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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2015 June 12; 290(24): 15313–15326.
Published online 2015 May 5. doi:  10.1074/jbc.M115.650051
PMCID: PMC4463470

Novel Antibodies Reactive with Sialyl Lewis X in Both Humans and Mice Define Its Critical Role in Leukocyte Trafficking and Contact Hypersensitivity Responses*An external file that holds a picture, illustration, etc.
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Abstract

Sialyl Lewis X (sLex) antigen functions as a common carbohydrate determinant recognized by all three members of the selectin family. However, its expression and function in mice remain undefined due to the poor reactivity of conventional anti-sLex monoclonal antibodies (mAbs) with mouse tissues. Here, we developed novel anti-sLex mAbs, termed F1 and F2, which react well with both human and mouse sLex, by immunizing fucosyltransferase (FucT)-IV and FucT-VII doubly deficient mice with 6-sulfo-sLex-expressing cells transiently transfected with an expression vector encoding CMP-N-acetylneuraminic acid hydroxylase. F1 and F2 specifically bound both the N-acetyl and the N-glycolyl forms of sLex as well as 6-sulfo-sLex, a major ligand for L-selectin expressed in high endothelial venules, and efficiently blocked physiological lymphocyte homing to lymph nodes in mice. Importantly, both of the mAbs inhibited contact hypersensitivity responses not only when administered in the L-selectin-dependent sensitization phase but also when administered in the elicitation phase in mice. When administered in the latter phase, F1 and F2 efficiently blocked rolling of mouse leukocytes along blood vessels expressing P- and E-selectin in the auricular skin in vivo. Consistent with these findings, the mAbs blocked P- and E-selectin-dependent leukocyte rolling in a flow chamber assay. Taken together, these results indicate that novel anti-sLex mAbs reactive with both human and mouse tissues, with the blocking ability against leukocyte trafficking mediated by all three selectins, have been established. These mAbs should be useful in determining the role of sLex antigen under physiological and pathological conditions.

Keywords: carbohydrate function, carbohydrate-binding protein, cellular immune response, glycosylation, immunology, leukocyte

Introduction

Sialyl Lewis X (sLex)2 antigen (sialic acid α2–3Galβ1-4(Fucα1–3)GlcNAcβ1-R) functions as a common carbohydrate determinant recognized by all three members of the selectin family. The selectins are a family of three C-type lectins that mediate rapid and reversible adhesive interactions between leukocytes and vascular endothelial cells under physiological flow (1,3). L-selectin is expressed on most leukocytes, whereas E- and P-selectin are expressed on activated endothelial cells. It is also known that P-selectin is expressed on activated platelets. However, none of the conventional anti-sLex monoclonal antibodies (mAbs), except for one mAb, react with the high endothelial venules (HEVs) in mouse or rat peripheral lymph nodes (PLNs), where L-selectin ligands are expressed (4, 5). This situation is probably due to the fact that a large proportion of the terminal sialic acid in WT mice is in the form of N-glycolylneuraminic acid (Neu5Gc), whereas most existing mAbs react with glycans modified with N-acetylneuraminic acid (Neu5Ac) (4). The exception is the anti-sLex mAb 2H5 (6), which is reactive with the HEVs in rat PLNs (7), but it also binds to cells lacking α1,3-fucosyltransferase (FucT), FucT-IV, and FucT-VII and may not be very specific for sLex (8). Therefore, although studies using FucT-IV and FucT-VII doubly deficient (DKO) mice revealed that α1,3-fucosylation of HEV ligands and leukocytes is critical for their interaction with selectins (9, 10), the tissue distribution and function of sLex in mouse tissues remain unclear.

We previously reported an efficient method of generating anti-carbohydrate mAbs using mice deficient in glycan-synthesizing enzyme, in which the glycan structures formed by the missing enzyme are highly antigenic (11, 12). By immunizing N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 DKO mice (13) using GlcNAc6ST-2-expressing cells that had been transfected with cDNAs encoding CMP-Neu5Ac hydroxylase (Cmah), which generates CMP-Neu5Gc from CMP-Neu5Ac (14), we were able to generate mAbs reactive with mouse tissues rich in Neu5Gc (11). Because both the cDNAs of various glycosyltransferases and sulfotransferases and a number of mutant mice deficient in those enzymes are now available, we hypothesized that the method should be widely applicable for the generation of various anti-carbohydrate mAbs.

