Conditions VCAM-1- Dependent Adhesion Under Flow 2,6-Linked Sialic Acid Inhibits Α Endothelial

Endothelial α2,6-Linked Sialic Acid Inhibits VCAM-1- Dependent Adhesion Under Flow Conditions

This information is current as Yasunori Abe, C. Wayne Smith, Julie P. Katkin, Lisa M. of September 24, 2021. Thurmon, Xudong Xu, Leonardo H. Mendoza and Christie M. Ballantyne J Immunol 1999; 163:2867-2876; ; http://www.jimmunol.org/content/163/5/2867 Downloaded from

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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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Endothelial ␣2,6-Linked Sialic Acid Inhibits VCAM-1- Dependent Adhesion Under Flow Conditions1

Yasunori Abe,* C. Wayne Smith,*† Julie P. Katkin,‡ Lisa M. Thurmon,* Xudong Xu,‡ Leonardo H. Mendoza,* and Christie M. Ballantyne2*§

We have previously shown that costimulation of endothelial cells with IL-1 ؉ IL-4 markedly inhibits VCAM-1-dependent adhesion under flow conditions. We hypothesized that sialic acids on the costimulated cell surfaces may contribute to the inhibition. Northern blot analyses showed that Gal␤1-4GlcNAc ␣2,6-sialyltransferase (ST6N) mRNA was up-regulated in cultured HUVEC by IL-1 or IL-4 alone, but that the expression was enhanced by costimulation, whereas the level of Gal␤1-4GlcNAc/Gal␤1- 3GalNAc ␣2,3-sialyltransferase (ST3ON) mRNA was unchanged. Removing both ␣2,6- and ␣2,3-linked sialic acids from IL-1 ؉ IL-4-costimulated HUVEC by sialidase significantly increased VCAM-1-dependent adhesion, whereas removing ␣2,3-linked sialic Downloaded from acid alone had no effect; adenovirus-mediated overexpression of ST6N with costimulation almost abolished the adhesion, which was reversible by sialidase. The same treatments of IL-1-stimulated HUVEC had no effect. Lectin blotting showed that VCAM-1 is decorated with ␣2,6- but not ␣2,3-linked sialic acids. However, overexpression of ␣2,6-sialyltransferase did not increase ␣2,6- linked sialic acid on VCAM-1 but did increase ␣2,6-linked sialic acids on other proteins that remain to be identified. These results -suggest that ␣2,6-linked sialic acids on a molecule(s) inducible by costimulation with IL-1 ؉ IL-4 but not IL-1 alone down regulates VCAM-1-dependent adhesion under flow conditions. The Journal of Immunology, 1999, 163: 2867–2876. http://www.jimmunol.org/

ascular cell adhesion molecule-1 is expressed on the sur- domain 1 of VCAM-1 is required, whereas both domains 1 and 4 faces of endothelial cells following stimulation with en- are involved in adhesion under static conditions (8). V dotoxin and cytokines such as IL-1, TNF, IL-4, and IL-4 is produced by Th2-type T cells, mast cells, basophils, and IL-13 (1, 2), as well as oxidized low-density lipoprotein (3, 4) and a subset of NK cells (13). It down-regulates endothelial cell ex- advanced glycation end products (5), which are postulated to me- pressions of E-selectin and ICAM-1 induced by IL-1 or TNF (14), diate atherosclerosis. VCAM-1 supports adhesion of leukocytes by and it enhances the expression of VCAM-1 (15, 16). However, we

␣ ␣ ␤ ␣ ␤ by guest on September 24, 2021 interacting with 4 integrins (i.e., 4 1 and 4 7), which are con- have previously shown that under flow conditions, adhesion of stitutively expressed on monocytes, lymphocytes, eosinophils, and monocytes and T cells to cultured endothelial cells stimulated with basophils but not on neutrophils in humans (1). For leukocytes to IL-1 and IL-4 in combination is almost abolished (6, 8). Thus, adhere to endothelial cells under physiological flow conditions, despite enhanced VCAM-1 expression, under physiological flow leukocytes must be captured from the bloodstream. Our laboratory conditions the net effect of the combination of IL-1 ϩ IL-4 on ␣ and others have shown that interactions between VCAM-1 and 4 endothelial cells is anti-adhesive. Although this might be partially integrins are necessary and sufficient to support primary capture of explained by down-regulation of E-selectin or other selectin-like mononuclear leukocytes under flow conditions in vitro (6–9). This molecules, we have found that VCAM-1-dependent adhesion un- is in contrast to adhesion of neutrophils under flow conditions, in der flow conditions is markedly inhibited (8). This latter finding is which selectins are indispensable for primary capture (1). A major of particular interest because the VCAM-1 expressed following isoform of VCAM-1 that is inducible on endothelial cells consists IL-1 ϩ IL-4 costimulation supports adhesion under static condi- ␣ of seven Ig-like domains, and the binding sites for 4 integrins tions as efficiently as VCAM-1 induced by IL-1 alone (8). These reside in domains 1 and 4 (10–12). For primary capture, only results strongly suggest that either the structure of VCAM-1 is somehow altered, interfering with the function of domain 1, or an antiadhesive mechanism(s) that works under flow conditions but not static conditions is up-regulated by the costimulation. *Speros Martel Section of Leukocyte Biology, Department of Pediatrics, †Department Sialic acids are 9-carbon monosaccharides that link to the ter- of Microbiology and Immunology, ‡Pulmonary Medicine, Department of Pediatrics, and §Section of Atherosclerosis, Department of Medicine, Baylor College of Medi- minal galactose, N-acetylgalactosamine, or other sialic acids in cine, Houston, TX 77030 carbohydrate chains that are attached to glycoproteins or glycolip- Received for publication February 11, 1999. Accepted for publication June 14, 1999. ids (17, 18). Because of their terminal location, sialic acids on the The costs of publication of this article were defrayed in part by the payment of page cell surface are among the first molecules encountered by other charges. This article must therefore be hereby marked advertisement in accordance cells coming in contact with the cell (17, 18). In addition, the first with 18 U.S.C. Section 1734 solely to indicate this fact. carbon of sialic acids is ionized at physiological pH (17, 18). This 1 This work was supported by National Institutes of Health Grants HL-42550 (to C.W.S. and C.M.B.), National Institutes of Health Grants ES-06091 and AI-19031 makes sialic acid the only sugar in glycoproteins that bears a net (to C.W.S), and an American Heart Association Established Investigator Award (to negative charge (17, 18). Because of their location and negative C.M.B.). charge, sialic acids have the potential to inhibit interactions be- 2 Address correspondence and reprint requests to Dr. Christie M. Ballantyne, Baylor tween molecules (17, 18). There are several reports that document College of Medicine, 6565 Fannin, M.S. A-601, Houston, TX 77030. E-mail address: [email protected] the antiadhesive nature of sialic acids (19–21).

