Glycosylation-dependent inhibition of cutaneous lymphocyte–associated antigen expression: implications in modulating lymphocyte migration to skin

Abstract

Constitutive E-selectin expression on dermal microvascular endothelial cells plays a critical role in mediating rolling adhesive interactions of human skin-homing T cells and in pathologic accumulation of lymphocytes in skin. The major E-selectin ligand on human skin-homing T cells is cutaneous lymphocyte-associated antigen (CLA), a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1) defined by monoclonal antibody HECA-452. Since HECA-452 reactivity, and not PSGL-1 polypeptide itself, confers the specificity of human T cells to enter dermal tissue, inhibition of HECA-452 expression is a potential strategy for modulating lymphocyte migration to skin. In this study, we examined the efficacy of several well-characterized metabolic inhibi- IntroductionTissue-specific migration of lymphocytes in neoplastic and inflammatory processes is critically dependent on the expression of cell membrane adhesion molecules that regulate adhesive interactions with target tissue endothelium. Skin disorders, including cutaneous T-cell lymphomas, cutaneous graft-versus-host disease (GVHD), and other inflammatory diseases (eg, psoriasis), are mediated by infiltrations of skin-homing T cells that express cutaneous lymphocyte-associated antigen (CLA). [1][2][3][4][5][6][7][8][9][10] CLA is a sialyl Lewis X-like carbohydrate epitope displayed on the mucinlike molecule Pselectin glycoprotein ligand-1 (PSGL-1) recognized by the rat monoclonal antibody HECA-452. 2,11,12 CLA functions as the P-selectin ligand and the principal E-and L-selectin ligand on human skin-homing T cells; specifically, the presence of HECA-452 epitopes on PSGL-1 directly correlates with the capacity of skin-homing memory T cells (including skin-disease-related lymphocytes) to bind E-selectin, which is constitutively expressed on dermal microvasculature. 2,11,13 CLA-E-selectin binding interactions mediate lymphocyte trafficking to skin, and therefore, posttranslational glycosylations on skin-homing leukocytes represent a potential therapeutic target for controlling cutaneous tropism.The ␣2,3 sialyltransferase, ST3Gal IV, and ␣1,3 fucosyltransferases, FucTIV and FucTVII, expressed in human skin-homing lymphocytes are responsible for synthesizing terminal sialofucosylations, recognized by HECA-452, that serve as E-selectinbinding determinants on PSGL-1. 14-23 These terminal sialyl Lewis X structures are expressed on poly-N-acetyllactosamines [(Gal␤1,4GlcNAc␤1,3) n ] found on core 2 serine/threonine (O)-linked glycans and asparagine (N)-linked glycans displayed by PSGL-1. [24][25][26][27][28][29][30] Although the majority of sialyl Lewis X epitopes appear to reside on PSGL-1 O-glycans, there are 3 potential sites for N-glycosylation and sialyl Lewis X decoration. 30 Consequently, interfering with the synthesis of O-glycans or N-glycans or of poly-N-acetyllactosamine structures on either O-or N-glycans (or both) would prevent the synthesis of HECA-452 epitopes and could dampen the E-selectin-binding function of PSGL-1 (CLA) on skin-homing leukocytes. The pu...

Skin-Homing Receptors on Effector Leukocytes Are Differentially Sensitive to Glyco-Metabolic Antagonism in Allergic Contact Dermatitis

“…To analyze whether CD43 and CD44 also function as E-selectin ligands on NK cells, we pretreated DX5 ϩ CD90 Ϫ NK cells with a robust protease, bromelain, before lysate preparation. With the exception of ESL-1, an E-selectin ligand characteristically localized in the Golgi (34), bromelain digestion cleaves all cell surface E-selectin glycoprotein ligands on leukocytes (20,33,37). By flow cytometry, we first confirmed that bromelain treatment eliminated PSGL-1, CD43 and CD44 expression (data not shown).…”

“…PBMC were first obtained from citrated-whole blood by density-gradient centrifugation as previously described (20). To prepare CD56 ϩ CD3 Ϫ NK cells, CD3 ϩ T cells were first depleted from PBMC using CD3 immunomagnetic beads (Miltenyi Biotec) and then CD56 ϩ cells were selected from CD3 Ϫ cells using CD56 immunomagnetic beads (Miltenyi Biotec).…”

“…KG1a cell lysate and lysates from murine and human leukocyte isolates were prepared as previously described (20). In some cases before lysate preparation, cells were treated with 0.1% bromelain (Sigma-Aldrich) for 30 min at 37°C.…”

“…T cell rolling on mouse E-selectin-Ig chimera was analyzed using the parallel-plate flow chamber under physiologic shear stress (20,35). To prepare E-selectin-Ig-coated plastic, E-selectin-Ig (50 ng/50 l 0.1M NaHCO 3 ) (pH 9.6) was pipetted on ten-twenty-nine petri dishes and allowed to adsorb overnight at 4°C.…”

“…These enzymes are responsible for generating sialyl Lewis X moieties on T cell membrane protein and lipid scaffolds, conferring ligand activity and the skin-homing designation of leukocytes (11)(12)(13)(14)(15)(16)(17)(18). Prior studies using metabolic inhibitors of selectin ligand biosynthesis, namely 4-fluorinated N-acetylglucosamine (4-F-GlcNAc), also show that preventing the formation of sialyl Lewis X moieties inhibits the recruitment of effector T cells to inflamed skin (19,20). Collectively, the functional importance of T cell E-and P-selectin ligands and FT4/7 highlights the potential benefit of targeting these factors as anti-inflammatory strategies.…”