In the present study, we generated novel anti-sLex mAbs, termed F1 and F2, which react with both human and mouse sLex, by immunizing FucT-IV and FucT-VII DKO mice with 6-sulfo-sLex-expressing cells transiently transfected with an expression vector encoding Cmah. These mAbs bound both the N-acetyl and the N-glycolyl forms of sLex as well as 6-sulfo-sLex, a major ligand for L-selectin expressed in the HEVs in PLNs, and efficiently blocked lymphocyte homing to PLNs and contact hypersensitivity (CHS) responses in mice. Interestingly, administration of the mAbs in the elicitation phase significantly inhibited the CHS responses and rolling of mouse leukocytes along blood vessels expressing P- and E-selectin in the inflamed ear, which could not be verified by the studies using FucT-IV and FucT-VII DKO mice that lacked selectin-mediated leukocyte trafficking in both the sensitization and the elicitation phases. These results demonstrate that our method is applicable to the generation of anti-carbohydrate mAbs to determine the biological function of glycans that have not been clarified by prior studies using gene-targeted mice.

Experimental Procedures

Mice

FucT-IV and FucT-VII DKO, GlcNAc6ST-1 and GlcNAc6ST-2 DKO, and core 1 β1,3-N-acetylglucosaminyltransferase (C1β3GnT) and core 2 β1,6-N-acetylglucosaminyltransferase-I (C2GnT-I) DKO mice were back-crossed at least 5 generations to C57BL/6 WT mice and maintained as described previously (9, 13, 15). C57BL/6 WT and BALB/c Slc-nu/nu mice were purchased from Japan SLC (Hamamatsu, Japan). The mice were treated in accordance with the guidelines of the Animal Research Committee of Hoshi University and University of Shizuoka.

Establishment of Anti-fucosylated Glycan mAbs

Anti-fucosylated glycan mAbs were generated as described previously (11) with certain modifications. In brief, CHO cells stably expressing human CD34, human C1β3GnT, human C2GnT-I, human FucT-VII, and mouse GlcNAc6ST-2 (CHO/CD34/C1/C2/FucT-7/GlcNAc6ST-2) were transiently transfected with mouse Cmah (14) using FuGENE 6 transfection reagent (Roche Applied Science). After 48 h of transfection, the cells were suspended in PBS and mixed with Imject Alum (Pierce) at a ratio of 1:1 and injected intraperitoneally into FucT-IV and FucT-VII DKO mice three times at 2-week intervals. Four days after the final immunization, lymphocytes from the spleens of the DKO mice were fused with P3X63Ag8.653 myeloma cells (American Type Culture Collection) in the presence of PEG solution (Mr 1,450; Sigma-Aldrich) and selected in the medium containing HAT (hypoxanthine, aminopterin, and thymidine) supplement (Invitrogen). The hybridoma supernatants that reacted with the HEVs of WT mice, but not with those of FucT-IV and FucT-VII DKO mice, were selected by immunofluorescence. Hybridomas secreting anti-fucosylated glycan mAbs were cloned by limiting dilution. The isotypes of the mAbs F1 and F2 thus obtained were determined to be mouse IgG1 (κ) using an isotyping kit (GE Healthcare). F1 and F2 were purified from the ascitic fluid using a caproic acid (6-aminohexanoic acid; Wako) precipitation method. In certain cases, purified antibodies and control mouse IgG (Sigma-Aldrich) were labeled with EZ-Link Sulfo-NHS-LC-Biotin (Pierce) according to the manufacturer's protocols.

Immunofluorescence

Acetone-fixed, frozen sections (7-μm) from mice that had been treated with or without 300 milliunits/ml sialidase (from Streptococcus 6646K, Seikagaku) for 6 h at 37 °C were incubated with PBS containing 3% BSA (Sigma-Aldrich) to block nonspecific binding sites and then with biotinylated F1, F2, MECA-79 (BioLegend), CSLEX1 (BD Biosciences), or HECA-452 (BioLegend). After washing, the sections were incubated with streptavidin-Alexa Fluor 594 (Invitrogen) and DAPI (Roche Applied Science) and mounted using Fluoromount (Diagnostic BioSystems). Paraffin-embedded human tonsil tissue sections (kindly provided by Drs. Yoko Ishihara and Toshio Nishikawa) were deparaffinized, boiled in 10 mm Tris/HCl buffer (pH 8.0) containing 1 mm EDTA for 20 min to retrieve antigens, and stained as described above. All images were obtained using a microscope (BZ-9000; KEYENCE).

