Activated T Cells CD43 Functions As a Ligand for E-Selectin On

CD43 Functions as a Ligand for E-Selectin on Activated T Cells Masanori Matsumoto, Kazuyuki Atarashi, Eiji Umemoto, Yuko Furukawa, Akiko Shigeta, Masayuki Miyasaka and This information is current as Takako Hirata of September 24, 2021. J Immunol 2005; 175:8042-8050; ; doi: 10.4049/jimmunol.175.12.8042 http://www.jimmunol.org/content/175/12/8042 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD43 Functions as a Ligand for E-Selectin on Activated T Cells1

Masanori Matsumoto,*† Kazuyuki Atarashi,* Eiji Umemoto,* Yuko Furukawa,*† Akiko Shigeta,† Masayuki Miyasaka,* and Takako Hirata2†

E-selectin, an inducible cell adhesion molecule expressed on endothelial cells, mediates the rolling on endothelium of leukocytes expressing E-selectin ligands, such as neutrophils and activated T cells. Although previous studies using mice lacking P-selectin glycoprotein ligand-1 (PSGL-1) have indicated that PSGL-1 on Th1 cells functions as an E-selectin ligand, the molecular nature of E-selectin ligands other than PSGL-1 remains unknown. In this study, we show that a 130-kDa glycoprotein was precipitated by an E-selectin-IgG chimera from mouse Th1 cells. This protein was cleaved by O-sialoglycoprotein endopeptidase and required sialic acid for E-selectin binding. The mAb 1B11, which recognizes the 130-kDa glycoform of CD43, recognized the 130-kDa band in the E-selectin-IgG precipitate. In addition, immunoprecipitation of the E-selectin-IgG precipitate with 1B11 depleted the Downloaded from 130-kDa protein, further confirming its identity as CD43. CD43 was also precipitated with E-selectin-IgG from cultured human T cells. E-selectin-dependent cell rolling on CD43 was observed under flow conditions using a CD43-IgG chimera generated in Chinese hamster ovary cells expressing ␣-1,3-fucosyltransferase VII and a core 2 ␤-1,6-N-acetylglucosaminyltransferase. These results suggest that CD43, when modified by a specific set of glycosyltranferases, can function as an E-selectin ligand and therefore potentially mediate activated T cell migration into inflamed sites. The Journal of Immunology, 2005, 175: 8042–8050. http://www.jimmunol.org/ eukocytes migrate from the blood into tissues through a nal allografts undergoing rejection (3). Although E-selectin is not multistep process regulated by a cascade of molecular in- normally detected in resting endothelial cells, it is constitutively L teractions between leukocytes and endothelial cells. This expressed in a subset of dermal vessels in noninflamed skin, sug- process is initiated by rapid and transient interactions that capture gesting that E-selectin is also involved in the cutaneous immuno- leukocytes from the bloodstream and allow them to roll on the surveillance system in the absence of pathologic inflammation (4). surface of endothelial cells under blood flow. These interactions E-selectin recognizes sialylated and fucosylated carbohydrate are primarily mediated by selectins, a family consisting of three structures such as the tetrasaccharide sialyl LewisX (sLeX)3. Gen- members, L-selectin (CD62L), E-selectin (CD62E), and P-selectin eration of the selectin-binding carbohydrates depends on the (CD62P). L-selectin is expressed on most leukocytes and mediates activity of certain glycosyltransferases, including ␣-1,3-fucosyl- by guest on September 24, 2021 the recruitment of lymphocytes into secondary lymphoid organs transferase VII (FucT-VII) and core 2 ␤-1,6-N-acetylglucosami- via interactions with L-selectin ligands on high endothelial nyltransferase (C2GnT). The presentation of the carbohydrate on venules, whereas E- and P-selectin are expressed on activated en- the protein backbone contributes to the specificity and affinity of dothelial cells and mediate the recruitment of neutrophils, mono- the selectin binding. To date, several glycoprotein ligands for E- cytes, and certain subsets of lymphocytes into sites of inflamma- selectin have been reported. These are P-selectin glycoprotein li- tion by interacting with specific ligands on the surface of these gand-1 (PSGL-1; CD162), E-selectin ligand-1 (ESL-1), L-selectin, leukocytes (1, 2). and CD44. PSGL-1 is a sialomucin that was identified as the major E-selectin expression is induced by a variety of inflammatory ligand for P-selectin on myeloid cells and subsets of lymphoid mediators, including IL-1, TNF-␣, IFN-␥, and LPS, in vitro and in cells (5). PSGL-1 can also serve as an E-selectin ligand on T cells most tissues. E-selectin expression is also associated in vivo with and neutrophils in vivo (6, 7). ESL-1 is a transmembrane glyco- a number of disease states. Notably, E-selectin is expressed in protein that was identified using a recombinant E-selectin-IgG chi- inflammatory skin lesions in psoriasis, contact dermatitis, and de- mera as the major E-selectin ligand on mouse neutrophils (8). The layed-type hypersensitivity, in arthritic joints, and in heart and re- specific glycoform expressed on myeloid cells carries sLeX on N- linked glycans and binds E-selectin. ESL-1, however, has never been demonstrated to mediate E-selectin-dependent rolling under *Laboratory of Immunodynamics, Department of Microbiology and Immunology, Osaka University Graduate School of Medicine, and †The 21st Century Center of in vivo conditions. L-selectin from human neutrophils and cultured Excellence Program, Research Institute for Microbial Diseases, Osaka University, T lymphoblasts, but not from mouse cells, is capable of binding Suita, Osaka, Japan E-selectin (9–12). CD44 is a hyaluronan-binding cell surface gly- Received for publication July 27, 2005. Accepted for publication October 6, 2005. coprotein that has recently been shown to mediate E-selectin-de- The costs of publication of this article were defrayed in part by the payment of page pendent rolling of neutrophils (13). In addition, several glycopro- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. teins from tumor cells have been shown to bind E-selectin (14). 1 This work was supported by a grant-in-aid from the 21st Century Center of Excel- lence Program of the Ministry of Education, Culture, Sports, Science and Technology, Japan, and a grant-in-aid for Scientific Research from the Japan Society for the Pro- motion of Science, Japan. 3 Abbreviations used in this paper: sLeX, sialyl LewisX; FucT-VII, ␣-1,3-fucosyl- 2 Address correspondence and reprint requests to Dr. Takako Hirata, The 21st transferase VII, C2GnT, core 2 ␤-1,6-N-acetylglucosaminyltransferase; PSGL-1, P- Century Center of Excellence Program, Research Institute for Microbial Diseases, selectin glycoprotein ligand-1; ESL-1, E-selectin ligand-1; CHO, Chinese hamster Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail ad- ovary; PNGase F, peptide:N-glycosidase F; OSGE, O-sialoglycoprotein dress: [email protected] endopeptidase.