Recently, Hanasaki et al. (22, 23) reported that Gal␤1-4Glc- was gel purified and cloned into pGEM-T Easy vector (Promega). Se- NAc␣2,6-sialyltransferase (ST6N)3 is up-regulated in endothelial quence analysis and database searches were performed by using the Mo- cells following stimulation with IL-1, TNF, and IL-4 and that lecular Biology Information Resources Center at Baylor College of Medicine. VCAM-1 molecules are decorated with ␣2,6-linked sialic acids. These reports prompted us to examine the following questions: 1) Northern blot analysis are sialic acids on the cell surfaces of HUVEC required for the Total RNA was isolated from HUVEC by acid guanidium thiocyanate- inhibition of VCAM-1-dependent adhesion by costimulation with ␮ ϩ phenol-chloroform extraction (28). Subsequently, 10 g of total RNA was IL-1 IL-4, 2) does overexpression of sialic acids lead to the electrophoresed in a 1% agarose/formaldehyde gel. The RNA was then inhibition of VCAM-1-dependent adhesion, and 3) if the above transferred to a charged nylon membrane (GeneScreenPlus, NEN, Boston, hypotheses are correct, is the inhibition due to increased sialylation MA). Equal loading of lanes was assessed by photographs of the ethidium of VCAM-1 molecules? bromide-stained gels as well as by probing of the membranes with human In this paper we demonstrate for the first time that ␣2,6-linked GAPDH cDNA (29). cDNA probes for human VCAM-1 (30) and ST6N (31) were gel purified with QIAEX II (Qiagen, Chatsworth, CA) and were sialic acids on endothelial cells can inhibit VCAM-1-dependent labeled with [32P]-dCTP using the random-hexamers (Prime-it II; Strat- adhesion of leukocytes under flow conditions. However, this in- agene, La Jolla, CA) according to the manufacturer’s protocol. The mem- hibitory effect is neither due to increased sialylation of VCAM-1 brane was prehybridized with QuikHyb solution (Stratagene), and hybrid- nor related to the total amount of sialic acids present on the cell ization was performed as previously described (29). To quantify radioactivity, ␤-emissions from respective bands were measured by the surfaces. Rather, to exhibit their inhibitory effect, sialic acids must Molecular Image Scanner (Bio-Rad, Hercules, CA) and the values were be associated with a molecule(s) that is inducible by stimulation normalized by those from the bands hybridized with GAPDH cDNA. Ra-

with IL-1 and IL-4 in combination but not IL-1 alone. We propose dioactivity was normalized and compared as follows: relative radioactivity Downloaded from a novel inducible mechanism on endothelial cells that negatively compared with unstimulated HUVEC ϭ [(counts from respective ST6N Ϭ regulates leukocyte adhesion under ﬂow conditions. band/counts from the GAPDH of the same sample lane) (counts from the ST6N band of unstimulated HUVEC/counts from the GAPDH band of unstimulated HUVEC)]. Materials and Methods Cell culture Detection of cell surface expression of ␣2,3- and ␣2,6-linked

HUVEC were harvested from 5 to 10 umbilical cords by collagenase di- sialic acids http://www.jimmunol.org/ gestion as previously described (24). Primary cultured cells were passaged Expression of ␣2,3- and ␣2,6-linked sialic acids on endothelial cell sur- with 0.05% trypsin and 0.53 mM EDTA (Life Technologies, Gaithersburg, faces was detected by cell-surface ELISA using lectins. HUVEC were MD) and seeded onto tissue culture dishes or flasks (Corning, Cambridge, seeded onto 1% gelatin-coated 96-well tissue culture plates (Becton Dick- MA) coated with 1% gelatin (Sigma, St. Louis, MO). The cells were grown inson, San Jose, CA). Two or 3 days after passage, cells were stimulated in M199 (Life Technologies) supplemented with 10% FCS (HyClone, Lo- with or without culture medium containing IL-1 (Genzyme, Cambridge, gan, UT), 10% CS (HyClone), 0.1 mg/ml heparin (Sigma), 50 ␮g/ml en- MA), IL-4 (Genzyme), and IL-1 ϩ IL-4 at 37°C with 5% CO for 22.5 h. dothelial cell growth supplement (Collaborative Research, Bedford, MA), 2 In some wells, 0.1 U/ml of sialidase from Arthrobacter ureafaciens (Boeh- and 1% penicillin-streptomycin (Life Technologies). First-passage ringer Mannheim, Indianapolis, IN), which cleaves both ␣2,3- and ␣2,6- HUVEC were used in all experiments. linked sialic acid (32), or 20 U/ml of recombinant sialidase L (V-LABS, Ramos cells (CRL-1596) and 293 cells (CRL-1573) were obtained from Covington, LA), which specifically cleaves ␣2,3-linked sialic acid (33), by guest on September 24, 2021 the American Type Culture Collection (Manassas, VA) and maintained in was added to the culture medium and incubated for another 90 min. After RPMI 1640 medium (Life Technologies) and Minimum Essential Medium washing four times with Dulbecco’s PBS (D-PBS), cells were fixed with (Life Technologies), respectively, containing 10% FCS and 1% D-PBS containing 2% paraformaldehyde and 0.2% glutaraldehyde for 20 penicillin-streptomycin. min. After monolayers were blocked with Tris-buffered saline containing RT-PCR and PCR cloning of ␣2,3-sialyltransferase cDNA 0.5% casein (Boehringer Mannheim), ␣2,6- and ␣2,3-linked sialic acids were detected by Sambucus nigra (SNA) and Maackia amurensis (MAA) Based on conserved motifs of three published human ␣2,3-sialyltrans- agglutinins labeled with digoxigenin (Boehringer Mannheim), respec- ferases (25–27), three pairs of oligonucleotides were synthesized at the tively, in Tris-buffered saline (pH 7.5) containing 1 mM CaCl2,1mM Child’s Health Research Center Core Laboratory, Baylor College of Med- MgCl2, and 1 mM MnCl2 (34, 35). Bound lectins were detected by alkaline icine. The sequences of sense and antisense primers were: Gal␤1-3(4)Glc- phosphatase-conjugated goat anti-digoxigenin Ab (Boehringer Mannheim) NAc ␣2,3-sialyltransferase (ST3N) (25), 5Ј-GCT GCC GCC GCT GCA and p-nitrophenyl phosphate (Sigma) as a substrate. The optical absorbance TCA TCG TGG GCA A (sense) and 5Ј-GAT GAA ATA TGG GTT GAG was measured at 405 nm by an automatic microplate reader (Cambridge GAT (antisense); Gal␤1-3GalNAc ␣2,3-sialyltransferase (ST3O) (26), 5Ј- Technology, Watertown, MA). Values of optical absorbance at 405 nm GCT GCC GGC GCT GCG CCG TTG TGG GCA A (sense) and 5Ј-GAT were subtracted with corresponding controls (wells in which lectins were GAA GGC TGG GTG GTA GAT (antisense); and Gal␤1-4GlcNAc/ not added). Gal␤1-3GalNAc ␣2,3-sialyltransferase (ST3ON) (27), 5Ј-GGT GCC GCC Ј GCT GTG TGG TCG TGG (sense) and 5 -CCG GCA ATG TGC ACC Flow adhesion assay AAG TCA CAG (antisense). Total RNA was isolated from unstimulated and IL-1-, IL-4-, and IL-1 ϩ IL-4-stimulated HUVEC by acid guanidium The adhesion of Ramos cells to HUVEC under hydrodynamic flow con- thiocyanate-phenol-chloroform extraction (28). Reverse transcriptase (RT) ditions was studied as previously described (8, 36). Briefly, cell monolay- reaction was performed by incubating 2 ␮g of total RNA with 1.5 ␮M ers in 35-mm tissue culture dishes were mounted in a parallel-plate flow antisense oligonucleotides and 1.5 mM dNTP (Promega, Madison, WI) in system. Ramos cells suspended at 106 cells/ml in 37°C D-PBS containing 10 ␮l of RT buffer (Promega) at 80°C for 5 min, followed by mixing with Ca2ϩ,Mg2ϩ, and 11 mM glucose were passed through the parallel plate 23 U of avian myeloblastosis virus (AMV)-RT (Promega) and 40 U of flow chamber at 1.0 dyne/cm2 under a phase contrast microscope (Diaphot RNase inhibitor (Promega) in 5 ␮l of RT buffer, and incubating at 42°C for TMD, Nikon, Garden City, NY) for 9 min at 37°C. At the end of the 2 h. Then, the RT products were diluted eight times with PCR buffer (Fish- perfusion, four different fields were videotaped at 15-s intervals. Video- er Scientific, Pittsburgh, PA), and the subsequent PCR was performed with taped images were analyzed with Optimas image analysis software (Bio- ␮ 0.2 M sense oligonucleotides, 0.2 mM dNTP, 2–5 mM MgCl2, and 5 scan, Edmonds, WA). The number of stably adherent cells was quantitated U/50 ␮lofTaq polymerase (Fisher Scientific) for 33 cycles. PCR product as the average number of leukocytes remaining on the monolayer in the four fields.

