Daniel K. Hsu

Galectin-3 and Galectin-1 Bind Distinct Cell Surface Glycoprotein Receptors to Induce T Cell Death

Galectins are a family of mammalian β-galactoside-binding proteins that positively and negatively regulate T cell death. Extracellular galectin-1 directly induces death of T cells and thymocytes, while intracellular galectin-3 blocks T cell death. In contrast to the antiapoptotic function of intracellular galectin-3, we demonstrate that extracellular galectin-3 directly induces death of human thymocytes and T cells. However, events in galectin-3- and galectin-1-induced cell death differ in a number of ways. Thymocyte subsets demonstrate different susceptibility to the two galectins: whereas galectin-1 kills double-negative and double-positive human thymocytes with equal efficiency, galectin-3 preferentially kills double-negative thymocytes. Galectin-3 binds to a complement of T cell surface glycoprotein receptors distinct from that recognized by galectin-1. Of these glycoprotein receptors, CD45 and CD71, but not CD29 and CD43, appear to be involved in galectin-3-induced T cell death. In addition, CD7 that is required for galectin-1-induced death is not required for death triggered by galectin-3. Following galectin-3 binding, CD45 remains uniformly distributed on the cell surface, in contrast to the CD45 clustering induced by galectin-1. Thus, extracellular galectin-3 and galectin-1 induce death of T cells through distinct cell surface events. However, as galectin-3 and galectin-1 cell death are neither additive nor synergistic, the two death pathways may converge inside the cell.

Critical role of galectin-3 in phagocytosis by macrophages

Galectin-3 is a member of a large family of animal lectins. This protein is expressed abundantly by macrophages, but its function in this cell type is not well understood. We have studied the effect of galectin-3 gene targeting on phagocytosis, a major function of macrophages. Compared with wild-type macrophages, galectin-3-deficient (gal3-/-) cells exhibited reduced phagocytosis of IgG-opsonized erythrocytes and apoptotic thymocytes in vitro. In addition, gal3-/- mice showed attenuated phagocytic clearance of apoptotic thymocytes by peritoneal macrophages in vivo. These mice also exhibited reduced IgG-mediated phagocytosis of erythrocytes by Kupffer cells in a murine model of autoimmune hemolytic anemia. Additional experiments indicate that extracellular galectin-3 does not contribute appreciably to the phagocytosis-promoting function of this protein. Confocal microscopic analysis of macrophages containing phagocytosed erythrocytes revealed localization of galectin-3 in phagocytic cups and phagosomes. Furthermore, gal3-/- macrophages exhibited a lower degree of actin rearrangement upon Fcgamma receptor crosslinkage. These results indicate that galectin-3 contributes to macrophage phagocytosis through an intracellular mechanism. Thus, galectin-3 may play an important role in both innate and adaptive immunity by contributing to phagocytic clearance of microorganisms and apoptotic cells.

Galectin-3 Induces Death of Candida Species Expressing Specific β-1,2-Linked Mannans

Lectins play a critical role in host protection against infection. The galectin family of lectins recognizes saccharide ligands on a variety of microbial pathogens, including viruses, bacteria, and parasites. Galectin-3, a galectin expressed by macrophages, dendritic cells, and epithelial cells, binds bacterial and parasitic pathogens including Leishmania major, Trypanosoma cruzi, and Neisseria gonorrhoeae. However, there have been no reports of galectins having direct effects on microbial viability. We found that galectin-3 bound only to Candida albicans species that bear β-1,2-linked oligomannans on the cell surface, but did not bind Saccharomyces cerevisiae that lacks β-1,2-linked oligomannans. Surprisingly, binding directly induced death of Candida species containing specific β-1,2-linked oligomannosides. Thus, galectin-3 can act as a pattern recognition receptor that recognizes a unique pathogen-specific oligosaccharide sequence. This is the first description of antimicrobial activity for a member of the galectin family of mammalian lectins; unlike other lectins of the innate immune system that promote opsonization and phagocytosis, galectin-3 has direct fungicidal activity against opportunistic fungal pathogens.