However, whether these glycoprotein ligands support physiologi- leader-IgG1 vector, provided by Dr. B. Seed (Massachusetts General Hos- cally relevant interactions with E-selectin is not yet certain. pital, Boston, MA), at the NheI and BamHI sites. To generate the human Studies using Ab blockade as well as gene targeting have dem- CD43-IgG construct, oligonucleotides that carry ApaI and EcoRV sites were first inserted into the CD5 leader-IgG1 vector, and then a fragment onstrated that E-selectin, together with P-selectin, plays a critical corresponding to the extracellular domain of human CD43 was ligated at role in the migration of neutrophils as well as certain subsets of T the ApaI and EcoRV sites. The mouse and human PSGL-1-IgG constructs cells into sites of inflammation (15–17). In a model of contact containing the entire extracellular domain were generated as previously hypersensitivity, E- and P-selectin cooperatively mediate Th1 cell described (26). The constructs were introduced by transient transfection into Chinese hamster ovary (CHO) cells stably expressing both human migration into the skin during the efferent phase (18). Using Ϫ/Ϫ FucT-VII and C2GnT (CD7II cells) (27), provided by Dr. R. McEver PSGL-1-deficient (PSGL-1 ) mice, we have shown that (Oklahoma Medical Research Foundation, Oklahoma City, OK), with PSGL-1 functions as the predominant P-selectin ligand as well as ESCORT V (Sigma-Aldrich). The chimeric proteins were purified from one of the E-selectin ligands on Th1 cells in this model (6). The culture supernatants using protein A-Sepharose as previously described identity of physiological E-selectin ligands other than PSGL-1 on (26). The expression plasmids for mouse E- and P-selectin-IgM chimeric proteins were provided by Dr. J. Lowe (University of Michigan Medical Th1 cells remains unknown. School, Ann Arbor, MI). COS-7 cells were transfected with the plasmids CD43 is a cell-surface sialoglycoprotein expressed by most he- using DEAE-dextran. Mouse E- and P-selectin-IgG chimeric proteins were mopoietic cells, including T cells (19). The extracellular domain of prepared as described previously (6). Ͼ both human and mouse CD43 contains 80 serine or threonine Preparation of mouse Th1 cells residues, most of which are glycosylated by O-linked glycans that ϩ are heavily sialylated, and has a rod-like structure that is predicted Splenic CD4 T cells were isolated by autoMACS (Miltenyi Biotec) using biotinylated anti-CD11b (M1/70; BD Biosciences), biotinylated anti-B220 to extend 45 nm from the lipid bilayer (20). Its extended structure Downloaded from (RA3-6B2; BD Biosciences), and biotinylated anti-CD8␣ (53-6.7; South- and negatively charged sialic acid residues may confer an antiad- ern Biotechnology Associates), followed by Streptavidin MicroBeads hesive function to this molecule. Indeed, CD43-deficient (Miltenyi Biotec). Alternatively, they were isolated with a BD IMag CD4 (CD43Ϫ/Ϫ) T cells show a marked increase in their in vitro pro- T Lymphocyte Enrichment set (BD Biosciences). The enriched population Ͼ ϩ liferative response and homotypic adhesion (21) and increased was 90% positive for CD4 staining. Purified CD4 T cells were cultured on 6-cm tissue-culture dishes coated with 10 ␮g/ml anti-CD3⑀ (145-2C11; tethering to L-selectin ligands in vivo and in vitro (22), indicating BD Biosciences) and 10 ␮g/ml anti-CD28 (37.51; BD Biosciences) for 2 that CD43 negatively regulates T cell activation and adhesion. days in the presence of 4 ng/ml IL-2 (R&D Systems), 8 ng/ml IL-12 (R&D http://www.jimmunol.org/ CD43Ϫ/Ϫ neutrophils also show enhanced rolling and adhesion Systems), and 0.2 ␮g/ml anti-IL-4 (11B11; BD Biosciences). The cells both in vivo and in vitro (23). Paradoxically, however, CD43 de- were then transferred to uncoated dishes and cultured for an additional ficiency reduces the recruitment of leukocytes into the peritoneal 4 days. cavity (23), and the administration of an anti-CD43 mAb inhibits Preparation of human T lymphoblasts T cell migration into secondary lymphoid organs (24), implicating Human blood from healthy adult donors was collected into tubes contain- CD43 in a proadhesive process. How CD43 mediates apparently ing EDTA, and the mononuclear fraction was isolated following centrifu- opposing functions remains unclear. gation through Ficoll-Paque PLUS (Amersham Biosciences). Mononuclear Two major glycoforms of human CD43 of 115 and 135 kDa cells were cultured in 24-well plates coated with 10 ␮g/ml anti-CD3 have been identified (19). Similarly, two major glycoforms of 115 (UCHT1; eBioscience) for 2 days in the presence of 5 ng/ml IL-2 (R&D by guest on September 24, 2021 Systems) in X-VIVO 15 medium (BioWhittaker). The cells were then and 130 kDa have been identified for mouse CD43 (25). The 115- transferred to uncoated dishes and expanded in X-VIVO 15 medium con- kDa glycoform is expressed on all T cells, whereas the larger gly- taining 5 ng/ml IL-2 for an additional 6 days. Human blood samples were coform is expressed predominantly on activated T cells. The O- obtained in accordance with protocols approved by the Ethics Review glycans attached to the larger glycoform consist of a branched Committee for Human Studies of the Research Institute for Microbial Dis- hexasaccharide core generated by C2GnT, which is up-regulated eases, Osaka University (Osaka, Japan). during T cell activation. In contrast, the O-glycans attached to the Flow cytometry 115-kDa glycoform consist of a tetrasaccharide core. All mAbs used for the flow cytometric analyses were purchased from BD In this study, we report that the 130-kDa glycoform of CD43 Biosciences. They included anti-CD4-FITC (RM4-5) and two anti-CD43 was precipitated from mouse Th1 cells using an E-selectin-IgG mAbs, 1B11-PE and S7-FITC. Cells were stained with mAbs for 30 min on chimera as an affinity matrix. CD43 was also precipitated with ice, washed, and analyzed on an EPICS XL flow cytometer (Beckman E-selectin-IgG from human T lymphoblasts. Furthermore, using a Coulter) or a FACSCalibur (BD Biosciences). To assess the selectin-IgM CD43-IgG chimera, we show that CD43, when appropriately mod- binding, cells were incubated with a COS-7 supernatant containing E-selectin-IgM, P-selectin-IgM, or control human IgM, washed, and then in- ified by a set of glycosyltransferases, supported E-selectin-depen- cubated with biotinylated anti-human IgM (American Qualex). The cells dent cell rolling under flow conditions. Taken together, our find- were then washed and stained with either streptavidin-PE (BD Bio- ings suggest that CD43 can function as a ligand for E-selectin on sciences) alone or streptavidin-allophycocyanin (BD Biosciences) together activated T cells and may potentially mediate activated T cell mi- with 1B11-PE and S7-FITC. gration into sites of inflammation. Precipitation with E-selectin-IgG PSGL-1Ϫ/Ϫ or wild-type Th1 cells or cultured human T cells were washed Materials and Methods three times with PBS and surface-biotinylated in PBS containing 0.5 mg/ml Mice sulfo-NHS-LC-biotin (Pierce) (2.5 ϫ 107 cells/ml) at room temperature for Ϫ Ϫ 30 min. The cells were then washed three times with PBS and lysed at a C57BL/6J mice were purchased from CLEA Japan. PSGL-1 / mice on a density of 3 ϫ 107 cells/ml in cold lysis buffer (1% Triton X-100, 50 mM C57BL/6J background were provided by Dr. B. Furie (Harvard Medical Tris (pH 7.4), 150 mM NaCl, 1 mM CaCl , and 1 mM PMSF) for 30 min. School, Boston, MA). All mice used were 6–12 wk of age. The mice were 2 Insoluble materials were pelleted at 15,000 ϫ g for 20 min. The superna- housed at the Institute of Experimental Animal Sciences at Osaka Univer- tant was aliquoted, and a fraction corresponding to 1 ϫ 107 cells was sity Medical School (Osaka, Japan). All studies and procedures were ap- incubated for 4 h with 50 ␮l of packed protein A-Sepharose. After removal proved by the Ethics Review Committee for Animal Experimentation of of the Sepharose beads, the lysate was incubated in the presence of 1 mM the Osaka University Graduate School of Medicine. ␮ CaCl2 with 20 l of protein A-Sepharose preloaded for4hat4°Cwith 50 Chimeric proteins ␮g of E-selectin-IgG or human IgG. After a 4-h incubation, the beads were washed five times with wash buffer (1% Triton X-100, 50 mM Tris (pH