3 Abbreviations used in this paper: ST6N, Gal␤1-4GlcNAc ␣2,6-sialyltransferase; Ad26st, adenovirus carrying ST6N cDNA; Ad5, type 5 adenovirus; Adgal, adenovirus Construction of a recombinant adenovirus carrying ST6N cDNA carrying ␤-galactosidase cDNA; D-PBS, Dulbecco’s PBS; MAA, Maackia amurensis agglutinin; MOI, multiplicity of infection; NCAM, neural cell adhesion molecules; A recombinant type 5 adenovirus (Ad5) vector expressing the ST6N gene SNA, Sambucus nigra agglutinin; ST3N, Gal␤1-3(4)GlcNAc ␣2,3-sialyltransferase; was constructed according to standard methods. Brieﬂy, an expression cas- ST3O, Gal␤1-3GalNAc ␣2,3-sialyltransferase; ST3ON, Gal␤1-4GlcNAc/Gal␤1- sette containing the ST6N cDNA (a generous gift of Dr. I. Stamenkovic, 3GalNAc ␣2,3-sialyltransferase; X-Gal, 5-bromo-4-chloro-3-indolyl ␤-D-galactoside. Harvard Medical School) under control of the human elongation factor-1␣ The Journal of Immunology 2869

promoter (EF-1␣ promoter) (37), and the bovine growth hormone polyad- enylation signal was subcloned into the shuttle plasmid p␦E1sp1B (Micro- bix Biosystems, Toronto, Canada), which contains the 5Ј Ad5 sequence with a deletion in the E1 region (38). The transformed shuttle plasmid DNA was cotransfected into Ad5-transformed 293 cells (39) with the cir- cular Ad5 genome plasmid pBHGE3 (40) (Microbix Biosystems) to gen- erate recombinant adenovirus carrying ST6N cDNA (Ad26st). Recombi- nant viruses were isolated by plaque purification and characterized by PCR. A plaque containing the correct recombinant vector was propagated to high titer by serial passage on 293 cells and purified on cesium chloride gradi- ents using standard methods (41, 42). The titer was determined by identi- fying the presence of viral cytopathic effect in 293 cells infected with serial dilutions of each preparation. The preparation used for these experiments had a titer of 2 ϫ 1011 infectious units/ml. The EF-1␣ promoter used in this construct has generated high levels of transgene expression from other recombinant vectors produced in our laboratory (43). Transduction of HUVEC with adenovirus vectors After HUVEC were grown to 80–90% confluence, they were incubated

with medium containing recombinant virus at 37°C with 5% CO2 for 24 h. For overexpression of ␣2,6-sialyltransferase and cytokine stimulation, cells

were ﬁrst incubated with Ad26st in medium at 37°C with 5% CO2 for 17 h, Downloaded from and then 10 U/ml of IL-1 and 10 ng/ml of IL-4, or 10 U/ml of IL-1 ϩ 10 ng/ml of IL-4 was added directly to the medium and the incubation was continued for another 24 h. Detection of ␤-galactosidase in HUVEC following transduction with adenovirus carrying ␤-galactosidase cDNA

Following transduction of HUVEC with recombinant adenovirus carrying http://www.jimmunol.org/ ␤-galactosidase cDNA (Adgal) for 24 h, the cells were stained with 5-bromo-4-chloro-3-indolyl ␤-D-galactoside (X-Gal) as follows. Cell monolayers were ﬁxed with 2% paraformaldehyde and 0.2% glutaraldehyde in D- PBS for 10 min. After rinsing twice with D-PBS containing 50 mM

glycine, cells were stained with 5 mM K4Fe(CN)6,5mMK3Fe(CN)6,1 mg/ml X-Gal (Sigma), 2 mM MgCl , 0.1% deoxycholic acid, and 0.2% 2 FIGURE 1. Northern blot analyses of ST6N and ST3ON mRNA ex- Nonidet P-40 in D-PBS at 37°C for 2 h. pression in HUVEC after cytokine stimulation. HUVEC monolayers were Lectin blotting of whole cell lysates and immunoprecipitated stimulated with 10 U/ml IL-1, 10 ng/ml IL-4, and 10 U/ml IL-1 ϩ 10 ng/ml ␮ IL-4 at 37°C with 5% CO2 for 22 h. Total RNA (10 g/lane) was hybrid-

VCAM-1 by guest on September 24, 2021 ized with cDNA probes for human ␣2,6 sialyltransferase, ST6N (ST6) (A, Lectin blotting of whole cell lysates and immunoprecipitated VCAM-1 was upper panel), and human ␣2,3 sialyltransferase, ST3ON (ST3) (B lower performed as previously described (22). After washing HUVEC monolay- panel) as described in Materials and Methods. For the detection of ers four times with ice-cold D-PBS, the cells were lysed with 1 ml of 20 GAPDH mRNA, ST6N and ST3ON probes were stripped from the filters mM Tris/HCl (pH 8.0), 150 mM NaCl containing 1% Nonidet P-40, 1 mM and the same filter was reprobed with GAPDH cDNA. EDTA, 1 mM PMSF (Sigma), 10 ␮g/ml aprotinin (Boehringer Mannheim), and 2 ␮g/ml of leupeptin (Boehringer Mannheim) at 4°C for 30 min. The lysates were centrifuged at 10,000 ϫ g at 4°C for 15 min to remove debris. For lectin blotting of whole cell lysates, protein concentration of cell ly- Results sates was measured by the BCA protein assay kit (Pierce, Rockford, IL), Expression of ␣2,6-sialyltransferase and ␣2,3-sialyltransferase ␮ and 20 g of total protein per lane was separated by 7.5% SDS-PAGE mRNA in HUVEC under reducing conditions and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) by standard methods. For immunopre- Human ST6N cDNA has been identified and cloned by three in- cipitation of VCAM-1, after preclearing with protein A-Sepharose (Pierce) dependent groups from different cell types (31, 45, 46). This en- at 4°C for 2 h, 400 ␮g of total protein was incubated with 15 ␮g/ml of anti-VCAM-1 mAb, 4B9 (a generous gift from Dr. Roy Lobb, Biogen) zyme is expressed in a wide variety of tissues, and the size of the (44), at 4°C for 2 h. The immune complexes were then absorbed with mRNA varies in cell types due to differences in the 5Ј untranslated protein A-Sepharose at 4°C for 2 h. The protein A-Sepharose beads were regions (22, 26, 46). As shown in Fig. 1A, ST6N mRNA was washed with lysis buffer, and the beads were boiled with Laemmli sample detected in unstimulated HUVEC; the size of the mRNA was ϳ4.2 buffer containing 100 mM dithiothreitol (Sigma). Supernatants were sep- kb, which is consistent with Hanasaki et al. (22). Because we have arated by 7.5% SDS-PAGE and transferred to nitrocellulose membranes. For detection of sialic acids, after blots were blocked with 0.5% casein, previously shown that the anti-adhesive effect of IL-1 ϩ IL-4 co- ␣2,6- and ␣2,3-linked sialic acids were detected by digoxigenin-labeled stimulation appears later than 8 h and remains at 24 h after incu- SNA (Boehringer Mannheim) (34) and MAA (Boehringer Mannheim) bation (8), we were interested in the levels of ST6N mRNA at (35), respectively. Bound lectins were detected by alkaline phosphatase- these time points. After 22 h of incubation, ST6N mRNA was conjugated sheep anti-digoxigenin Ab (Boehringer Mannheim). For detection of VCAM-1, after blots were blocked with 3% BSA in Tris-buffered up-regulated by either IL-1 or IL-4 alone. However, up-regulation saline, VCAM-1 was probed with goat anti-VCAM-1 sera (R&D Systems, was further enhanced by the combination of IL-1 ϩ IL-4 (see Fig. Minneapolis, MN) and alkaline phosphatase-conjugated rabbit anti-goat 1A). The relative radioactivities of bands identified with ST6N IgG (Pierce). Bound alkaline phosphatase-conjugated Abs were detected cDNA compared with those of unstimulated HUVEC were 1.25 Ϯ by bromochloroindoyl phosphate/nitro blue tetrazolium chloride (Boehr- 0.35 (IL-1), 1.64 Ϯ 0.27 (IL-4), and 2.27 Ϯ 0.48 (IL-1 ϩ IL-4). inger Mannheim). To examine which ␣2,3-sialyltransferase is expressed in human Statistics endothelial cells, we performed RT-PCR using primers synthe- ␣ Results are presented as mean Ϯ SD. Statistical assessments were made sized according to three published human 2,3-sialyltrans- using Student’s t test or one-way ANOVA with Bonferroni multiple com- ferases—ST3N, ST3O, and ST3ON—which use glycoproteins as parisons. The p values that exceeded 0.05 were not considered significant. acceptor substrates. Although we tried using different annealing 2870 SIALIC ACID INHIBITS VCAM-1-MEDIATED ADHESION