Glycan Array Analysis

Glycan array analysis was performed at the Consortium for Functional Glycomics using microarray slides (printed array version 5.0) containing 611 different glycans, as described previously (11).

Flow Cytometric Analysis

Cells were incubated with biotinylated F1, F2, mouse IgG, CSLEX1, or HECA-452, followed by streptavidin-Alexa Fluor 647 (Invitrogen). For the analysis of mouse leukocytes induced in the peritoneal cavity by thioglycollate medium (Thermo Fisher Scientific), as described below, cells were incubated with APC-Cy7-anti-mouse CD45 (BioLegend) and FITC-anti-Gr-1 (BioLegend) together with biotinylated F1, F2, mouse IgG, CSLEX1, or HECA-452 mAbs. The cells were analyzed by flow cytometry using a FACSCanto II cell analyzer (BD Biosciences). For mouse leukocyte staining, CD45+Gr-1high mouse granulocytes were gated and analyzed for their reactivity with various anti-sLex mAbs. The data were acquired and analyzed with FACSDiva software (BD Biosciences) and FlowJo software (Tree Star, Inc.).

Transient Transfection and Flow Cytometry

The CHO-K1 cell-derived mutant line Lec1 (16) was transiently transfected with pcDNA3.1/EGFP together with various combinations of pCDM8/human FucT-VII, pcDNA3/human C1β3GnT, pcDNA3/human C2GnT-I, and pcDNA3.1/Zeo using a pipette-type electroporator (Neon Transfection System; Invitrogen) according to the protocol provided by the manufacturer. A total of 72 h after transfection, the cells were washed with PBS, dispersed in 1 mm EDTA-PBS, and incubated with biotinylated F1 or F2 or with mouse IgG, followed by incubation with streptavidin-Alexa Fluor 647 (Invitrogen). The cells were analyzed by flow cytometry using a FACSCanto II system, as described above.

ELISA

The wells of a 96-well ELISA plate (Costar EIA/RIA Half Area Plate 3690, Corning, Inc.) were first coated overnight with 1 μg/ml sialyl N-acetyllactosamine (LacNAc)-polyacrylamide (PAA), Lex-PAA, sLex-PAA, or 6-sulfo-sLex-PAA (GlycoTech Co.) in PBS at 4 °C. After blocking with Blocking One (1:5 dilution; Nacalai Tesque, Inc.), 0.5 μg/ml F1, 0.5 μg/ml F2, 0.5 μg/ml control mouse IgG1 (MOPC-21, BioLegend), 1 μg/ml HECA-452 (rat IgM), or 5 μg/ml CSLEX1 (mouse IgM) in PBS containing 0.1% BSA, 0.05% Tween 20 (buffer A) was added to the wells and incubated for 1 h at room temperature. After washing, HRP-goat anti-mouse IgG (1:2,000 dilution; Zymed Laboratories Inc.), HRP-goat anti-rat IgM (1:2,000 dilution; Southern Biotech), or HRP-goat anti-mouse IgM (1:500 dilution; Zymed Laboratories Inc.) in buffer A was added to the samples treated with F1, F2, or control mouse IgG; HECA-452; or CSLEX1, respectively, and incubated for 1 h. After washing with buffer A, 1-Step Ultra TMB-ELISA HRP substrate (Thermo Fisher Scientific) was added. The reaction was terminated by the addition of 2 m H2SO4, and the optical density at 450 nm was measured using a 96-well spectrometer (Sunrise Rainbow RC-R, TECAN).

