Cell-Specific Variation in E-Selectin Ligand Expression among Human Peripheral Blood Mononuclear Cells: Implications for Immunosurveillance and Pathobiology This information is current as of September 29, 2021. Mariana Silva, Ronald Kam Fai Fung, Conor Brian Donnelly, Paula Alexandra Videira and Robert Sackstein J Immunol 2017; 198:3576-3587; Prepublished online 22 March 2017; doi: 10.4049/jimmunol.1601636 Downloaded from http://www.jimmunol.org/content/198/9/3576 Supplementary http://www.jimmunol.org/content/suppl/2017/03/22/jimmunol.160163 http://www.jimmunol.org/ Material 6.DCSupplemental References This article cites 89 articles, 35 of which you can access for free at: http://www.jimmunol.org/content/198/9/3576.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • No Triage! 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The Journal of Immunology Cell-Specific Variation in E-Selectin Ligand Expression among Human Peripheral Blood Mononuclear Cells: Implications for Immunosurveillance and Pathobiology Mariana Silva,*,†,‡,x Ronald Kam Fai Fung,†,‡,{ Conor Brian Donnelly,†,‡,x Paula Alexandra Videira,*,‖ and Robert Sackstein†,‡,x Both host defense and immunopathology are shaped by the ordered recruitment of circulating leukocytes to affected sites, a process initiated by binding of blood-borne cells to E-selectin displayed at target endothelial beds. Accordingly, knowledge of the expression and function of leukocyte E-selectin ligands is key to understanding the tempo and specificity of immunoreactivity. In this study, we performed E-selectin adherence assays under hemodynamic flow conditions coupled with flow cytometry and Western blot analysis to elucidate the function and structural biology of glycoprotein E-selectin ligands expressed on human PBMCs. Circulating mono- Downloaded from cytes uniformly express high levels of the canonical E-selectin binding determinant sialyl Lewis X (sLeX) and display markedly greater adhesive interactions with E-selectin than do circulating lymphocytes, which exhibit variable E-selectin binding among CD4+ and CD8+ T cells but no binding by B cells. Monocytes prominently present sLeX decorations on an array of protein scaffolds, including P-selectin glycoprotein ligand-1, CD43, and CD44 (rendering the E-selectin ligands cutaneous lymphocyte Ag, CD43E, and hematopoietic cell E-selectin/L-selectin ligand, respectively), and B cells altogether lack E-selectin ligands. Quanti- X tative PCR gene expression studies of glycosyltransferases that regulate display of sLe reveal high transcript levels among http://www.jimmunol.org/ circulating monocytes and low levels among circulating B cells, and, commensurately, cell surface a(1,3)-fucosylation reveals that acceptor sialyllactosaminyl glycans convertible into sLeX are abundantly expressed on human monocytes yet are relatively deficient on B cells. Collectively, these findings unveil distinct cell-specific patterns of E-selectin ligand expression among human PBMCs, indicating that circulating monocytes are specialized to engage E-selectin and providing key insights into the molecular effectors mediating recruitment of these cells at inflammatory sites. The Journal of Immunology, 2017, 198: 3576–3587. he ability of patrolling blood-borne effector cells to matory reactions (1). Under physiologic blood flow conditions, converge efficiently at discrete anatomic sites drives both circulating leukocytes extravasate at inflammatory sites via a by guest on September 29, 2021 T immunosurveillance and the immunobiology of inflam- multistep process that is initiated by cellular tethering and rolling contacts on affected endothelium (2). These step one, initial ad- hesive interactions are principally dictated by engagement of *Centro de Estudos de Doenc¸as Cro´nicas, NOVA Medical School/Faculdade de 2+ Cieˆncias Me´dicas, Universidade Nova de Lisboa, 1169-056 Lisbon, Portugal; vascular selectins, E- and P-selectin, which are Ca -dependent †Department of Dermatology, Brigham and Women’s Hospital, Boston, MA 02115; lectins that bind glycosylated coreceptors (ligands) expressed on ‡Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115; xProgram of Excellence in Glycosciences, Harvard Medical School, Boston, MA blood-borne cells, resulting in shear-resistant adherence of the 02115; {Medical Training and Administration Unit, Royal