FIGURE 2. Binding of SNA and MAA lectins to HUVEC and the effects of sialidase. HUVEC were seeded onto 96-well tissue culture plates. After cells were grown to conﬂuence, the monolayers were stimulated with 10 U/ml IL-1, 10 ng/ml IL-4, or 10 U/ml ϩ IL-1 10 ng/ml IL-4 at 37°C with 5% CO2 for 24 h. In some wells, 0.1 U/ml of sialidase from A. ureafaciens was added to the culture medium and incubated for the last 90 min. Sialic acids on the cell surfaces were detected as described in Materials and Methods. Values represent mean absorbance at 405 nm Ϯ SD from triplicate experiments. SNA lectin detects ␣2,6- linked sialic acids (A), and MAA lectin detects ␣2,3- linked sialic acids (B). Downloaded from

temperatures and Mg2ϩ concentrations, PCR product was only ob- cause any visible change in the monolayers under phase contrast tained with primers made from the sequence of ST3ON. The PCR microscopy. Although incubation for 90 min at neutral pH in cul-

product was a single band on gel electrophoresis, and this 493-bp ture medium might not be optimal for sialidase, this was sufficient http://www.jimmunol.org/ PCR product was cloned into pGEM-T Easy vector. Four clones to remove sialic acids from surfaces of HUVEC monolayers as randomly picked were sequenced and found to be identical with shown in Fig. 2. Under flow conditions, costimulation of HUVEC ϩ ␣ the published sequence of ST3ON cDNA (27). Northern blot anal- with IL-1 IL-4 markedly inhibited 4 integrin/VCAM-1-depen- ysis using this clone as a probe demonstrated that ST3ON mRNA dent adhesion compared with stimulation with IL-1 alone (Fig. was constitutively expressed in HUVEC (Fig. 1B). The size of the 3B), although the surface expression of VCAM-1 was higher in the RNA was ϳ2.0 kb, which is consistent with a previous report (27). costimulated HUVEC, as was seen in our previous report (8). However, unlike ST6N, the levels of ST3ON mRNA were not Treatment of IL-1 ϩ IL-4-costimulated HUVEC with sialidase regulated by IL-1, IL-4, or IL-1 ϩ IL-4 (see Fig. 1B). significantly increased adhesion of Ramos cells, and this increase

was completely inhibited by the anti-VCAM-1 mAb 4B9 (44) and by guest on September 24, 2021 Surface expression of ␣2,3- and ␣2,6-linked sialic acids on ␣ the anti- 4 integrin mAb HP2/1 (47) (Fig. 3A). In contrast, the HUVEC same treatment of either IL-1- or IL-4-stimulated HUVEC failed to MAA and SNA lectins recognize ␣2,3- and ␣2,6-linked sialic ac- increase the adhesion of Ramos cells (see Fig. 3A). Thus, sialic ids, respectively (34, 35). In spite of the increased expression of acids are required for the inhibition of VCAM-1-dependent adhe- ST6N, the binding of SNA lectin to HUVEC stimulated with IL-1, sion in IL-1 ϩ IL-4-costimulated HUVEC. However, this is not IL-4, and IL-1 ϩ IL-4 for 24 h was not significantly different from related solely to the total amount of sialic acids on cell surfaces, unstimulated HUVEC (Fig. 2A). After treatment of HUVEC with because removing a high percentage of sialic acids from the sur- sialidase from A. ureafaciens, which cleaves all three linkages of faces of HUVEC stimulated with either IL-1 or IL-4 had no sig- sialic acids (32), the binding of SNA lectin was markedly reduced nificant effect on adhesion. (see Fig. 2A). The reduction was similar among different cytokine stimulations. This suggests that up-regulation of ST6N has little ␣ effect on total amount of ␣2,6-linked sialic acids on cell surfaces. Effects of general versus 2,3-linkage-specific sialidase on lectin Similarly, MAA binding to HUVEC was not affected by stimula- binding and on Ramos cell adhesion under flow conditions tion with IL-1, IL-4, or IL-1 ϩ IL-4 for 24 h. This binding was Because sialidase isolated from A. ureafaciens cleaves both ␣2,6- almost eliminated by sialidase (Fig. 2B). and ␣2,3-linked sialic acids as shown in Fig. 2, the increased adhesion of Ramos cells following the treatment of IL-1 ϩ IL-4- Effects of sialidase treatment of HUVEC on Ramos cell costimulated HUVEC with the sialidase may have been due to the adhesion under flow conditions removal of ␣2,6- or ␣2,3-linked sialic acids. Although to our Ramos cells are able to tether and adhere stably to cytokine-stim- knowledge sialidase specific to ␣2,6-linked sialic acid is not ulated HUVEC under shear stresses up to 2.0 dyne/cm2 (8). This known, it has been reported that sialidase from Macrobdella tethering and stable adhesion are mediated exclusively by interac- decora leech (sialidase L) specifically cleaves ␣2,3-linked sialic ␣ ␣ tions between 4 integrins on Ramos cells and VCAM-1 on acids (33). We examined whether this sialidase cleaves 2,3- HUVEC (8). To examine the role of sialic acids on the surfaces of linked sialic acids from the surfaces of HUVEC under the same ␣ HUVEC in 4/VCAM-1-dependent adhesion under flow condi- conditions used for sialidase from A. ureafaciens. Confluent mono- tions, we studied Ramos cell adhesion at 1.0 dyne/cm2 to HUVEC layers of HUVEC grown in 96-well plates were costimulated with that were stimulated with cytokines and then incubated with or IL-1 (10 U/ml) and IL-4 (10 ng/ml) for 24 h; in some wells, 20 without sialidase. Because most Ramos cells that tethered stably U/ml of recombinant sialidase L or 0.1 U/ml of sialidase from A. adhered, we examined only the number of stably adherent Ramos ureafaciens was added to the culture medium and incubated for the cells. Treatment of HUVEC with sialidase after 22.5 h of cytokine last 90 min. ␣2,6- and ␣2,3-linked sialic acids on the surfaces of stimulation, for the last 90 min of cytokine stimulation, did not HUVEC were detected by the binding of SNA and MAA lectins, The Journal of Immunology 2871