Lymphocyte Homing Assay

A lymphocyte homing assay was performed as described previously, with certain modifications (11, 13, 17). In brief, mesenteric lymph node (MLN) lymphocytes and splenocytes from WT mice were labeled with 5 μm carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen). After washing, 2.0 × 107 cells in 200 μl of PBS were injected intravenously into recipient mice. One hour after the injection, the number of CFSE+ cells in cell suspensions prepared from the recipient lymphoid organs was determined by flow cytometry. For lymphocyte homing inhibition experiments, mice were preinjected intravenously with purified F1 or F2 (200 μg/mouse) 2 h before being injected with CFSE-labeled lymphocytes.

Modified Stamper-Woodruff Cell-binding Assay

A modified Stamper-Woodruff cell-binding assay was performed as described previously (11).

In Vitro Leukocyte Rolling Assay

To analyze L-selectin-dependent leukocyte rolling, CHO/CD34/C1/C2/FucT-7/GlcNAc6ST-2 cells cultured as monolayers in 35-mm culture dishes (Corning Inc.) were incubated with or without 10 μg/ml purified F1, F2, or control mouse IgG for 10 min at room temperature. After incubation, the dishes were equipped with a parallel plate flow chamber (GlycoTech Co.) according to the manufacturer's instructions. Freshly prepared leukocytes from the spleens of WT mice were suspended in buffer B (20 mm HEPES-NaOH, 0.15 m NaCl, 1 mm CaCl2, and 1 mm MgCl2, pH 7.4) containing 0.1% BSA at 1 × 106 cells/ml and introduced into the flow chamber at a wall shear stress of 2, 1.5, 1, or 0.5 dynes/cm2 using the syringe pump Model 11 Plus (Harvard Apparatus Co.). For L-selectin inhibition experiments, the leukocyte suspension was preincubated with 10 μg/ml MEL-14 (eBioscience) in buffer B containing 0.1% BSA for 10 min at 4 °C. To analyze P- or E-selectin-dependent leukocyte rolling, CHO-K1 cells stably expressing human P-selectin were established by co-transfecting the plasmids hCD62-P/pCMV6-XL5 (OriGENE) and pBApo-CMV/Pur (TaKaRa) at a ratio of 20:1 using FuGENE 6 transfection reagent and by performing selection in 6 μg/ml puromycin (TaKaRa) by standard procedures. After cloning the cells by limiting dilution, cells expressing P-selectin were selected by flow cytometry after staining the cells with Alexa Fluor 647-labeled anti-human CD62P (AbD SeroTec). CHO-K1 cells stably expressing human P-selectin thus obtained and those expressing human E-selectin, established previously (18), were cultured as monolayers in 35-mm culture dishes and used in the rolling assay described above. In brief, human promyelocytic leukemia HL-60 cells (American Type Culture Collection) that had been preincubated with or without F1, F2, or control mouse IgG for 10 min at 4 °C were allowed to roll on CHO-K1 cells expressing P- or E-selectin. Images were taken with a CCD camera (model ADT-40S; Flovel Co., Ltd.) attached to an inverted microscope (×20 objective; Olympus CKX41).

CHS

The CHS responses were measured as described previously, with certain modifications (11, 13). In brief, 25 μl of 1% oxazolone (Sigma-Aldrich) in acetone/olive oil (4:1, v/v) was applied to the shaved forelegs of mice on day 0. On day 5, the ears were treated with 20 μl of 1% oxazolone (10 μl/side of the pinna). In Schedule 1, 200 μl of 0.5 mg/ml mAb in PBS or PBS alone was intravenously injected on days −1, 0, and 1. In Schedule 2, the same amount of mAb or PBS alone was intravenously injected on day 5, 30 min before the 1% oxazolone treatment was applied to the ear. Ear swelling was measured using a thickness gauge before and 24 h after the treatment. Paraffin-embedded tissue sections from the ears of the oxazolone-treated mice were stained with hematoxylin-eosin (Sigma-Aldrich) and observed using a microscope (BZ-9000; KEYENCE).

In Vivo Leukocyte Rolling Assay

To observe leukocyte rolling in the skin, hair removal cream (Veet HPS-a, Reckitt Benckiser Japan Ltd.) was applied to both sides of the ear. After 5 min, the ear was gently cleaned with gauze. CHS responses were then elicited as described above. To observe in vivo leukocyte rolling, 0.5 mg/ml rhodamine 6G in 200 μl of saline was injected intravenously to label leukocytes fluorescently in the mouse peripheral blood 0.5 or 14 h after the challenge with 1% oxazolone on day 5. The mice were then anesthetized and mounted on the microscope stage. The ear was flattened and held in place by glass slides, and leukocyte rolling along the vein in the inflamed ear was observed with a CCD camera installed on an inverted fluorescence microscope (CKX41, Olympus) and was recorded for 3 min.

