Dendritic Cell Maturation Results in Pronounced Changes in Glycan Expression Affecting Recognition by Siglecs and Galectins This information is current as of September 23, 2021. Marieke Bax, Juan J. García-Vallejo, Jihye Jang-Lee, Simon J. North, Tim J. Gilmartin, Gilberto Hernández, Paul R. Crocker, Hakon Leffler, Steven R. Head, Stuart M. Haslam, Anne Dell and Yvette van Kooyk J Immunol 2007; 179:8216-8224; ; Downloaded from doi: 10.4049/jimmunol.179.12.8216 http://www.jimmunol.org/content/179/12/8216 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2008/03/07/179.12.8216.DC1 Material References This article cites 70 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/179/12/8216.full#ref-list-1 Why The JI? Submit online. by guest on September 23, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Dendritic Cell Maturation Results in Pronounced Changes in Glycan Expression Affecting Recognition by Siglecs and Galectins1 Marieke Bax,2* Juan J. Garcı´a-Vallejo,2* Jihye Jang-Lee,† Simon J. North,† Tim J. Gilmartin,‡ Gilberto Herna´ndez,‡ Paul R. Crocker,§ Hakon Leffler,¶ Steven R. Head,‡ Stuart M. Haslam,† Anne Dell,† and Yvette van Kooyk3* Dendritic cells (DC) are the most potent APC in the organism. Immature dendritic cells (iDC) reside in the tissue where they capture pathogens whereas mature dendritic cells (mDC) are able to activate T cells in the lymph node. This dramatic functional change is mediated by an important genetic reprogramming. Glycosylation is the most common form of posttranslational mod- ification of proteins and has been implicated in multiple aspects of the immune response. To investigate the involvement of Downloaded from glycosylation in the changes that occur during DC maturation, we have studied the differences in the glycan profile of iDC and mDC as well as their glycosylation machinery. For information relating to glycan biosynthesis, gene expression profiles of human monocyte-derived iDC and mDC were compared using a gene microarray and quantitative real-time PCR. This gene expression profiling showed a profound maturation-induced up-regulation of the glycosyltransferases involved in the expression of LacNAc, core 1 and sialylated structures and a down-regulation of genes involved in the synthesis of core 2 O-glycans. Glycosylation changes during DC maturation were corroborated by mass spectrometric analysis of N- and O-glycans and by flow cytometry using plant http://www.jimmunol.org/ lectins and glycan-specific Abs. Interestingly, the binding of the LacNAc-specific lectins galectin-3 and -8 increased during mat- uration and up-regulation of sialic acid expression by mDC correlated with an increased binding of siglec-1, -2, and -7. The Journal of Immunology, 2007, 179: 8216–8224. endritic cells (DC)4 are the most potent APC in the im- signaling cascade leading to the migration of DC to the neighbor- mune system. They reside as immature DC (iDC) in the ing lymph nodes. In these lymph nodes, the DC arrive as mature D peripheral tissues, where they sense for pathogens (1). DC (mDC), ready to interact with a naive lymphocyte carrying the Pathogen recognition often results in the activation of iDC via appropriate TCR. mDC are characterized by the expression of high by guest on September 23, 2021 TLR present on their membrane or in intracellular compartments levels of MHC class II, costimulatory molecules, chemokines, and (2). The interaction of TLRs with their ligands elicits a complex cytokines, in contrast to iDC, which show low expression of these molecules and high levels of Ag-uptake receptors. Thus, DC suffer *Department of Molecular Cell Biology and Immunology, Vrije Universiteit Univer- a dramatic change in phenotype and functionality upon maturation. sity Medical Center, Amsterdam, The Netherlands; †Division of Molecular Bio- This change is mediated by the modulation of a wide array of sciences, Imperial College, London, United Kingdom; ‡DNA Microarray Core Fa- molecules and ensures the development of a potent and specific cility, The Scripps Research Institute, La Jolla, CA 92037; §Wellcome Trust Biocentre, University of Dundee, Dundee, United Kingdom; and ¶Section Microbi- immune response. As a result, mDC have a limited Ag uptake and ology, Immunology, and Glycobiology, Department of Laboratory Medicine, Lund processing capacity, whereas Ag presentation and T cell costimu- University, Lund, Sweden lation are promoted (3). Received for publication March 29, 2007. Accepted for