Overexpression of human ␣2,6-sialyltransferase in HUVEC by adenovirus vector To investigate whether overexpression of ST6N in HUVEC inhibits VCAM-1-dependent adhesion, ST6N cDNA was introduced into HUVEC by means of a recombinant adenovirus vector. To confirm the expression of ST6N mRNA by the virus, HUVEC were incubated with increasing concentrations of Ad26st for 24 h, and Northern blot analysis was performed. The transcript length of ST6N expressed by Ad26st was 2.7 kb and was expressed at high levels at multiplicity of infection (MOI) Ն100 infectious units (Fig. 5A). Wild-type ST6N mRNA, which has a longer untranslated region and is ϳ4.2 kb, was not evident because of the very short exposure time of the x-ray film (4 h). Because HUVEC constitutively express ␣2,6-linked sialic acids and ST6N, it is difficult to distinguish the expression of ST6N induced by gene transfer from that of wild type in each cell. There- fore, instead of ST6N, we examined the expression of ␤-galactosidase after the cells were transduced with increasing concentrations of recombinant Ad5 carrying ␤-galactosidase cDNA for 24 h. Downloaded from Expression was confirmed by visual inspection of infected monolayers after exposure to X-Gal. In this study, 100% of cells expressed the transgene following infection at an MOI of 100 (data not shown). To determine whether transfection of the adenovirus vector it- self may induce expression of cell adhesion molecules, Northern http://www.jimmunol.org/ blot analysis of VCAM-1 mRNA was performed (Fig. 5B). Al- though VCAM-1 mRNA was up-regulated by the virus at concentrations Ͼ500 MOI, the level of mRNA expression was not dif- FIGURE 3. Effects of sialidase on Ramos cell adhesion to HUVEC un- ferent from controls at concentrations Ͻ250 MOI (see Fig. 5B). der flow conditions. HUVEC were seeded onto 35-mm culture dishes. Af- Based on these results, we selected an MOI of 100 for use in ter cells were grown to confluence, the monolayers were stimulated with 10 U/ml IL-1, 10 ng/ml IL-4, and 10 U/ml IL-1 ϩ 10 ng/ml IL-4 at 37°C with subsequent experiments, similar to that previously published for HUVEC (48). 5% CO2 for 24 h. In some experiments, 0.1 U/ml sialidase from A. ure-

afaciens was added to the culture medium and incubated for the last 90 by guest on September 24, 2021 min. For blocking of VCAM-1, 20 ␮g/ml of mAb 4B9 was added to the Effects of overexpression of human ␣2,6-sialyltransferase in culture medium for 20 min immediately before assay and to the perfusion HUVEC on Ramos cell adhesion under flow conditions buffer to maintain saturation during assay. For blocking of ␣ integrins, 4 To examine the effects of overexpression of ST6N on VCAM-1- Ramos cells were preincubated with 10 ␮g/ml of HP2/1 at room temper- ature for 10 min immediately before assay. Adhesion assays were per- dependent adhesion, adhesion of Ramos cells to HUVEC stimu- formed at 1.0 dyne/cm2 as described in Materials and Methods. For each lated with cytokines in association with overexpression of ST6N cytokine stimulation, the number of stably adherent Ramos cells after siali- was studied under flow conditions. In HUVEC stimulated with dase treatment of HUVEC was compared with the number of Ramos cells either IL-1 or IL-4 alone for 24 h, overexpression of ST6N did not stably adherent to HUVEC without sialidase to provide percent adhesion have a significant effect on Ramos cell adhesion (Fig. 6A). Al- (A). The actual number of stably adherent Ramos cells is shown in B. though the adhesion of Ramos cells to IL-1 ϩ IL-4-costimulated Values represent the mean Ϯ SD from four to eight experiments except HUVEC was significantly inhibited compared with IL-1 stimula- -p Ͻ 0.001 as compared with the tion alone, the inhibition was not complete and VCAM-1-depen ,ء .(blocking studies using Abs (n ϭ 2 group not treated with sialidase. dent adhesion was still observed (see Fig. 3) (8). However, overexpression of ST6N in IL-1 ϩ IL-4-costimulated HUVEC led to further inhibition of Ramos cell adhesion, and adhesion was nearly eliminated (Fig. 6, B and C), whereas transduction of HUVEC with respectively, as described in Materials and Methods. As shown in Adgal at the same MOI had no effect (Fig. 6B). This inhibition was Fig. 4B, sialidase L significantly reduced the level of ␣2,3-linked reversed by removing sialic acids from HUVEC (see Fig. 6, B and sialic acid on IL-1 ϩ IL-4-costimulated HUVEC, whereas it did C), and the adhesion was completely VCAM-1 dependent (see Fig. not change the level of ␣2,6-linked sialic acid (Fig. 4A). To de- 6, B and C). termine whether removing ␣2,3-linked sialic acid from IL-1 ϩ IL-4-costimulated HUVEC increases Ramos cell adhesion, Lectin blotting of VCAM-1 immunoprecipitated from HUVEC ϩ HUVEC were costimulated with IL-1 IL-4 for 24 h and treated We wished to determine whether VCAM-1 induced by IL-1, IL-4, with sialidases for the last 90 min, and the adhesion of Ramos cells and IL-1 ϩ IL-4 is decorated by ␣2,6-linked sialic acids and to HUVEC was compared under flow conditions (Fig. 4C). Re- whether overexpression of ST6N up-regulates the levels of ␣2,6- moval of ␣2,3-linked sialic acids by sialidase L treatment of IL- linked sialic acids on VCAM-1. VCAM-1 molecules immunopre- 1 ϩ IL-4-costimulated HUVEC did not show any increase in cipitated from HUVEC stimulated with IL-1 and IL-1 ϩ IL-4 Ramos cell adhesion, whereas treatment with sialidase from A. showed two distinct bands in SDS-PAGE. The lower band and ureafaciens markedly increased the adhesion. These results sug- upper band correspond to the precursor and mature forms of gest that ␣2,6-linked sialic acid is required for the inhibition of VCAM-1, respectively (Fig. 7, A and B) (49). The amount of VCAM-1-dependent adhesion by IL-1 and IL-4. VCAM-1 immunoprecipitated from IL-1 ϩ IL-4-costimulated 2872 SIALIC ACID INHIBITS VCAM-1-MEDIATED ADHESION

FIGURE 4. Effects of general vs ␣2,3-linkage-specific sialidase on lectin binding and on Ramos cell adhesion under flow conditions. A and B, SNA and Downloaded from MAA lectin binding. HUVEC were seeded onto 96-well tissue culture plates. After cells were grown to confluence, the monolayers were stimulated with ϩ 10 U/ml IL-1 10 ng/ml IL-4 at 37°C with 5% CO2, for 24 h. In some wells, 20 U/ml of sialidase L or 0.1 U/ml of sialidase from A. ureafaciens was added to the culture medium and incubated for the last 90 min. Sialic acids on the cell surfaces were detected as described in Materials and Methods. Values represent mean absorbance at 405 nm Ϯ SD from triplicate experiments. SNA lectin detects ␣2,6-linked sialic acids (A), and MAA lectin detects ␣2,3-linked sialic acids (B). C, Ramos cell adhesion. HUVEC were seeded onto 35-mm culture dishes. After cells were grown to confluence, the monolayers ϩ were stimulated with 10 U/ml IL-1 10 ng/ml IL-4 at 37°C with 5% CO2 for 24 h. In some experiments, 20 U/ml of sialidase L or 0.1 U/ml sialidase from A. ureafaciens was added to the culture medium and incubated for the last 90 min. Adhesion assays were performed at 1.0 dyne/cm2 as described http://www.jimmunol.org/ in Materials and Methods. The number of stably adherent Ramos cells after sialidase treatment of HUVEC was compared with the number of Ramos cells ;p Ͻ 0.0001 ,ء .stably adherent to HUVEC without sialidase to provide percent adhesion. Values represent the mean Ϯ SD from four separate experiments †, p Ͻ 0.005 as compared with the group not treated with sialidase.