RT-PCR

Total RNA was purified from the ears of C57BL/6 WT mice using TRIzol reagent (Invitrogen). cDNA was synthesized using a PrimeScript RT-PCR kit (TaKaRa) and subjected to RT-PCR. The primers used were as follows: P-selectin, 5′-GCTTCAGGACAATGGACATG-3′ and 5′-ACTCCGTATGTTCCTAGGTG-3′; E-selectin, 5′-CCTCTGACAGAGGAAGCTCAGAACT-3′ and 5′-TCCACTCTCCAGAGGACGTACACCG-3′; β-actin, 5′-TGGAATCCTGTGGCATCCATGAAAC-3′ and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′. The PCR cycle (94 °C for 30 s, 61 °C for 30 s, and 72 °C for 60 s) was repeated 50 times for P-selectin. The PCR cycle (94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s) was repeated 50 times for E-selectin. The PCR cycle (94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s) was repeated 30 times for β-actin.

Statistical Analysis

Dunnett's test (for Figs. 7E, ,88 (A and C), and and99 (B and C)) or Student's t test (for Fig. 7, A–C) was used for the determination of statistically significant differences between experimental groups. All results are expressed as the mean ± S.D.

FIGURE 7.
Inhibition of lymphocyte homing by F1 and F2. A–C, CFSE-labeled lymphocytes (2.0 × 107 cells) were injected into the tail vein of WT (A), GlcNAc6ST-1 and GlcNAc6ST-2 DKO (B), or C1β3GnT and C2GnT-I DKO mice (C). One hour after ...
FIGURE 8.
Inhibition of CHS responses by F1 and F2. Ear swelling 24 h after challenge with oxazolone in WT mice intravenously injected three times with F1, F2, or S2 (100 μg/mouse) or with PBS on days −1, 0, and 1 (Schedule 1; A) or once with F1, ...
FIGURE 9.
Inhibition of leukocyte rolling along the blood vessels in the inflamed ear by F1 and F2. A, rolling of leukocytes along the blood vessels in the inflamed ear was analyzed at various time points after the 1% oxazolone challenge. B and C, F1, F2, or S2 ...

Results

Generation of Anti-sLex mAbs

To obtain anti-fucosylated glycan mAbs that recognize glycan epitopes on mouse HEVs, FucT-IV, and FucT-VII DKO mice were immunized with CHO cells stably expressing CD34, C1β3GnT, C2GnT-I, FucT-VII, and GlcNAc6ST-2 (CHO/CD34/C1/C2/FucT-7/GlcNAc6ST-2 cells) that had been transiently transfected with an expression vector encoding Cmah (14). The splenocytes of the immunized mice were used to generate hybridomas by cell fusion with mouse myeloma cells. The culture supernatants were screened for their immunoreactivity with the HEVs of WT mice and for their lack of immunoreactivity with the HEVs of FucT-IV and FucT-VII DKO mice deficient in sLex biosynthesis in the HEVs (9). As a result, two independent hybridoma clones, secreting the anti-sLex mAbs F1 and F2 (mouse IgG1, κ), were established.

To determine the carbohydrate-binding specificity of the mAbs F1 and F2, immunofluorescence staining using frozen sections of PLNs from various KO mice was performed (Fig. 1A). F1 and F2 bound well to the HEVs of the PLNs in GlcNAc6ST-1 and GlcNAc6ST-2 DKO (13) and C1β3GnT and C2GnT-I DKO (15) mice. These mAbs did not bind to PLN HEVs in FucT-IV and FucT-VII DKO mice (9). In contrast, the mAb MECA-79, which recognizes sulfated extended core 1 O-glycans (19), bound to the HEVs in FucT-IV and FucT-VII DKO but not to those in the other two strains of mutant mice. The staining intensity of F1 and F2, but not that of MECA-79, was significantly diminished after sialidase treatment of the tissue sections (Fig. 1B). Therefore, these results indicate that sialylation and α1,3-fucosylation, but not sulfation, are required for the binding of F1 and F2 to HEVs.