Prince Alfred Hospital, circulating cells to target endothelial beds (3, 4). Importantly, the ‖ Sydney, New South Wales 2050, Australia; and Unidade de Cieˆncias Biomoleculares expression of E- and P-selectin critically varies among mammals: Aplicadas, Departamento de Cieˆncias da Vida, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal in rodents, the inflammatory cytokines IL-1b and TNF-a each ORCIDs: 0000-0002-3974-9209 (R.K.F.F.); 0000-0001-5987-2485 (P.A.V.). upregulate transcription of mRNA encoding P-selectin and E-selectin; however, in primates, the P-selectin promoter lacks the Received for publication September 20, 2016. Accepted for publication February 22, 2017. pertinent response elements for these cytokines and only E-selectin This work was supported by National Institutes of Health/National Heart Lung Blood is inducibly expressed (5). Thus, in human immunobiology, endo- Institute Grant P01 HL107146 (Program of Excellence in Glycosciences; to R.S.), the thelial expression of E-selectin dominates in mediating recruitment Team Jobie Fund (to R.S.), a Fulbright Commission fellowship (to P.A.V.), and Portuguese Foundation for Science and Technology Fellowship SFRH/BD/81860/ of leukocytes at inflammatory sites, and, accordingly, those cells 2011 (to M.S.). possessing the most potent E-selectin ligands serve as primary Address correspondence and reprint requests to Dr. Robert Sackstein, Harvard Insti- sentinels of host defense and principal effectors of both immediate tutes of Medicine, 77 Avenue Louis Pasteur, Room 671, Boston, MA 02115. E-mail and sustained inflammatory processes. address: [email protected] E-selectin binds to sialofucosylated glycan determinants deco- The online version of this article contains supplemental material. rating specific glycoproteins and glycolipids. These glycans con- Abbreviations used in this article: CHO, Chinese hamster ovary; CHO-E, Chinese tain an a(2,3)-linked sialic acid substitution on galactose and an hamster ovary cells transfected with full-length human E-selectin cDNA; CHO-M, mock-transfected CHO cells; CLA, cutaneous lymphocyte Ag; E-Ig, E-selectin–Ig a(1,3)-linked fucose modification on N-acetylglucosamine, pro- chimera; FT, fucosyltransferase; HCELL, hematopoietic cell E-selectin/L-selectin totypically displayed as the terminal lactosaminyl tetrasacharide ligand; HSPC, hematopoietic stem and progenitor cell; MPO, myeloperoxidase; X MPO-EL, E-selectin–reactive glycoform of myeloperoxidase; PNGase-F, peptide- known as sialyl Lewis X (sLe ; CD15s) (3, 6–8). Although both X N-glycosidase F; PSGL-1, P-selectin glycoprotein ligand-1; qRT-PCR, quantitative glycoproteins and glycolipids can display sLe , glycoproteins, by X real-time PCR; sLe , sialyl Lewis X; ST3, a(2,3)-sialyltransferase; ST6, a(2,6)- virtue of their extended configuration beyond the cell membrane, sialyltransferase. mediate more efficient primary contacts between flowing cells and Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 the endothelium and thereby play a predominant role in initial www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601636 The Journal of Immunology 3577 binding to endothelial E-selectin under hemodynamic shear stress with the following Abs: FITC-conjugated anti-human/mouse CLA, PE- (9). On human cells, three principal E-selectin ligands have been conjugated anti-human CD14, allophycocyanin-conjugated anti-human identified: cutaneous lymphocyte Ag (CLA), hematopoietic cell CD16, and PE-Cy7–conjugated anti-human HLA-DR (all from Bio- Legend). Monocytes were identified by their characteristic forward scatter/ E-selectin/L-selectin ligand (HCELL), and CD43E, which are side scatter proprieties and HLA-DR expression; subsets of monocytes X 2 heavily sLe -decorated glycoforms of P-selectin glycoprotein were defined as classical (CD14++CD16 ), intermediate (CD14++CD16+), ligand-1 (PSGL-1; CD162), CD44, and CD43, respectively (10). and nonclassical (CD14+CD16++) (Supplemental Fig. 1) (20). Among human leukocytes, neutrophils and monocytes are the E-selectin reactivity was determined using a recombinant mouse E-selectin–human Fc Ig chimera (E-selectin–Ig chimera [E-Ig]) (R&D earliest cells to colonize inflammatory sites (11, 12), with later 2+ 2+ + + Systems) in Dubelcco’s PBS containing Ca /Mg , as previously de- entry of T cells (CD4 prior to CD8