Modulation of Innate and Adaptive Immunity by Galectin Family Members Open Access

Stowell, Sean Robinson (2009)

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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


Abstract Cover Page
Cover Page
Table of Contents
List of Figures and Tables

Chapter 1: Introduction...1

Figure Legends...11

Chapter 2: Galectin-1 induces reversible phosphatidylserine exposure at the plasma membrane.


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

Figure Legends...36

Chapter 3: Ligand Reduces Galectin-1 Sensitivity to Oxidative Inactivation by Enhancing Dimer Formation.


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

Figure Legends...66

Chapter 4: Immunoregulatory Galectin-1 Exhibits Diffuse Expression in Adult Tissue with Cytosolic Organization in Striated Muscle Tissues


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

Figure Legends...89

Chapter 5: Galectins-1, -2, and -4 Induce Surface Exposure of Phosphatidylserine in Activated Human Neutrophils but Not Activated T cells


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

Figure Legends...117

Chapter 6: Differential Roles of Galectin-1 and Galectin-3 in Regulating Leukocyte Viability and Cytokine Secretion


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

Figure Legends...151

Chapter 7: Human Galectin-1 Recognition of Poly-N-Acetyllactosamine and Chimeric Polysaccharides


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

Figures and Table...171
Figure Legends...177

Chapter 8: Galectins-1, -2, and -3 Exhibit Differential Recognition of Sialylated Glycans and Blood Group Antigens


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

Figures and Tables...204
Figure Legends...217

Chapter 9: Innate immune lectin provides defense against molecular mimicry


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

Figure Legends...234

Chapter 10: Summary and Future Directions...236

Materials and Methods...256


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