FIGURE 1.
Binding of the mAbs F1, F2, and MECA-79 to PLN sections from various gene-targeted mice. A, frozen sections of PLNs from WT, GlcNAc6ST-1 and GlcNAc6ST-2 DKO, FucT-IV and FucT-VII DKO, or C1β3GnT and C2GnT-I DKO mice were incubated with biotinylated ...

The above mentioned results obtained using C1β3GnT and C2GnT-I DKO mice suggest that F1 and F2 can interact with sLex on N-glycans. In support of this notion, treatment of the PLN tissue sections of C1β3GnT and C2GnT-I DKO mice with N-glycosidase F as described previously (11) reduced the staining intensity with these mAbs.3 Furthermore, both of the mAbs specifically bound stable transfectants expressing 6-sulfo-sialyl sLex on their N-glycans (CHO/CD34/FucT-7/GlcNAc6ST-2) as well as those expressing 6-sulfo-sLex on both N- and O-glycans (CHO/CD34/C1/C2/FucT-7/GlcNAc6ST-2) (Fig. 2A). To examine whether these mAbs can also interact with sLex on O-glycans and glycolipids, transient transfection of CHO-K1 cell-derived mutant Lec1 cells (16) and flow cytometric analysis were performed. CHO-K1 cells lack C1β3GnT and C2GnT-I. In addition, Lec1 cells lack N-acetylglucosaminyltransferase-I, which forms complex- and hybrid-type N-glycans (16). Therefore, transient transfection of Lec1 cells with a human FucT-VII expression vector together with an EGFP expression vector results in the expression of sLex only on glycolipids. As shown in Fig. 2B, Lec1 cells transiently transfected as such (FucT-7) reacted with both F1 and F2, indicating that both of these mAbs can interact with sLex on glycolipids. Further co-transfection of the Lec1 cells with C1β3GnT (C1/FucT-7) or C2GnT-I (C2/FucT-7) resulted in a significant increase in the cells reactive with the mAbs. These results indicate that F1 and F2 can interact not only with sLex on N-glycans but also with that on glycolipids and O-glycans.

FIGURE 2.
Binding of the mAbs F1 and F2 to sLex-expressing cells. A, CHO-K1, CHO/CD34/FucT-7/GlcNAc6ST-2, and CHO/CD34/C1/C2/FucT-7/GlcNAc6ST-2 cells were stained with biotinylated F1, F2, or control mouse IgG (0.5 μg/ml), followed by Alexa Fluor 647-conjugated ...

Glycan Array Analysis of F1 and F2

To determine the fine carbohydrate-binding specificities of F1 and F2, glycan array screening was performed (Fig. 3 and supplemental Table S1). As shown in Fig. 3, both F1 and F2 interacted with sLex terminated with Neu5Ac (Glycan 256) as well as with that terminated with Neu5Gc (Glycan 283). Consistent with immunofluorescence studies, F1 and F2 also bound well to 6-sulfo-sLex (Glycan 253), which is abundantly expressed in PLN HEVs. In contrast, F1 and F2 failed to interact with the Lex structure (Glycan 153) or with α2,3-sialylated LacNAc (Glycan 261). In addition, these mAbs did not bind to other related glycans lacking sialic acid (Glycans 170, 80, 291, 130, 73, and 114). Importantly, both of the mAbs failed to interact with an isomer of sLex, or sLea (Glycan 240), bearing β1,3-linked galactose and α1,4-linked fucose residues. In addition, these mAbs bound to terminal sLex on a di-LacNAc backbone (Glycan 258) but not to VIM-2 antigen bearing α1,3-linked fucose attached to the internal GlcNAc residue (Glycan 219). Both of the mAbs bound well to sLex on the GlcNAcβ1–2Man present in N-glycans (Glycan 535) but only weakly or not at all to sLex on the core 2-branched O-glycans (Glycan 330), suggesting that the core 1 branch of the O-glycan chain may interfere with the accessibility of the terminal sLex on the core 2 branch to the mAbs. A comparison between F1 and F2 suggests that the two mAbs have similar binding specificities, although F2 appears to bind sLex more avidly than F1 does (Glycans 256, 283, 253, and 535).