publication October 5, 2007. To limit Ag uptake, C-type lectin receptor (CLR) expression on The costs of publication of this article were defrayed in part by the payment of page the cell surface of DC is down-regulated during maturation. CLRs charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. constitute an important family of pattern recognition receptors ex- 1 This work was supported primarily by a Netherlands Organization of Scientific pressed on DC (4) and are well-characterized as Ag-uptake recep- Research Pioneer grant (to Y.v.K.) and in part by National Institute of General Med- tors for glycosylated structures (5) found on pathogens. Except for ical Sciences–The Consortium for Functional Glycomics GM62116. M.B. was sup- the recognition of pathogens, CLR have been implicated in several ported by a Vrije Universiteit Medical Center Institute for Cancer and Immunology PhD student grant, P.R.C. was supported by the Wellcome Trust, and H. L. was other functions, such as cell migration (6) and intercellular com- supported by the Swedish Research Council. A.D. is a Biotechnology and Biological munication (7). One of the best studied CLRs is the DC-specific Sciences Research Council Professorial Fellow. ICAM-3-grabbing nonintegrin, also known as DC-SIGN (8). Be- 2 M.B. and J.J.G.-V. contributed equally to this article. sides a pattern recognition receptor for HIV-1 (9), CMV (10), 3 Address correspondence and reprint requests to Dr. Y. van Kooyk, Department of Schistosoma mansoni (11), and other pathogens (5, 12), DC-SIGN Molecular Cell Biology and Immunology, Vrije Universiteit University Medical Cen- ter, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail address: has been shown to also recognize glycan epitopes on endogenous [email protected] ligands, such as ICAM-2 on endothelial cells (6), ICAM-3 on T 4 Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; mDC, cells (8), or Mac1 on neutrophils (13). Interaction of DC-SIGN mature DC; CLR, C-type lectin receptor; DC-SIGN, DC-specific ICAM-3-grabbing with its ligands regulates DC precursor migration into peripheral nonintegrin; MS, mass spectrometry; CRD, carbohydrate recognition domain; Ct, cycle threshold; Siglec, sialic acid-binding Ig superfamily lectin; MAA, Maackia tissues, stabilizes the DC-T cell contact surface in the immuno- amurensis agglutinin; SNA, Sambucus nigra agglutinin; RCA, Ricinus communis logical synapse (14), and allows neutrophils to induce DC matu- agglutinin. ration, respectively. Another CLR involved in intercellular com- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 munication is the macrophage galactose-type C-type lectin, which www.jimmunol.org The Journal of Immunology 8217 binds to specific CD45 glycoforms on T cells (7) to down-regulate (Promega) following the manufacturer’s guidelines. Cells (0.5 ϫ 106) were effector T cell functions. Other members of the lectin superfamily washed twice with ice-cold PBS, pelleted, and lysed in 500 ␮l of lysis buffer. Lysates were incubated with biotin-labeled oligo(dT) for 5 min at that play an important role in the immune system are siglecs (15) 20 37°C and then 50 ␮l of the mix was transferred to streptavidin-coated tubes and galectins (16). The sialic acid-binding Ig superfamily lectins and incubated for 5 min at 37°C. After washing three times with 250 ␮lof ␮ ϫ (siglecs) are a class of Ig superfamily proteins which show binding washing buffer, 30 l of the reverse transcription mix (5 mM MgCl2,1 activity to specific glycan structures containing sialic acid (17). reverse transcription buffer, 1 mM dNTP, 0.4 U of recombinant RNasin Many siglecs have molecular features of inhibitory receptors, in- RNase inhibitor, 0.4 U of reverse transcriptase, 0.5 ␮g of random hexamers in nuclease-free water) were added to the tubes and incubated for 10 min cluding conserved tyrosine-based motifs. This is the case of si- at room temperature followed by 45 min at 42°C. To inactivate AMV glec-2 (CD22), involved in the control of the BCR signaling (18), reverse transcriptase and separate mRNA from the streptavidin-biotin com- and the siglec-3 (CD33) related siglecs, known to relay inhibitory plex, samples were heated at 99°C for 5 min, transferred to microcentrifuge signals or to inhibit activatory pathways when cross-linked with tubes and incubated in ice for 5 min, diluted 1/2 in nuclease-free water, and Ϫ activating receptors (19–22). Galectins have a binding specificity stored at 20°C until analysis.