Modulation of Innate and Adaptive Immunity by Galectin FamilyMembers Público
Stowell, Sean Robinson (2009)
Abstract
Modulation of Innate and Adaptive Immunity by Galectin Family Members
Regulation of leukocyte viability is a key mechanism responsible for inflammatory resolution. Many factors, including members of the TNF and galectin families, appear to regulate fundamental activities of leukocytes involved in both innate and adaptive immunity. However, the mechanisms responsible for this regulation remain enigmatic. Therefore, in this study we sought to clarify the mechanisms underlying the immunomodulatory activities of galectins. Our studies demonstrate varied effects of galectin on different leukocyte populations as well as innate antimicrobial activity. Several galectins, including Galectin-1 (Gal-1), galectin-2 (Gal-2), galectin-3 (Gal-3), galectin-4 (Gal-4) and galectin-8 (Gal-8) induce phosphatidylserine (PS) exposure, a common feature of apoptotic cell death, in activated neutrophils independently of cell death. Induction of PS exposure sensitizes cells to phagocytic removal, yet fails to induce alterations in cell viability. This process, preaparesis, prepares live cells for removal, in contrast with pathways induced by other immunomodulatory factors such as TNF family members that induce apoptotic cell removal. In addition to effects in neutrophils, galectins also regulate T cells. Gal-3 signals T cell apoptosis while other galectin family members signal secretion of the cytokine IL-10. Unlike TNF family members, which regulate leukocyte turnover through protein-protein interactions, galectins uniquely recognize cell surface carbohydrates. Selective modification of these carbohydrate ligands significantly and differentially alters cellular sensitivity to galectin signaling of leukocytes. In addition to regulating leukocyte function, several galectins, including galectin-8, appear to also possess critical innate immune capacity, directly killing blood group B positive pathogens independent of complement. Taken together, these studies demonstrate that galectins possess critical activities in both innate and adaptive immunity.
Table of Contents
TABLE OF CONTENTS
Abstract Cover Page Abstract Cover Page Acknowledgements Table of Contents List of Figures and Tables
Chapter 1: Introduction...1
Figures...7 Figure Legends...11
Chapter 2: Galectin-1 induces reversible phosphatidylserine exposure at the plasma membrane.
Introduction...13 Results
Gal-1 induces reversible PS exposure...16 PS reversion does not represent PS-positive cellular removal...16 Cells previously positive for PS remain sensitive to restimulation by Gal-1...17 Gal-1 induces PS exposure through a caspase-independent process...18 Gal-1-induced PS exposure resides in punctate microdomains...18 iGal-1 induces sustained PS exposure without altering cell division...20
Discussion...22 Figures...27 Figure Legends...36
Chapter 3: Ligand Reduces Galectin-1 Sensitivity to Oxidative Inactivation by Enhancing Dimer Formation.
Introduction...42 Results
mGal-1 induces Ca2+ flux yet fails to induce PS exposure in HL60 cells...44 Gal-1 and mGal-1 display similar glycan recognition properties...44 mGal-1 displays enhanced sensitivity to oxidative inactivation...45 Prolonged incubation of HL60 cells with Gal-1 results in PS reversion...46 Ligand shifts Gal-1 monomer-dimer equilibrium in favor of dimerization...47 Carboxymethylation protects Gal-1 from oxidative inactivation...48 Carboxymethylation enhances Gal-1 and mGal-1 signaling in HL60 cells...49 Gal-1 signals PS exposure independent of cellular aggluatination...50
Discussion...52 Figures...57 Figure Legends...66
Chapter 4: Immunoregulatory Galectin-1 Exhibits Diffuse Expression in Adult Tissue with Cytosolic Organization in Striated Muscle Tissues
Introduction...71 Results
Monoclonal antibody, αGal-1, displays specificity for hGal-1 in multiple formats...73 αGal-1 recognizes Gal-1 bound to ligand...74 Gal-1 displays broad expression in adult tissue...75 Gal-1 exhibits diffuse cytosolic localization in liver, brain and stomach but organized localization in striated tissue...75
Discussion...79 Figures...82 Figure Legends...89
Chapter 5: Galectins-1, -2, and -4 Induce Surface Exposure of Phosphatidylserine in Activated Human Neutrophils but Not Activated T cells
Introduction...92 Results
Gal-2 and Gal-4 induce PS exposure in MOLT-4 and HL60 cells...95 Leukocytes induced to expose PS by Gal-2 and Gal-4 fail to display other known features of programmed cell death...95 Activated neutrophils, but not activated T cells, are sensitive to galectin-induced PS exposure...97 In the presence of DTT Gal-1 induces PS exposure of activated T cells...98
Discussion...102 Figures...106 Figure Legends...117
Chapter 6: Differential Roles of Galectin-1 and Galectin-3 in Regulating Leukocyte Viability and Cytokine Secretion
Introduction...122 Results
Gal-1 and Gal-3 induce PS exposure in activated neutrophils...125 Gal-3 induces PS exposure in activated T cells...126 iGal-1 and Gal-3 induce PS exposure in the absence of apoptosis in activated neutrophils...127 Gal-3, but not Gal-1, induces apoptosis in activated T cells...128 Gal-3 induces PS exposure independently of apoptosis in T leukemic cell lines...129 iGal-1 does not modulate apoptosis induced by Gal-3 in activated T cells...130 Gal-3 induces mobilization of intracellular Ca2+ in activated T cells...131 iGal-1 and Gal-3 signal additive PS exposure in neutrophils...132 Gal-1 and Gal-3 bind to distinct microdomains on activated T cells...132 Gal-1 and Gal-3 display similar affinities for T cell ligands...133 iGal-1 retains biological activity toward T cells...133
Discussion...135 Figures...141 Figure Legends...151
Chapter 7: Human Galectin-1 Recognition of Poly-N-Acetyllactosamine and Chimeric Polysaccharides
Introduction...160 Results
Preparation of neoglycoproteins...162 dGal-1 displays higher binding to poly-N-acetyllactosamine than N-acetyllactosamine...162 dGal-1 exhibits similar affinity to poly-N-acetyllactosamine regardless of internal N-acetyllactosamine modification...164 dGal-1 does not independently recognize internal LN units within PL chains...165
Discussion...167 Figures and Table...171 Figure Legends...177
Chapter 8: Galectins-1, -2, and -3 Exhibit Differential Recognition of Sialylated Glycans and Blood Group Antigens
Introduction...181 Results
Gal-1, Gal-2, and Gal-3 differentially recognize O- and N glycans...185 Gal-1, Gal-2, and Gal-3 exhibit differential recognition of LacNAc derivatives...186 Gal-1, Gal-2, and Gal-3 recognize polyLacNAc structures...188 Gal-1, Gal-2, and Gal-3 display differential recognition of polyLacNAc glycans and chimera polyLacNAc glcans...189 Gal-1, Gal-2, and Gal-3 differentially recognize sialylated cell surface glycans...191 Gal-1, Gal-2, and Gal-3 display differential recognition of cell surface polyLacNAc...194
Discussion...198 Figures and Tables...204 Figure Legends...217
Chapter 9: Innate immune lectin provides defense against molecular mimicry
Introduction...222 Results
Gal-8 and Gal-3 specifically recognize blood group B positive E. coli...224 Gal-8 kills blood group B positive E. coli...224 Gal-8 kills blood group B positive E. coli entirely through its C-terminal blood group binding domain...226 Gal-8 specifically kills blood group B positive E. coli...227
Discussion...228 Figures...230 Figure Legends...234
Chapter 10: Summary and Future Directions...236
Materials and Methods...256
References...277
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