receptor in macrophages 24 and by other receptors. [25][26][27] However, PS exposure in leukocytes can occur independently of apoptosis. 28,29 Thus, factors that induce PS exposure independently of apoptosis may be involved in leukocyte turnover.Recently, we showed that galectin-1 (Gal-1), a prototypical homodimeric member (subunit ϳ 14.5 kDa) of the galectin family, which has immunomodulatory functions, [30][31][32][33][34] can induce PS exposure in activated, but not resting, neutrophils, independently of cell death, while concomitantly rendering them sensitive to phagocytic recognition and removal. 35 We also found that the signaling pathway of Gal-1 in activated neutrophils is unique and involves elevations of cytosolic Ca 2ϩ and mobilization of PS through the actions of Src kinases and phospholipase C␥. 36 However, there are many conflicting reports about galectin's effects on leukocytes. Several groups have reported that Gal-1 induces apoptosis along with PS exposure in vitro of activated T lymphocytes and several T-leukemic cell lines. [37][38][39] It has also been reported that human Gal-2, a protein structurally related to Gal-1, ind...

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Human galectin-1 is a dimeric carbohydrate binding protein (Gal-1) (subunit 14.6 kDa) widely expressed by many cells but whose carbohydrate binding specificity is not well understood. Because of conflicting evidence regarding the ability of human Gal-1 to recognize N-acetyllactosamine (LN, Galbeta4GlcNAc) and poly-N-acetyllactosamine sequences (PL, [-3Galbeta4GlcNAcbeta1-]n), we synthesized a number of neoglycoproteins containing galactose, N-acetylgalactosamine, fucose, LN, PL, and chimeric polysaccharides conjugated to bovine serum albumin (BSA). All neoglycoproteins were characterized by MALDI-TOF. Binding was determined in ELISA-type assays with immobilized neoglycoproteins and apparent binding affinities were estimated. For comparison, we also tested the binding of these neoglycoconjugates to Ricinus communis agglutinin I, (RCA-I, a galactose-binding lectin) and Lycopersicon esculentum agglutinin (LEA, or tomato lectin), a PL-binding lectin. Gal-1 bound to immobilized Galbeta4GlcNAcbeta3Galbeta4Glc-BSA with an apparent K(d) of approximately 23 micro M but bound better to BSA conjugates with long PL and chimeric polysaccharide sequences (K(d)'s ranging from 11.9 +/- 2.9 microM to 20.9 +/- 5.1 micro M). By contrast, Gal-1 did not bind glycans lacking a terminal, nonreducing unmodified LN disaccharide and also bound very poorly to lactosyl-BSA (Galbeta4Glc-BSA). By contrast, RCA bound well to all glycans containing terminal, nonreducing Galbeta1-R, including lactosyl-BSA, and bound independently of the modification of the terminal, nonreducing LN or the presence of PL. LEA bound with increasing affinity to unmodified PL in proportion to chain length. Thus Gal-1 binds terminal beta4Gal residues, and its binding affinity is enhanced significantly by the presence of this determinant on long-chain PL or chimeric polysaccharides.

Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell surface exposure of phosphatidylserine (PS), a ligand that targets cells for phagocytic removal, in the absence of apoptosis. Gal-1 monomer-dimer equilibrium appears to modulate Gal-1-induced PS exposure, although the mechanism underlying this regulation remains unclear. Here we show that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired dimerization and fails to induce cell surface PS exposure while retaining the ability to recognize carbohydrates and signal Ca 2؉ flux in leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1 dimerization. Continual incubation of leukocytes with Gal-1 resulted in gradual oxidative inactivation with concomitant loss of cell surface PS, whereas rapid oxidation prevented mGal-1 from inducing PS exposure. Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to signal sustained PS exposure in leukocytes and mGal-1 to signal both Ca 2؉ flux and PS exposure. Taken together, these results demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidative inactivation and provides a mechanism whereby ligand partially protects Gal-1 from oxidation.Immunological homeostasis relies on efficient contraction of activated leukocytes following an inflammatory episode. Several factors, including members of the galectin and tumor necrosis factor families (1, 2), regulate leukocyte turnover by inducing apoptotic cell death. In contrast, several galectin family members, in particular galectin-1 (Gal-1), 2 uniquely regulate neutrophil turnover by inducing phosphatidylserine (PS) exposure, which normally sensitizes apoptotic cells to phagocytic removal (3, 4), independent of apoptosis, a process recently termed preaparesis (5).Previous studies suggested that dimerization may be required for Gal-1-induced PS exposure, as a mutant form of Gal-1 (mGal-1) containing two point mutations within the dimer interface, C2S and V5D (C2S,V5D), displays impaired Gal-1 dimerization and fails to induce PS exposure (6). However, the manner in which monomer-dimer equilibrium regulates Gal-1 signaling remains unclear. Previous studies suggest that dimerization may be required for efficient cross-linking of functional receptors or the formation of signaling lattices (7-9). Consistent with this, monomeric mutants of several other galectins fail to induce PS exposure or signal leukocytes (4, 8). Gal-1 signaling of PS exposure requires initial signaling events, such as mobilization of intracellular Ca 2ϩ followed by sustained receptor engagement (10). Although mGal-1 fails to induce PS exposure (6), whether mGal-1 can induce these initial signaling events remains unknown (10).In addition to directly regulating signaling, monomer-dimer equilibrium may ...

Galectins are an evolutionarily ancient family of glycan-binding proteins (GBPs) and are found in all animals. Although they were discovered over 30 years ago, ideas about their biological functions continue to evolve. Current evidence indicates that galectins, which are the only known GBPs that occur free in the cytoplasm and extracellularly, are involved in a variety of intracellular and extracellular pathways contributing to homeostasis, cellular turnover, cell adhesion, and immunity. Here we review evolving insights into galectin biology from a historical perspective and explore current evidence regarding biological roles of galectins.