HUVEC was higher than from IL-1 stimulation alone, as reported the above mechanisms. This inducible mechanism on endothelial previously (see Fig. 7B) (8). VCAM-1 immunoprecipitated from cells negatively regulates leukocyte adhesion under ﬂow condi- ϩ ␣

HUVEC costimulated with IL-1 IL-4 was decorated with 2,6- tions. For the following reasons, we concluded that at least part of by guest on September 24, 2021 but not ␣2,3-linked sialic acids (see Fig. 7A), and ␣2,6-linked this mechanism is attributable to ␣2,6-linked sialic acids that are sialic acids were also detected on VCAM-1 molecules induced by associated with a molecule(s) inducible by costimulation of IL-1 (see Fig. 7B). However, overexpression of ST6N did not in- HUVEC with IL-1 ϩ IL-4: 1) removing both ␣2,3- and ␣2,6- crease the levels of ␣2,6-linked sialic acids on VCAM-1 induced linked sialic acids increased VCAM-1-dependent adhesion in IL- by either IL-1 or IL-1 ϩ IL-4 (see Fig. 7B). 1 ϩ IL-4-costimulated HUVEC but not IL-1-stimulated HUVEC; 2) removing ␣2,3-linked sialic acid without removing ␣2,6-linked Lectin blotting of whole cell lysates sialic acid from IL-1 and IL-4-costimulated HUVEC did not affect Because the level of ␣2,6-linked sialic acid on VCAM-1 was un- VCAM-1-dependent adhesion; 3) ␣2,6-sialyltransferase was changed despite the overexpression of ␣2,6-sialyltransferase highly up-regulated in IL-1 ϩ IL-4-costimulated HUVEC, mRNA by adenovirus vector, we examined whether the increased whereas ␣2,3-sialyltransferase was not regulated by the cytokines; ␣2,6-sialytransferase mRNA resulted in an increased level of and 4) overexpression of ␣2,6-sialyltransferase inhibited VCAM- ␣2,6-linked acid on other proteins. Detergent lysates of HUVEC 1-dependent adhesion in IL-1 ϩ IL-4-costimulated HUVEC but stimulated with IL-1 ϩ IL-4 had numerous proteins that were de- not IL-1-stimulated HUVEC. tected by SNA lectin (Fig. 8). Although we did not see any dif- Although our results and others suggest that ␣2,6-linked sialic ference in ␣2,6-linked sialic acids on VCAM-1, we did see an acids are common among glycoproteins expressed by HUVEC increase of ␣2,6-linked sialic acids on some other proteins, which (22), little is known about their function. A great deal of evidence are larger than 90 kDa, following overexpression of ST6N in IL- suggests that sialic acids support cell adhesion (18). Sialic acids ϩ 1 IL-4-costimulated HUVEC (see Fig. 8). This finding suggests and their related structures bind to numerous microbial lectins that that transduction of HUVEC with Ad26st not only increased ST6N support adhesion of microorganisms to their hosts (18). Selectins mRNA but also increased enzyme activity, which resulted in an and sialoadhesin family molecules mediate cell-cell adhesion by ␣ increase in 2,6-linked sialic acids on at least several proteins. binding to sialic acids and their related structures in a ligand and receptor-specific manner (18). Several reports indicate that ␣2,6- Discussion linked sialic acids may serve as a ligand for CD22, which is ex- Published evidence suggests that adhesion of mononuclear leuko- pressed by mature B cells to support adhesion of these lymphocytes to endothelial cells is mediated by cell adhesion molecules, cytes (50–52). A recent study in ST6N-deficient mice suggests ␣ including 4 integrins and VCAM-1, in a ligand and receptor- that ST6N is essential in immune responses of B cells (53). Ramos specific manner (1). Adhesion of leukocytes can be regulated by cells are a B cell line that is reported to express CD22, although the levels of expression and activation state of cell adhesion mol- unlike mature B cells, most of the CD22 in Ramos cells is present ecules (1). In this study, we have demonstrated a novel regulatory in the cytoplasm instead of on the cell surface (54). However, our mechanism of leukocyte adhesion to endothelial cells distinct from previous report and the current study clearly indicate that the The Journal of Immunology 2873 Downloaded from http://www.jimmunol.org/

FIGURE 5. Expression of ST6N mRNA following transduction of HUVEC with Ad26st and the effect on the expression of VCAM-1 mRNA. A; After HUVEC were grown to 80–90% confluence, the monolayers were incubated with medium alone, Adgal at 500 MOI, or increasing concentrations of Ad26st in HUVEC medium at 37°C with 5% CO2 for 24 h. Total RNA was extracted and hybridized with ST6N cDNA probe. High levels of the 2.7-kb transcript of ST6N (ST6) expressed by Ad26st were observed for MOI of Ն100. Endogenous ST6N mRNA, which is 4.2 kb in length, by guest on September 24, 2021 was not evident because of the very short exposure time of the x-ray film (4 h) for this figure. B; Incubation of HUVEC monolayers with medium alone, Adgal at 250 MOI and 500 MOI, or increasing concentrations of Ad26st or 10 U/ml IL-1. Total RNA was extracted and hybridized with VCAM-1 cDNA probe as described in Materials and Methods. Increased FIGURE 6. Effects of overexpression of ST6N on adhesion of Ramos levels of VCAM-1 mRNA were not observed at MOI of Ͻ500 compared cells to HUVEC under flow conditions. A; HUVEC were seeded onto with controls. 35-mm culture dishes. After cells were grown to 80–90% confluence, they were incubated with or without 100 MOI of Ad26st for 17 h. Then, 10 U/ml IL-1, 10 ng/ml IL-4, or 10 U/ml IL-1 ϩ 10 ng/ml IL-4 were added adhesion of Ramos cells to cytokine-stimulated HUVEC under to the culture medium for 24 h. Adhesion assays were performed at 1.0 ␣ dyne/cm2. Values represent percent adhesion of Ramos cells to ST6N- both static and flow conditions is dependent exclusively on 4 integrins and VCAM-1 (8). There is no evidence that Ramos cells overexpressed HUVEC compared with those without the overexpression. adhere to HUVEC in a CD22 and ␣2,6-linked sialic acid-depen- B; HUVEC were incubated with or without 100 MOI of Ad26st or Adgal for 17 h. Then, 10 U/ml IL-1 ϩ 10 ng/ml IL-4 were added to the culture dent manner in our system, and in fact, overexpression of ST6N in medium for 24 h. In some experiments, IL-1 ϩ IL-4-costimulated HUVEC HUVEC led to reduced Ramos cell adhesion to costimulated were treated with 0.1 U/ml sialidase from A. ureafaciens for the last 90 min HUVEC. of the cytokine stimulation. For blocking of VCAM-1, 20 ␮g/ml of mAb A few reports suggest that sialic acids may inhibit intercellular 4B9 was added to the culture medium and to the perfusion buffer. The interactions as well. For instance, the rosette formation of T cells number of adherent cells in each condition was compared to that in the with erythrocytes can be enhanced by removing sialic acids from control to provide a percentage. Values represent mean Ϯ SD from three Ͻ ء ϭ ␤ T cells (19). The mucin-like molecule CD43 inhibits 2 integrin- to five experiments except blocking studies using 4B9 (n 2). , p dependent adhesion of T cells to ICAM-1, and this inhibitory effect 0.001 compared with control; †, p Ͻ 0.001 compared with the group with is in part attributable to sialic acids (21). Polysialic acid, a diverse Ad26st without sialidase. C; Images were taken from one representative set ϩ polymer of sialic acids attached to neural cell adhesion molecules of experiments, which correspond to the conditions described in B: IL-1 IL-4 costimulation alone (N), overexpression of ST6N and IL-1 ϩ IL-4 (NCAM), negatively regulates NCAM-dependent cell adhesion costimulation (ST), sialidase treatment following overexpression of ST6N (20). and IL-1 ϩ IL-4 costimulation (SA), and blocking of VCAM-1 and siali- In eukaryotic cells, sialic acids are attached to terminal galactose dase treatment following overexpression and IL-1 ϩ IL-4 costimulation or N-acetylgalactosamine with ␣2,3- or ␣2,6-linkages (55). Trans- (SB). The white round cells are Ramos cells attached to HUVEC, which fer of sialic acids and formation of these linkages are catalyzed by are seen as dark cells in the background; the white blurs are the paths of a family of sialyltransferases (55). Sialyltransferases transfer flowing Ramos cells. CMP-sialic acids to acceptor substrates, namely galactose and N-acetylgalactosamine, on carbohydrate chains that are attached to 2874 SIALIC ACID INHIBITS VCAM-1-MEDIATED ADHESION