To generate the mouse CD43-IgG construct, a fragment corresponding to 7.4), 150 mM NaCl, and 1 mM CaCl2). Proteins bound to E-selectin-IgG the extracellular domain was ampliﬁed by PCR and ligated into the CD5 were eluted with elution buffer (5 mM EDTA, 50 mM Tris (pH 7.4), and 8044 CD43 AS A T CELL E-SELECTIN LIGAND

0.05% Triton X-100). Eluted materials were separated by SDS-PAGE un- Results der nonreducing conditions and transferred to an Immobilon-P membrane PSGL-1-deficient Th1 cells roll on E-selectin under flow (Millipore). Membranes were blotted with HRP-conjugated streptavidin (Zymed Laboratories). The membranes were also blotted with a polyclonal conditions anti-mouse PSGL-1 Ab (28), a polyclonal anti-mouse ESL-1 provided by We showed previously that Th1 cells from wild-type mice bind to Dr. B. Furie, an anti-mouse CD43 mAb 1B11 (BD Biosciences), an anti- both P-selectin-IgG and E-selectin-IgG chimeric proteins immo- mouse CD44 mAb KM201 (29), an anti-human PSGL-1 mAb KPL-1 (BD Ϫ/Ϫ Biosciences), or an anti-human CD43 mAb L60 (BD Biosciences), fol- bilized on 96-well microtiter plates, whereas PSGL-1 Th1 cells lowed by the appropriate HRP-conjugated secondary Abs (all from Amer- do not bind to P-selectin-IgG but bind measurably to E-selectin- ican Qualex). IgG (6). Consistent with this result, flow cytometric assays using 6 To perform Western blot analysis of whole-cell extract, 1 ϫ 10 freshly Ϫ/Ϫ ϩ selectin-IgM chimeric proteins showed that PSGL-1 Th1 cells isolated CD4 T or Th1 cells were lysed in lysis buffer. The lysate was resolved by SDS-PAGE under nonreducing and reducing conditions and did not bind P-selectin-IgM but bound E-selectin-IgM, albeit at a transferred to an Immobilon-P membrane. For Western blot analysis of slightly lower level than wild-type cells (Fig. 1A). We also con- mouse CD43-IgG chimeric proteins, 50 ng of protein was separated by firmed that unstimulated CD4ϩ T cells from both wild-type and SDS-PAGE. Membranes were probed with an anti-CD43 mAb, S7 or PSGL-1Ϫ/Ϫ mice fail to bind P-selectin-IgM and E-selectin-IgM 1B11, followed by HRP-conjugated anti-rat IgG. To immunoprecipitate and immunodeplete CD43, eluted materials or total cell lysates were in- (Fig. 1A). To investigate whether Th1 cells would interact with cubated for 2 h with 10 ␮l of protein G-Sepharose preloaded with 5 ␮gof E-selectin in the absence of PSGL-1 in a more physiological set- 1B11, L60, or control rat or mouse IgG. The incubation with Ab-loaded ting, we tested Th1 cells for their ability to roll on E-selectin under beads was repeated. The supernatant and beads were analyzed by Western flow conditions. When wild-type Th1 cells were infused into cap- blotting. illary tubes coated with selectin-IgG chimeras at 1 dyn/cm2, they Downloaded from rolled on E-selectin-IgG as well as on P-selectin-IgG (Fig. 1B). Enzyme treatment The addition of EDTA completely abolished the rolling, confirm- To remove sialic acid, E-selectin-IgG precipitates or immunoprecipitates ing that this was a calcium-dependent interaction. PSGL-1Ϫ/Ϫ were incubated with 1 U/ml neuraminidase from Clostridium perfringens cells did not roll on P-selectin-IgG. In contrast, they rolled on (Sigma-Aldrich) in 50 mM sodium phosphate (pH 5.0) at 37°C for 1 h. To N N E-selectin-IgG, although the number of rolling cells was reduced remove -glycan chains, samples were treated with 250 U/ml peptide: - Ϫ/Ϫ glycosidase F (PNGase F; Calbiochem) in 50 mM sodium phosphate (pH by ϳ60%, compared with wild-type cells (Fig. 1B). PSGL-1 http://www.jimmunol.org/ 7.5) at 37°C overnight. Samples were also treated with 120 ␮g/ml O- sialoglycoprotein endopeptidase (OSGE; Cederlane Laboratories) at 37°C for 1 h. In some experiments, enzyme-treated samples were reprecipitated with E-selectin-IgG before subjected to SDS-PAGE.