Critical Role for Galectin-3 in Airway Inflammation and Bronchial Hyperresponsiveness in a Murine Model of Asthma

Galectin-3 is a member of a beta-galactoside-binding animal lectin family. Previous in vitro studies have demonstrated that galectin-3 is involved in a number of activities; however, the roles of this lectin in physiological and pathological processes in vivo remain to be elucidated. Herein, we show, in a murine model of ovalbumin (OVA)-induced asthma that 1) peribronchial inflammatory cells expressed large amounts of galectin-3; 2) bronchoalveolar lavage fluid from OVA-challenged mice contained significantly higher levels of galectin-3 compared to control mice; and 3) macrophages in bronchoalveolar lavage fluid were the major cell type that contained galectin-3. We investigated the role of galectin-3 in the allergic airway response by comparing galectin-3-deficient (gal3(-/-)) mice and wild-type (gal3(+/+)) mice. OVA-sensitized gal3(-/-) mice developed fewer eosinophils and lower goblet cell metaplasia, after airway OVA challenge compared to similarly treated gal3(+/+) mice. In addition, the OVA-sensitized gal3(-/-) mice developed significantly less airway hyperresponsiveness after airway OVA challenge compared to gal3(+/+) mice. Finally, gal3(-/-) mice developed a lower Th2 response, but a higher Th1 response, suggesting that galectin-3 regulates the Th1/Th2 response. We conclude that galectin-3 may play an important role in the pathogenesis of asthma and inhibitors of this lectin may prove useful for treatment of this disease.

Galectins-3 and -7, but not Galectin-1, Play a Role in Re-epithelialization of Wounds

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study, using cornea as a model, we demonstrate for the first time the importance of carbohydrate-binding proteins galectins-3 and -7 in re-epithelialization of wounds. In two different models of corneal wound healing, re-epithelialization of wounds was significantly slower in galectin-3-deficient (gal3−/−) mice compared with wild-type (gal3+/+) mice. In contrast, there was no difference in corneal epithelial wound closure rates between galectin-1-deficient and wild-type mice. Quantitation of the bromodeoxyuridine-labeled cells in gal3+/+ and gal3−/− corneas revealed that corneal epithelial cell proliferation rate is not perturbed in gal3−/− corneas. Exogenous galectin-3 accelerated re-epithelialization of wounds in gal3+/+ mice but, surprisingly, not in the gal3−/− mice. Gene expression analysis using cDNA microarrays revealed that healing corneas of gal3−/− mice contain markedly reduced levels of galectin-7 compared with those of gal3+/+ mice. More importantly, unlike galectin-3, galectin-7 accelerated re-epithelialization of wounds in both gal3−/− and gal3+/+ mice. In corresponding experiments, recombinant galectin-1 did not stimulate the corneal epithelial wound closure rate. The extent of acceleration of re-epithelialization of wounds with both galectin-3 and galectin-7 was greater than that observed in most of the published studies using growth factors. These findings have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.

Galectin-3 Is a Negative Regulator of Lipopolysaccharide-Mediated Inflammation

Galectin-3 is a β-galactoside-binding lectin that plays an important role in inflammatory diseases. It also interacts with the surface carbohydrates of many pathogens, including LPS. However, its role in infection is not fully understood. Data presented herein demonstrate for the first time that galectin-3 is a negative regulator of LPS-induced inflammation. Galectin-3 is constitutively produced by macrophages and directly binds to LPS. Galectin-3-deficient macrophages had markedly elevated LPS-induced signaling and inflammatory cytokine production compared with wild-type cells, which was specifically inhibited by the addition of recombinant galectin-3 protein. In contrast, blocking galectin-3 binding sites by using a neutralizing Ab or its ligand, β-lactose, enhanced LPS-induced inflammatory cytokine expression by wild-type macrophages. In vivo, mice lacking galectin-3 were more susceptible to LPS shock associated with excessive induction of inflammatory cytokines and NO production. However, these changes conferred greater resistance to Salmonella infection. Thus, galectin-3 is a previously unrecognized, naturally occurring, negative regulator of LPS function, which protects the host from endotoxin shock but, conversely, favors Salmonella survival.

Galectin-3 regulates T-cell functions.