FIGURE 7. ␣2,6-linked sialic acids on VCAM-1 following the overexpression of ST6N. A; After HUVEC were grown to conﬂuence, the cells Downloaded from were costimulated with 10 U/ml IL-1 ϩ 10 ng/ml IL-4 for 24 h. Then, VCAM-1 was immunoprecipitated with anti-VCAM-1 mAb 4B9 from detergent lysates, and ␣2,6- and ␣2,3-linked sialic acids on VCAM-1 were detected by SNA and MAA lectin respectively as described in Materials and Methods. VCAM-1 was also detected by a polyclonal anti-VCAM-1 Ab. B; After HUVEC were grown to 80–90% conﬂuence, they were in- FIGURE 8. Effects of the overexpression of ST6N on SNA binding to proteins expressed by HUVEC. After HUVEC were grown to 80–90%

cubated with or without 100 MOI of Ad26st for 17 h. Then, 10 U/ml IL-1 http://www.jimmunol.org/ or 10 U/ml IL-1 ϩ 10 ng/ml IL-4 were added to the culture medium and confluence, they were incubated with or without 100 MOI of Ad26st at ϩ the cells were incubated for another 24 h. The amount of ␣2,6-linked sialic 37°C with 5% CO2 for 17 h. Then, 10 U/ml IL-1 or 10 U/ml IL-1 10 acids on VCAM-1 was not increased after incubation with Ad26st. ng/ml IL-4 were added to the culture medium and the cells were incubated at 37°C with 5% CO2 for another 24 h. The cells were lysed, and SNA lectin blotting, which detects ␣2,6-linked sialic acids, showed increased binding of SNA to several high-molecular weight proteins. glycoproteins and glycolipids (55). So far, a number of sialyltransferases have been identified, which differ in specificities for acceptor substrates and linkages they create (55). In N-linked acids from IL-1 ϩ IL-4-costimulated HUVEC by sialidase in- glycoproteins, sialic acids are attached to Gal␤1-4GlcNAc or creased VCAM-1-dependent adhesion under flow conditions, by guest on September 24, 2021 Gal␤1-3GlcNAc with ␣2,6 and ␣2,3 linkages (55). In humans, whereas the same treatment of IL-1-stimulated HUVEC did not. ST6N (31, 45, 46), ST3N (25), and ST3ON (27) have been iden- Although the level of ST6N in costimulated HUVEC was ex- tified. ST6N creates ␣2,6 linkages to Gal␤1-4GlcNAc, whereas tremely high compared with IL-1 stimulation alone, the levels of ST3N and ST3ON create ␣2,3 linkages to Gal␤1-4GlcNAc in N- total ␣2,6-linked sialic acids on the cell surfaces were similar. linked glycoproteins. ST3N also uses Gal␤1-3GlcNAc as an ac- Therefore, the inhibitory effect of sialic acids on VCAM-1-depen- ceptor substrate. In O-linked glycoproteins, sialic acids are at- dent adhesion under flow conditions does not appear to be related tached to Gal␤1-3GalNAc and GalNAc. In humans, ST3O (26) solely to the total amount of sialic acids on the cell surfaces. In and ST3ON (27) have been identified. Both ST3O and ST3ON contrast with sialidase from A. ureafaciens, sialidase L cleaves catalyze the formation of ␣2,3 linkages to Gal␤1-3GalNAc. Al- ␣2,3-linked sialic acid without changing the level of ␣2,6-linked though GalNAc ␣2,6-sialyltransferase (ST6O), which creates ␣2,6 sialic acid, and removing ␣2,3-linked sialic acid alone from IL- linkages to GalNAc, has been cloned in rats (56), to our knowledge 1 ϩ IL-4-costimulated HUVEC by using this enzyme did not in- the human counterpart of this enzyme has not yet been identified. crease VCAM-1-dependent adhesion. Therefore, ␣2,6-linked sialic Hanasaki et al. (22, 23) reported that the activity and mRNA acid is required for the inhibition of VCAM-1-dependent adhesion levels of ST6N, which creates ␣2,6 linkages of sialic acids to N- by IL-1 ϩ IL-4 costimulation. To examine further whether in- linked and possibly O-linked glycoproteins, are up-regulated in creased expression of ST6N might be mechanistically related to HUVEC by IL-1, TNF, and IL-4. We found that either IL-1 or IL-4 the inhibition of VCAM-1-dependent adhesion, we overexpressed up-regulates ST6N mRNA in HUVEC, and the combination of the ST6N in HUVEC. We chose adenovirus-mediated gene transfer two cytokines further enhances the expression of ST6N. Western for the following reasons: 1) because HUVEC are primary cultured blot analysis of whole cell lysates using MAA lectin (data not cells, stably transfected HUVEC cannot be established unless cells shown) and the binding of MAA lectin (see Fig. 2B) to the surfaces are transformed to become immortal; and 2) transient gene transfer suggested that there are glycoproteins in HUVEC that are deco- by conventional methods is not efficient enough to determine the rated with ␣2,3-linked sialic acids. Therefore, we sought to iden- effect in a flow adhesion assay. Overexpression of ST6N inhibited tify the enzyme(s) responsible for the formation of ␣2,3 linkages Ramos cell adhesion only when ST6N was overexpressed in to glycoproteins in HUVEC. The only transcript identified by PCR HUVEC costimulated with IL-1 ϩ IL-4. These results suggest that was ST3ON, which uses both N- and O-linked glycoproteins as to exhibit the inhibitory effect, ␣2,6-linked sialic acids must be acceptor substrates (27). In contrast to ST6N, ST3ON mRNA was associated with a molecule(s) that is inducible by IL-1 ϩ IL-4 but constitutively expressed in HUVEC and was not regulated by the not IL-1 alone. cytokines we tested. VCAM-1 has seven potential N-linked glycosylation sites (57), Sialidase isolated from A. ureafaciens cleaves all three linkages and ϳ30% of the molecular weight of VCAM-1 expressed in of sialic acids from carbohydrate chains (32). Removal of sialic HUVEC is attributed to N-linked carbohydrates (49). Our previous The Journal of Immunology 2875 report demonstrated that VCAM-1 immunoprecipitated from adhesion molecules in cultured human and rabbit arterial endothelial cells. HUVEC after stimulation with IL-1, IL-4, or IL-1 ϩ IL-4 migrated J. Clin. Invest. 90:1138. 4. Khan, B. V., S. S. Parthasarathy, R. W. Alexander, and R. M. Medford. 1995. similarly in SDS-PAGE (8). This result suggests that there is no Modified low density lipoprotein and its constituents augment cytokine-activated marked difference in glycosylation among these VCAM-1 mole- vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J. Clin. Invest. 95:1262. cules, although the possibility of minor modification of carbohy- 5. Schmidt, A. M., O. Hori, J. X. Chen, J. F. Li, J. Crandall, J. Zhang, R. Cao, drate chains could not be excluded. Because ST6N mRNA in IL- S. D. Yan, J. Brett, and D. Stern. 