Cell adhesion assays The cell adhesion assays were performed as described previously (6). In brief, CD43-IgG, PSGL-1-IgG, or control human IgG (Sigma-Aldrich) (10 ␮g/ml) was immobilized on 96-well plates (Sumilon H; Sumitomo

Bakelite) at 4°C overnight, and the plates were blocked with 1% BSA in by guest on September 24, 2021 PBS at 37°C for 2 h. CHO cells expressing mouse E-selectin (CHO-E cells), provided by Dr. B. Furie, or parental CHO cells resuspended in

HBSS containing 2 mM CaCl2 or 5 mM EDTA were added to the plate and incubated for 20 min at 4°C with rotation (100 rpm). In some experiments, the cells were preincubated with an anti-E-selectin mAb 9A9, provided by Dr. B. Wolitzky and colleagues (30), or control rat IgG (Zymed Labora- tories) before being added to the plate. After the plates were washed three times with HBSS containing 2 mM CaCl2 or 5 mM EDTA, the number of bound cells was determined by photographing the cells and counting them.

Cell adhesion assays under flow conditions Cell adhesion assays under flow conditions were performed according to the method of Nandi et al. (31) with slight modifications. E-selectin-IgG, P-selectin-IgG, CD43-IgG, PSGL-1-IgG, or control human IgG (1 ␮g/ml) was immobilized on the inside walls of glass capillaries (inner diameter, 0.69 mm; Drummond Scientific) at 4°C overnight. The capillaries were then blocked with 3% BSA for1hatroom temperature. The capillaries were mounted on the stage of an inverted microscope (Diaphot 300; Nikon) FIGURE 1. PSGL-1-deficient Th1 cells bind and roll on E-selectin un- ϫ with a 4 objective. At this magnification, all cells rolling at a fixed po- der flow conditions. A, P- and E-selectin-IgM binding of unstimulated sition of the capillaries could be monitored. Mouse Th1 cells, CHO-E cells, ϩ Ϫ Ϫ CD4 T and Th1 cells from wild-type and PSGL-1 / mice. Cells were or parental CHO cells were resuspended at 1 ϫ 106 cells/ml in HBSS incubated with P- and E-selectin-IgM (open histogram) or control human containing either CaCl2 or EDTA and infused into the capillaries at a shear force of 1 dyn/cm2. The rate of flow was controlled by a PHD 2000 syringe IgM (shaded histogram). Bound selectin IgM was detected using biotinyl- pump (Harvard Apparatus). In some experiments, the CHO-E cells were ated anti-human IgM and streptavidin-PE. B, Rolling of wild-type and Ϫ Ϫ preincubated with the anti-E-selectin mAb 9A9 or control rat IgG. Three PSGL-1 / Th1 cells on P- and E-selectin-IgG. Cells were infused into minutes after the start of infusion, cell images were recorded with a cell- capillaries coated with P-selectin-IgG, E-selectin-IgG, or human IgG in the viewing system (SRM-100; Nikon) and video recorder (JVC BR-S600; presence of calcium or EDTA at a shear stress of 1 dyn/cm2. The number Victor), and the number of rolling cells passing through a fixed plane (at of rolling cells was determined. C, Resistance of wild-type and PSGL-1Ϫ/Ϫ three-quarters of the capillary tube from the entrance) perpendicular to the Th1 cells rolling on E-selectin-IgG to detachment by shear stress. Cells capillary axis was counted. The results are expressed as the number of were infused into E-selectin-IgG-coated capillaries and allowed to accu- rolling cells per minute. The cells that rolled stably along the wall of the mulate at a shear stress of 1 dyn/cm2 for 6 min. The shear stress was then glass capillary tube for at least 3 s were considered to be rolling cells in this 2 assay. For the detachment assays, cells were infused at 1 dyn/cm2 for 6 min increased every 20 s to 128 dyn/cm . The number of cells remaining bound and the shear stress was increased stepwise every 20 s until it reached 128 at the end of each interval was determined and expressed as a percentage dyn/cm2. At the end of each shear stress treatment, the number of cells that of the cells accumulated before applying increasing shear stress. One of remained bound was determined. three similar independent experiments is shown. The Journal of Immunology 8045