Summary:  Galectin‐3 is absent in resting CD4+ and CD8+ T cells but is inducible by various stimuli. These include viral transactivating factors, T‐cell receptor (TCR) ligation, and calcium ionophores. In addition, galectin‐3 is constitutively expressed in human regulatory T cells and CD4+ memory T cells. Galectin‐3 exerts extracellular functions because of its lectin activity and recognition of cell surface and extracellular matrix glycans. These include cell activation, adhesion, induction of apoptosis, and formation of lattices with cell surface glycoprotein receptors. Formation of lattices can result in restriction of receptor mobility and cause attenuation of receptor functions. Consistent with the presence of galectin‐3 in intracellular locations, several functions have been described for this protein inside T cells. These include inhibition of apoptosis, promotion of cell growth, and regulation of TCR signal transduction. Studies of cell surface glycosylation have led to convergence of glycobiology and galectin biology and provided new clues on how galectin‐3 may participate in the regulation of cell surface receptor activities. The rapid expansion of the field of galectin research has positioned galectin‐3 as a key regulator in T‐cell functions.

Galectin-3 negatively regulates TCR-mediated CD4+ T-cell activation at the immunological synapse

We have investigated the function of endogenous galectin-3 in T cells. Galectin-3-deficient (gal3−/−) CD4+ T cells secreted more IFN-γ and IL-4 than gal3+/+CD4+ T cells after T-cell receptor (TCR) engagement. Galectin-3 was recruited to the cytoplasmic side of the immunological synapse (IS) in activated T cells. In T cells stimulated on supported lipid bilayers, galectin-3 was primarily located at the peripheral supramolecular activation cluster (pSMAC). Gal3+/+ T cells formed central SMAC on lipid bilayers less effectively and adhered to antigen-presenting cells less firmly than gal3−/− T cells, suggesting that galectin-3 destabilizes the IS. Galectin-3 expression was associated with lower levels of early signaling events and phosphotyrosine signals at the pSMAC. Additional data suggest that galectin-3 potentiates down-regulation of TCR in T cells. By yeast two-hybrid screening, we identified as a galectin-3-binding partner, Alix, which is known to be involved in protein transport and regulation of cell surface expression of certain receptors. Co-immunoprecipitation confirmed galectin-3-Alix association and immunofluorescence analysis demonstrated the translocation of Alix to the IS in activated T cells. We conclude that galectin-3 is an inhibitory regulator of T-cell activation and functions intracellularly by promoting TCR down-regulation, possibly through modulating Alixs function at the IS.

Galectins in acute and chronic inflammation

Galectins are animal lectins that bind to β‐galactosides, such as lactose and N‐acetyllactosamine, in free form or contained in glycoproteins or glycolipids. They are located intracellularly or extracellularly. In the latter they exhibit bivalent or multivalent interactions with glycans on cell surfaces and induce various cellular responses, including production of cytokines and other inflammatory mediators, cell adhesion, migration, and apoptosis. Furthermore, they can form lattices with membrane glycoprotein receptors and modulate receptor properties. Intracellular galectins can participate in signaling pathways and alter biological responses, including apoptosis, cell differentiation, and cell motility. Current evidence indicates that galectins play important roles in acute and chronic inflammatory responses, as well as other diverse pathological processes. Galectin involvement in some processes in vivo has been discovered, or confirmed, through studies of genetically engineered mouse strains, each deficient in a given galectin. Current evidence also suggests that galectins may be therapeutic targets or employed as therapeutic agents for these inflammatory responses.

Endogenous Galectin-3 Is Localized in Membrane Lipid Rafts and Regulates Migration of Dendritic Cells

This study reveals a function of endogenous galectin-3, an animal lectin recognizing beta-galactosides, in regulating dendritic cell motility both in vitro and in vivo, which to our knowledge is unreported. First, galectin-3-deficient (gal3(-/-)) bone marrow-derived dendritic cells exhibited defective chemotaxis compared to gal3(+/+) cells. Second, cutaneous dendritic cells in gal3(-/-) mice displayed reduced migration to draining lymph nodes upon hapten stimulation compared to gal3(+/+) mice. Moreover, gal3(-/-) mice were impaired in the development of contact hypersensitivity relative to gal3(+/+) mice in response to a hapten, a process in which dendritic cell trafficking to lymph nodes is critical. In addition, defective signaling was detected in gal3(-/-) cells upon chemokine receptor activation. By immunofluorescence microscopy, we observed that galectin-3 is localized in membrane ruffles and lamellipodia in stimulated dendritic cells and macrophages. Furthermore, galectin-3 was enriched in lipid raft domains under these conditions. Finally, we determined that ruffles on gal3(-/-) cells contained structures with lower complexity compared to gal3(+/+) cells. In view of the participation of membrane ruffles in signal transduction and cell motility, we conclude that galectin-3 regulates cell migration by functioning at these structures.