1995. Advanced glycation endproducts inter- 1 ϩ IL-4-costimulated HUVEC was highly up-regulated and acting with their endothelial receptor induce expression of vascular cell adhesion ␣ molecule-1 (VCAM-1) in cultured human endothelial cells and in mice: a po- VCAM-1 molecules were decorated with 2,6-linked sialic acids, tential mechanism for the accelerated vasculopathy of diabetes. J. Clin. Invest. we examined the hypothesis that costimulation of HUVEC with 96:1395. IL-1 ϩ IL-4 increases ␣2,6-linked sialic acids on VCAM-1, which 6. Jones, D. A., L. V. McIntire, C. W. Smith, and L. J. Picker. 1994. A two-step ␣ adhesion cascade for T cell/endothelial cell interactions under flow conditions. hampers the ability of VCAM-1 to interact with 4 integrins under J. Clin. Invest. 94:2443. flow conditions. This hypothesis was not supported by the exper- 7. Alon, R., P. D. Kassner, M. W. Carr, E. B. Finger, M. E. Hemler, and ϩ T. A. Springer. 1995. The integrin VLA-4 supports tethering and rolling in flow imental results, because overexpression of ST6N in IL-1 IL-4- on VCAM-1. J. Cell Biol. 128:1243. costimulated HUVEC 1) did not increase ␣2,6-linked sialic acids 8. Abe, Y., C. M. Ballantyne, and C. W. Smith. 1996. Functions of domain 1 and ␣ on VCAM-1; 2) did reduce adhesion of Ramos cells to HUVEC; 4 of vascular cell adhesion molecule-1 in 4 integrin-dependent adhesion under ␣ static and flow conditions are differentially regulated. J. Immunol. 157:5061. and 3) led to increased 2,6-linked sialic acids on other proteins. 9. Kukreti, S., K. Konstantopoulos, C. W. Smith, and L. V. McIntire. 1997. Mo- These results suggest that the inhibitory effect of ␣2,6-linked sialic lecular mechanisms of monocyte adhesion to interleukin-1␤-stimulated endothe- acid may be due to the expression of ␣2,6-linked sialic acids on lial cells under physiologic flow conditions. Blood 89:4104. 10. Cybulsky, M. I., J. W. Fries, A. J. Williams, P. Sultan, V. M. Davis, some other molecule(s) that remains to be identified. M. A. Gimbrone, Jr., and T. Collins. 1991. Alternative splicing of human Downloaded from There are reports that certain cell surface molecules are able to VCAM-1 in activated vascular endothelium. Am. J. Pathol. 138:815. inhibit cell adhesion; the molecules that have so far been identified 11. Vonderheide, R. H., and T. A. Springer. 1992. Lymphocyte adhesion through very late antigen 4: evidence for a novel binding site in the alternatively spliced are mucins such as leukosialin (CD43) on T cells (21), episialin ␣ domain of vascular cell adhesion molecule 1 and an additional 4 integrin and epiglycanin on epithelial cells (58, 59), and polysialic acids, counter-receptor on stimulated endothelium. J. Exp. Med. 175:1433. which are attached to NCAM on neuronal cells (20). These mol- 12. Osborn, L., C. Vassallo, B. G. Browning, R. Tizard, D. O. Haskard, C. D. Benjamin, I. Dougas, and T. Kirchhausen. 1994. Arrangement of domains, ecules have two features in common: 1) they are all very large, and and amino acid residues required for binding of vascular cell adhesion molecule-1 http://www.jimmunol.org/ ␣ ␤ 2) they have a high sialic acid content. One of the proposed mech- to its counter-receptor VLA-4 ( 4 1). J. Cell Biol. 124:601. 13. Paul, W. E., and R. A. Seder. 1994. Lymphocyte responses and cytokines. Cell anisms for sialic acid inhibition of cell adhesion is the negative 76:241. charge, which generates repulsive forces (18). However, a negative 14. Thornhill, M. H., and D. O. Haskard. 1990. IL-4 regulates endothelial cell acti- charge per se does not explain the anti-adhesive effect in our ex- vation by IL-1, tumor necrosis factor, or IFN-␥. J. Immunol. 145:865. 15. Masinovsky, B., D. Urdal, and W. M. Gallatin. 1990. IL-4 acts synergistically periments, because neither removing sialic acids from IL-1-stim- with IL-1␤ to promote lymphocyte adhesion to microvascular endothelium by ulated HUVEC nor overexpressing them on IL-1-stimulated induction of vascular cell adhesion molecule-1. J. Immunol. 145:2886. HUVEC had a significant effect on adhesion. In addition, most 16. Thornhill, M. H., S. M. Wellicome, D. L. Mahiouz, J. S. Lanchbury, U. Kyan-Aung, and D. O. Haskard. 1991. Tumor necrosis factor combines with negative charges on endothelial cells are attributable to glycosami- IL-4 or IFN-␥ to selectively enhance endothelial cell adhesiveness for T cells: the noglycans rather than sialic acids (60). Because it is reported that contribution of vascular cell adhesion molecule-1-dependent and -independent by guest on September 24, 2021 the anti-adhesive effect of the mucins can be abrogated by capping binding mechanisms. J. Immunol. 146:592. 17. Schauer, R., K. So¨rge, G. Reuter, P. Roggentin, and L. Shaw. 1995. Biochemistry these molecules with Abs (58, 59), spatial relationships between and role of sialic acids. In Biology of the Sialic Acids. A. Rosenberg, ed. Plenum adhesion and anti-adhesion molecules appear to be critical in their Press, New York, p. 7. 18. Varki, A. 1997. Sialic acids as ligands in recognition phenomena. FASEB J. anti-adhesive effects. 11:248. In summary, ␣2,6-linked sialic acids play an important role in 19. Plunkett, M. L., M. E. Sanders, P. Selvaraj, M. L. Dustin, and T. A. Springer. the anti-adhesive mechanism by which costimulation of HUVEC 1987. Rosetting of activated human T lymphocytes with autologous erythrocytes. ϩ Definition of the receptor and ligand molecules as CD2 and lymphocyte function- with IL-1 IL-4 negatively regulates leukocyte adhesion under associated antigen 3 (LFA-3). J. Exp. Med. 165:664. flow conditions. We propose a novel inducible mechanism by 20. Acheson, A., J. L. Sunshine, and U. Rutishauser. 1991. NCAM polysialic acid which ␣2,6-linked sialic acids associated with an endothelial cell can regulate both cell-cell and cell-substrate interactions. J. Cell Biol. 114:143. 21. Ardman, B., M. A. Sikorski, and D. E. Staunton. 1992. CD43 interferes with molecule(s) negatively regulates leukocyte adhesion under flow T-lymphocyte adhesion. Proc. Natl. Acad. Sci. USA 89:5001. conditions. 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