Th1 cells rolling on E-selectin-IgG exhibited significant resistance carrying N-linked carbohydrates for selectin binding. To examine to detachment by shear stress, albeit reduced, compared with wild- whether the 130-kDa protein precipitated with E-selectin-IgG is a type cells, with 60% remaining bound at 32 dyn/cm2 (Fig. 1C). In member of the sialomucin family, we treated the E-selectin-IgG this experiment, all cells remaining bound rolled until they finally precipitate from PSGL-1Ϫ/Ϫ Th1 cells with OSGE. The 130-kDa detached. These data confirm that, although PSGL-1 functions as band was no longer detected after OSGE treatment (Fig. 3), sug- an E-selectin ligand, there are ligands other than PSGL-1 that can gesting that the 130-kDa protein is an OSGE-cleavable protein and mediate Th1 cell interactions with E-selectin under physiological most likely a sialomucin. The presence of sialic acid was also shear flow. shown by the shift in the apparent molecular mass after sialidase treatment (Fig. 3). Treatment with PNGase F showed a small shift Several proteins are precipitated with E-selectin-IgG from Th1 of the 130-kDa protein (Fig. 3), suggesting that this protein is also cells N-glycosylated. Only a small amount of the 130-kDa protein, To identify E-selectin ligands on Th1 cells, we used E-selectin-IgG which migrated near 150 kDa after the sialidase treatment, could bound to protein A-Sepharose as an affinity matrix. Wild-type and be reprecipitated with E-selectin-IgG (Fig. 3). In contrast, this pro- PSGL-1Ϫ/Ϫ Th1 cells were surface-biotinylated, and detergent ex- tein was reprecipitated after PNGase F treatment, suggesting that tracts of these cells were incubated with E-selectin-IgG bound to N-glycans are not required for E-selectin binding. Together, these protein A-Sepharose. The proteins that bound to E-selectin-IgG results suggest that the 130-kDa protein is likely to be a sialomucin were eluted with EDTA and subjected to Western blotting with and to require sialic acid for E-selectin binding. HRP-conjugated streptavidin. Two major bands, which migrated around 130 and 270 kDa under nonreducing conditions, were pre- The 130-kDa glycoform of CD43 is expressed on Th1 cells Downloaded from cipitated from wild-type cells with E-selectin-IgG in the presence CD43 is a major sialomucin expressed on T cells. Two major of calcium (Fig. 2A). A minor band migrating around 150 kDa was forms of mouse CD43, a 115-kDa and a 130-kDa glycoform, have also inconsistently detected. No bands were detected when deter- been identified (25). The 115-kDa glycoform, which is recognized gent extracts were incubated with control human IgG in the pres- by the anti-CD43 mAb S7, is expressed on all T cells, whereas the ence of calcium or with E-selectin-IgG in the presence of EDTA, 130-kDa glycoform, which is recognized by the mAb 1B11, is confirming the specificity of the binding. When the precipitation expressed preferentially on activated T cells (25). We examined http://www.jimmunol.org/ experiments were performed using PSGL-1Ϫ/Ϫ cells, two bands the expression of the S7 and 1B11 epitopes on unstimulated CD4ϩ around 130 and 150 kDa, but not a 270-kDa band, were detected. T cells and Th1 cells by flow cytometry. Unstimulated CD4ϩ T These results suggested that the 270-kDa band precipitated with cells from wild-type mice expressed the S7 epitope but not the E-selectin-IgG from wild-type cells represented PSGL-1. Indeed, a 1B11 epitope, whereas Th1 cells expressed both (Fig. 4A). PSGL- 270-kDa band was detected by Western blotting the E-selectin-IgG 1Ϫ/Ϫ cells showed a similar expression pattern (Fig. 4A). Western precipitate from wild-type cells using anti-PSGL-1 Abs (Fig. 2B). blot analysis also showed the expression of the 115-kDa glycoform Besides PSGL-1, ESL-1 has been identified as an E-selectin recognized by S7 in both unstimulated CD4ϩ T cells and Th1 ligand on mouse neutrophils (8). Whether mouse T cells express cells, while the 130-kDa glycoform detected by 1B11 was ex- ESL-1 has not been reported. We tested whether the 130- or 150- pressed only in Th1 cells (Fig. 4B). by guest on September 24, 2021 kDa band detected both in wild-type and PSGL-1Ϫ/Ϫ cells might The 130-kDa glycoform of CD43 was immunoprecipitated from represent ESL-1. Western blots of the E-selectin-IgG precipitate Th1 cells with 1B11 (Fig. 4C). Treatment of the immunoprecipi- probed with anti-ESL-1 Abs showed a band at 150 kDa both in tates with OSGE resulted in a complete loss of the band (Fig. 4C), wild-type and PSGL-1Ϫ/Ϫ cells (Fig. 2C), suggesting that the 150- confirming that it is an OSGE-sensitive sialomucin. Sialidase treat- kDa component or at least part of it represents ESL-1. ment of the immunoprecipitate resulted in a decrease in electro- phoretic mobility, thus confirming the presence of sialic acid. The 130-kDa protein is cleaved by OSGE and requires sialic acid to bind E-selectin Most selectin ligands identified to date are sialomucins and require O-linked carbohydrates modified with sLeX-like structures for selectin binding. Some selectin ligands such as ESL-1 require sLeX-

FIGURE 3. The 130-kDa protein is an OSGE-cleavable protein and re- FIGURE 2. Several proteins were precipitated with E-selectin-IgG from quires sialic acid to bind E-selectin. PSGL-1Ϫ/Ϫ Th1 cells were surface- mouse Th1 cells. Wild-type and PSGL-1Ϫ/Ϫ Th1 cells were surface-bioti- biotinylated and their detergent lysates were incubated with E-selectin-IgG nylated and their detergent extracts were incubated with E-selectin-IgG bound to protein A-Sepharose. Eluted materials were either untreated (E-IgG) or control human IgG (hIgG) bound to protein A-Sepharose in the (lanes 1 and 3) or treated with OSGE (lane 2), sialidase (lanes 4 and 5), or presence of calcium or EDTA. Bound proteins were eluted with EDTA, PNGase F (lanes 6 and 7). Half of each sample was then reprecipitated with separated by SDS-PAGE under nonreducing conditions, and subjected to E-selectin-IgG (lanes 3, 5, and 7). Samples were separated by SDS-PAGE Western blotting with HRP-conjugated streptavidin (SA-HRP) (A), anti- under nonreducing conditions and subjected to Western blotting with HRP- PSGL-1 Ab (B), or anti-ESL-1 Ab (C). conjugated streptavidin. 8046 CD43 AS A T CELL E-SELECTIN LIGAND Downloaded from http://www.jimmunol.org/

FIGURE 5. The 130-kDa protein contains CD43. A, Western blot of the E-selectin-IgG precipitate with HRP-conjugated streptavidin (SA-HRP) and the anti-CD43 mAb 1B11. PSGL-1Ϫ/Ϫ Th1 cells were surface-bioti- FIGURE 4. The 130-kDa glycoform of CD43 is expressed on mouse nylated, lysed, and incubated with E-selectin-IgG bound to protein ϩ Th1 cells. A, Expression of S7 and 1B11 epitopes on unstimulated CD4 A-Sepharose. Bound proteins were eluted with EDTA and subjected to by guest on September 24, 2021 T and Th1 cells. Cells were stained with the indicated mAbs (open histo- Western blotting with HRP-conjugated streptavidin or the anti-CD43 mAb gram) or isotype controls (shaded histogram), and analyzed by flow cy- 1B11. B, Depletion of the 130-kDa protein from the E-selectin-IgG pre- tometry. B, Western blotting of lysates from CD4ϩ T and Th1 cells. Total cipitate by 1B11. The E-selectin-IgG precipitate was immunoprecipitated cell lysates of freshly isolated CD4ϩ T and Th1 cells were separated by with either 1B11 or control rat IgG. The unbound supernatants (sup) and SDS-PAGE under nonreducing conditions and blotted using S7 and 1B11. the first (pel1) and second (pel2) precipitates were analyzed by Western C, Digestion of immunoprecipitated CD43 with OSGE, sialidase, and PN- blotting using HRP-conjugated streptavidin. C, Expression of the S7 and Gase F. CD43 was immunoprecipitated from surface-biotinylated PSGL- 1B11 epitopes on Th1 cells and E-selectin-binding activities. Cells were 1Ϫ/Ϫ Th1 cells using 1B11. The immunoprecipitate was treated with incubated with E-selectin-IgM or control human IgM. The cells were then OSGE, sialidase, or PNGase F separated by SDS-PAGE under nonreduc- incubated with biotinylated anti-human IgM followed by streptavidin-al- ing and reducing conditions, and subjected to Western blotting with HRP- lophycocyanin, 1B11-PE, and S7-FITC, and analyzed by flow cytometry. conjugated streptavidin. The percentage of cells in each region is indicated for each quadrant on the right of the dot plot. E-selectin-IgM binding of 1B11high and 1B11low pop- ulations is also shown, and the percentage of cells with high E-selectin- binding activities is indicated in each histogram. Treatment with PNGase F also resulted in a shift, indicating the presence of N-linked carbohydrates. This pattern of shift in elec- trophoretic mobility after enzyme treatment resembled that ob- cells was immunoprecipitated with 1B11, the 130-kDa band was served with the 130-kDa protein precipitated with E-selectin-IgG, mostly depleted from the unbound supernatant, but it was present raising the possibility that the 130-kDa component of the E-selec- in the immunoprecipitate (Fig. 5B). In contrast, the 130-kDa band tin-IgG precipitate may represent CD43. was not detected when control rat IgG was used for the immunoprecipitation; it remained in the supernatant (Fig. 5B). The specific The 130-kDa component of the E-selectin-IgG precipitate depletion of the 130-kDa band by 1B11 strongly indicates that represents CD43 CD43 made up most or all of the 130-kDa band. To examine whether CD43 was represented by the 130-kDa com- The 130-kDa glycoform of CD43 that the mAb 1B11 recognizes ponent of the E-selectin-IgG precipitate, we subjected the E-selec- carries core 2-branched O-glycans (25). Because most selectin li- tin-IgG precipitate from PSGL-1Ϫ/Ϫ Th1 cells to Western blotting gands carry core 2 O-glycans containing the sLeX moiety, we per- with the anti-CD43 mAb 1B11. The 1B11 mAb specifically rec- formed flow cytometric analyses to determine whether the expres- ognized the 130-kDa band in the E-selectin-IgG precipitate, sug- sion of the 1B11 epitope was correlated with that of E-selectin gesting that the 130-kDa band contained CD43 (Fig. 5A). The ligands. Most Th1 cells expressed the 1B11 epitope, and the cells identity of the 130-kDa component was further confirmed by im- with high E-selectin-binding activities were enriched in the munoprecipitation and immunodepletion assays with the 1B11 1B11high population, compared with the 1B11low population, but mAb. When the E-selectin-IgG precipitate from PSGL-1Ϫ/Ϫ Th1 no correlation was observed between the expression of the S7 The Journal of Immunology 8047 epitope and E-selectin-binding activity (Fig. 5C). However, not all human CD43-IgG chimeric proteins in CHO-derived CD7II cells 1B11high cells had E-selectin-binding activity, suggesting that only stably expressing both FucT-VII and C2GnT (27), which together part of the 130-kDa glycoform of CD43 is a selectin-binding form. generate core 2 O-glycans with sLeX-like structures on the chimeric proteins. The mouse and human PSGL-1-IgG chimeric pro- CD43 is an E-selectin ligand on human T lymphoblasts teins were generated similarly. The proteins were also made in To investigate whether human T cell CD43 would function as an CHO cells that did not express FucT-VII or C2GnT, and used as E-selectin ligand, we prepared T lymphoblasts from PBMCs of controls. The mouse CD43-IgG generated in CD7II cells expressed healthy donors by stimulating them with anti-CD3 and culturing the 1B11 epitope but not the S7 epitope, whereas the control them in the presence of IL-2 in X-VIVO 15 medium. As shown CD43-IgG expressed the S7 epitope but not the 1B11 epitope (Fig. previously (32), these cells had high E-selectin-binding activity 7A). As shown in Fig. 7B, although the parental CHO cells did not (data not shown). The cells were then surface-biotinylated, and bind to any of the chimeric proteins, the CHO-E cells bound to the their detergent extracts were precipitated with E-selectin-IgG. PSGL-1-IgG and CD43-IgG of both mouse and human origin gen- Three major bands, migrating at around 130, 180, and 270 kDa erated in the CD7II cells, but not to the control PSGL-1-IgG and under nonreducing conditions, were detected (Fig. 6A). A 270-kDa CD43-IgG. The binding was almost completely abrogated by the band was detected by Western blotting the E-selectin-IgG precip- addition of EDTA (data not shown), confirming that it was cal- itate with anti-PSGL-1 Abs (Fig. 6B), suggesting that PSGL-1 was cium-dependent. The binding of CHO-E cells to the CD7II- at least one component of the 270-kDa band of the E-selectin-IgG generated CD43-IgG and PSGL-1-IgG was also inhibited by precipitate. To determine whether CD43 contributed to the 130- pretreating the cells with the anti-E-selectin mAb 9A9, but not kDa component of the E-selectin-IgG precipitate from cultured with control rat IgG, verifying that the binding was dependent Downloaded from human T cells, we subjected the E-selectin-IgG precipitate to on E-selectin (Fig. 7C). Western blotting with the anti-human CD43 mAb L60, which spe- We next investigated whether CD43 could mediate E-selectin- cifically recognized the 130-kDa component (Fig. 6C). In addition, dependent cell rolling under flow conditions. When CHO-E cells L60 but not control mouse IgG, largely depleted the 130-kDa band were infused into capillary tubes coated with the chimeric proteins from the E-selectin-IgG precipitate (Fig. 6D), indicating that on under a shear stress of 1 dyn/cm2, the CHO-E cells rolled on the cultured human T cells too, CD43 is an E-selectin ligand. CD43-IgG and the PSGL-1-IgG generated in CD7II cells, but not http://www.jimmunol.org/ on the control chimeric proteins (Fig. 7D). The rolling of CHO-E CD43 mediates E-selectin-dependent cell rolling under flow cells was completely inhibited by pretreating the cells with the conditions anti-E-selectin blocking mAb 9A9 but not with control rat IgG, We next investigated whether CD43 could mediate E-selectin-de- verifying that the rolling was dependent on E-selectin (Fig. 7E). pendent cell adhesion. To this end, we first prepared mouse and Taken together, these data suggest that CD43, when appropriately modified by a set of glycosyltransferases, functions as an E-selectin ligand under flow conditions.

Discussion by guest on September 24, 2021 In this study, we identified a 130-kDa glycoprotein ligand for E- selectin on mouse Th1 cells using an E-selectin-IgG chimera as an affinity probe, and identified this glycoprotein as the 130-kDa glycoform of CD43. Human CD43 also represented the 130-kDa glycoprotein precipitated with an E-selectin-IgG chimera from activated human T cells. Furthermore, using CD43-IgG chimeric proteins generated in CHO cells expressing FucT-VII and C2GnT, we showed that CD43 could support E-selectin-dependent cell rolling under flow, indicating that CD43 can serve as an E-selectin ligand on activated T cells and potentially mediate the migration of activated T cells to sites of inflammation. The identity and physiological contributions of E-selectin ligands are not well established. We showed previously that the PSGL-1 expressed on mouse Th1 cells functions as an E-selectin ligand (6). Our study shows that PSGL-1 is indeed precipitated from mouse Th1 cells with an E-selectin-IgG chimera, confirming biochemically that PSGL-1 is an E-selectin ligand. We have also reported that ESL-1, which was identified as an E-selectin ligand FIGURE 6. CD43 is an E-selectin ligand expressed on cultured human on mouse neutrophils (8), is also precipitated with E-selectin-IgG T cells. A–C, Western blots of the E-selectin-IgG precipitate from cultured from mouse Th1 cells, suggesting that ESL-1 can serve as an E- human T cells. The cells were surface-biotinylated and their detergent ex- selectin ligand on T cells as well. In addition to these two ligands, tracts were incubated with E-selectin-IgG (E-IgG) or control human IgG we report that a 130-kDa glycoprotein is another E-selectin ligand (hIgG) bound to protein A-Sepharose in the presence of calcium or EDTA. expressed on mouse Th1 cells, and we identified it as CD43. We Bound proteins were eluted with EDTA and subjected to Western blotting also showed that CD43 was the 130-kDa protein species precipi- with HRP-conjugated streptavidin (SA-HRP) (A), the anti-human PSGL-1 tated with E-selectin-IgG from cultured human T cells. Note that mAb KPL-1 (B), or the anti-human CD43 mAb L60 (C). D, Depletion of the 130-kDa protein from the E-selectin-IgG precipitate. The precipitate a band of similar size has been described as a major component of was immunoprecipitated with either the anti-CD43 mAb L60 or control the E-selectin-IgG precipitate from cultured human T cells (12). mouse IgG. The unbound supernatants (sup) and the first (pel1) and second The same study also precipitated a band of 70–80 kDa using E- (pel2) precipitates were analyzed by Western blotting using HRP-conju- selectin IgG, which they identified as L-selectin (12), but our pre- gated streptavidin. cipitation experiments with E-selectin-IgG did not show a band of 8048 CD43 AS A T CELL E-SELECTIN LIGAND Downloaded from http://www.jimmunol.org/

FIGURE 7. CD43 mediates E-selectin-dependent cell rolling under flow conditions. A, Expression of S7 and 1B11 epitopes on mouse CD43-IgG generated either in CHO-derived CD7II cells that express both FucT-VII and C2GnT or control CHO cells. Mouse CD43-IgG generated in CD7II or control CHO cells was separated by SDS-PAGE and blotted using S7 and 1B11. B, Adhesion of CHO-E cells to CD43- and PSGL-1-IgG chimeras. CHO-E or control CHO cells were added to 96-well plates coated with mouse or human CD43- or PSGL-1-IgG, generated either in CD7II or control CHO cells. The plates were rotated for 20 min, the unbound cells were removed, and the number of bound cells was determined. Values are means Ϯ SD from triplicate wells. C, Effect of the anti-E-selectin mAb 9A9 on CHO-E cell adhesion to CD7II-generated CD43- or PSGL-1-IgG chimeras. CHO-E cells were incubated by guest on September 24, 2021 with or without the anti-E-selectin mAb 9A9 or control rat IgG for 30 min and then added to the wells. Values are means Ϯ SD from triplicate wells. D, Rolling of CHO-E cells on CD43- and PSGL-1-IgG chimeras. CHO-E and control CHO cells were infused into capillaries coated with mouse or human CD43- or PSGL-1-IgG that had been generated either in CD7II or control CHO cells, at a shear stress of 1 dyn/cm2. The number of rolling cells was determined. E, Effect of the anti-E-selectin mAb 9A9 on CHO-E cell rolling on mouse or human CD43- or PSGL-1-IgG chimeras generated in CD7II cells. CHO-E cells were incubated with or without the anti-E-selectin mAb 9A9 or control rat IgG for 30 min and then infused into the capillaries. similar size. This difference is likely to be due to variations in the Interestingly, both antiadhesive and proadhesive functions have culture conditions: in the other study, lymphocytes were activated been described for several sialomucins, such as CD34, podoca- by two cycles of anti-CD3 stimulation, but in our study, the cells lyxin, and endomucin, which can serve as L-selectin ligands when were stimulated only once. In addition, CD44, a hyaluronan-bind- appropriately modified (41–43). ing cell surface molecule, has recently been shown to function as The ability of CD43 to act as an E-selectin ligand is regulated by an E-selectin ligand on mouse neutrophils (13). Although mouse cell-specific posttranslational modifications. E-selectin recognizes Th1 cells express high levels of CD44, we did not detect any bands sialylated and fucosylated carbohydrate structures represented by in Western blots of the E-selectin-IgG precipitate probed with the sLeX. The biosynthesis of sLeX requires the sequential action of anti-CD44 mAb KM201 (data not shown), suggesting that CD44 several glycosyltransferases including ␣-1,3-fucosyltransferases. may not act as an E-selectin ligand on mouse Th1 cells. FucT-VII plays a dominant role in selectin ligand generation in T The function of CD43 remains elusive (33). Because of its ex- ϩ cells (44). Although FucT-VII is not expressed in naive CD4 T tended structure and strong negative charge, CD43 is thought to cells, its mRNA is augmented when the cells are activated in the function as an antiadhesive molecule, providing a repulsive barrier presence of the Th1-polarizing cytokine IL-12 (45). In addition, around a cell. Targeted disruption of CD43 in cell lines and mice the expression level of C2GnT, which generates core 2 structures, leads to increased cell adhesion and proliferation (21, 34) as well as increased T cell migration to secondary lymphoid organs in vivo increases upon T cell activation, regardless of the presence of po- (22). In contrast, the expression of CD43 on T cell lines enhances larizing cytokines (45). The expression of the 130-kDa glycoform their adhesion to APCs, suggesting a proadhesive role for CD43 of CD43 detected by 1B11 depends on the activity of C2GnT, but high (35). Indeed, several potential ligands for CD43 have been re- not FucT-VII, which explains why not all 1B11 cells have E- ported, including ICAM-1 (36), galectin-1 (37), and sialoadhesin selectin-binding activity. Our data suggest that in mouse Th1 cells (38). In this study, we showed that CD43 on activated T cells can and cultured human T cells that express both FucT-VII and serve as a proadhesive ligand for E-selectin. Tumor-derived CD43 C2GnT, CD43 is one of the core proteins modified by these en- has also been shown to bind E-selectin (14, 39, 40). It needs to be zymes to carry selectin-binding fucosylated core 2 O-glycans. clarified whether any of these potential ligands function in vivo. Thus, although the 130-kDa glycoform of CD43 that carries core The Journal of Immunology 8049

2 O-glycans has been implicated in an antiadhesive process (46), 14. Fernandez-Rodriguez, J., O. Dwir, R. Alon, and G. C. Hansson. 2001. Tumor cell MUC1 and CD43 are glycosylated differently with sialyl-Lewis A and X epitopes fucosylation of this glycoform may switch the molecule to a proad- and show variable interactions with E-selectin under physiological flow condi- hesive selectin ligand. tions. Glycoconj. J. 18: 925–930. In vitro adhesion assays have shown that CD43, when appro- 15. Frenette, P. S., T. N. Mayadas, H. Rayburn, R. O. Hynes, and D. D. Wagner. 1996. Susceptibility to infection and altered hematopoiesis in mice deficient in priately modified by FucT-VII and C2GnT, can support cell teth- both P- and E-selectins. Cell 84: 563–574. ering and rolling under flow conditions. The molecules that sup- 16. Bullard, D. C., E. J. Kunkel, H. Kubo, M. J. Hicks, I. Lorenzo, N. A. Doyle, ␣ C. M. Doerschuk, K. Ley, and A. L. Beaudet. 1996. Infectious susceptibility and port tethering and rolling, such as L-selectin, PSGL-1, and 4 severe deficiency of leukocyte rolling and recruitment in E-selectin and P-selectin integrins, are typically concentrated at the tip of folds or processes, double mutant mice. J. Exp. Med. 183: 2329–2336. called microvilli, on the cell surface. CD43 is also localized on 17. Staite, N. D., J. M. Justen, L. M. Sly, A. L. Beaudet, and D. C. Bullard. 1996. microvilli (47), favoring the view that CD43 is an adhesion mol- Inhibition of delayed-type contact hypersensitivity in mice deficient in both E- ecule mediating tethering and rolling interactions. ESL-1 is also selectin and P-selectin. Blood 88: 2973–2979. 18. Austrup, F., D. Vestweber, E. Borges, M. Lohning, R. Brauer, U. Herz, H. Renz, located on microvilli, albeit mostly on their sides (48), whereas R. Hallmann, A. Scheffold, A. Radbruch, and A. Hamann. 1997. P- and E-selectin CD44 is located on the planar cell surface (49). It is interesting to mediate recruitment of T-helper-1 but not T-helper-2 cells into inflammed tissues. speculate that the difference in the localization of each E-selectin Nature 385: 81–83. 19. Fukuda, M., and S. Tsuboi. 1999. Mucin-type O-glycans and leukosialin. Bio- ligand may confer on them distinct roles in E-selectin-mediated chim. Biophys. Acta 1455: 205–217. adhesive processes. 20. Cyster, J. G., D. M. Shotton, and A. F. Williams. 1991. The dimensions of the T In conclusion, our study provides evidence for the role of CD43 lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation. EMBO J. 10: 893–902. as an E-selectin ligand on activated T cells. The identification of 21. Manjunath, N., M. Correa, M. Ardman, and B. Ardman. 1995. Negative regula- CD43 as one of the E-selectin ligands, in addition to previously tion of T-cell adhesion and activation by CD43. Nature 377: 535–538. Downloaded from identified ligands, such as PSGL-1 and ESL-1, will allow the clar- 22. Stockton, B. M., G. Cheng, N. Manjunath, B. Ardman, and U. H. von Andrian. 1998. Negative regulation of T cell homing by CD43. ification of the distinct and redundant roles of these ligands in Immunity 8: 373–381. various inflammatory conditions in vivo. 23. Woodman, R. C., B. Johnston, M. J. Hickey, D. Teoh, P. Reinhardt, B. Y. Poon, and P. Kubes. 1998. The functional paradox of CD43 in leukocyte recruitment: a study using CD43-deficient mice. J. Exp. Med. 188: 2181–2186. Acknowledgments 24. McEvoy, L. M., H. Sun, J. G. Frelinger, and E. C. Butcher. 1997. Anti-CD43

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