FIGURE 3.
Glycan array analysis of F1 and F2. Version 5.0 of the printed array of the Consortium for Functional Glycomics was probed with 10 μg/ml F1 or F2. Bars, mean ± S.D. (error bars) of four measurements. Glycan numbers are shown in parentheses ...

Binding of F1 and F2 to Human and Mouse HEVs and Leukocytes

To compare the reactivity of F1 and F2 with that of the previously reported anti-sLex mAbs CSLEX1 (20) and HECA-452 (21), immunofluorescence studies were performed using human tonsil and mouse PLN tissue sections (Fig. 4A). In human tonsils, F1, F2, and HECA-452, but not CSLEX1, bound to MECA-79+ HEVs as well as infiltrated leukocytes. In contrast, F1 and F2, but not CSLEX1 and HECA-452, bound to mouse MECA-79+ HEVs.

FIGURE 4.
Reactivity of various anti-sLex mAbs with human and mouse HEVs and leukocytes. A, paraffin-embedded tissue sections of human tonsils and frozen sections of mouse PLNs were stained with biotinylated anti-sLex mAbs (1.0 μg/ml), followed by streptavidin-Alexa ...

Flow cytometric analysis of human promyelocytic leukemia HL-60 cells and mouse leukocytes expressing human and mouse sLex, respectively, indicated that all four anti-sLex mAbs examined bound to HL-60 cells (Fig. 4B). In contrast, F1 and F2, but not CSLEX1 and HECA-452, bound to CD45+Gr-1high mouse granulocytes.

To determine the reasons for the differential reactivity of the mAbs described above, ELISA using immobilized PAA-oligosaccharides was performed (Fig. 5A). Consistent with the results of the glycan array screening, F1 and F2 bound sLex- and 6-sulfo-sLex-PAA. HECA-452 also bound to these oligosaccharides with certain cross-reactivity with Lex-PAA. CSLEX1 bound only to sLex without sulfation, which is probably the reason why CSLEX1 did not bind HEVs in human tonsil sections. To determine the preference of the mAbs for terminal sialic acid species, flow cytometric analysis using CHO cells stably expressing P-selectin glycoprotein ligand 1 (PSGL-1), C2GnT-I, and FucT-VII (CHO/PSGL/C2/FucT-7) and CHO cells additionally expressing Cmah (CHO/PSGL/C2/FucT-7/Cmah) was performed (Fig. 5B). F1 and F2, but not HECA-452, reacted with cells expressing Cmah. This result is consistent with a previous report showing the nonreactivity of HECA-452 with the N-glycolyl form of sLex (4). In addition, the reactivity of CSLEX1 was only slightly diminished in the presence of Cmah, as described previously (4), which is probably because the lack of reactivity of CSLEX1 with mouse leukocytes is mainly due to other sialic acid modifications, such as O-acetylation, as suggested previously (4). These results, together with the results in Fig. 3, indicate that F1 and F2 bind both Neu5Ac- and Neu5Gc-terminated sLex, whereas the other two mAbs prefer Neu5Ac-terminated sLex.

FIGURE 5.
Binding of anti-sLex mAbs to synthetic glycans and to transfectants expressing PSGL-1 modified with the Neu5Ac or Neu5Gc form of sLex. A, the binding of anti-sLex mAbs and control mouse IgG to a plate coated with PAA coupled with various synthetic glycans ...

Inhibition of Selectin-mediated Leukocyte Rolling by F1 and F2

sLex is recognized by all three members of the selectin family. To examine the ability of F1 and F2 to block selectin-mediated leukocyte rolling under physiological flow conditions, a parallel plate flow chamber assay was performed. As shown in Fig. 6A, CHO/CD34/C1/C2/FucT-7/GlcNAc6ST-2 cells supported L-selectin-dependent rolling of mouse leukocytes under physiological shear stress. The rolling was markedly blocked by F1 and F2. Rolling of HL-60 cells on P- and E-selectin transfectants was also significantly blocked by F1 and F2 (Fig. 6, B and C).

FIGURE 6.
Inhibition of selectin-dependent leukocyte rolling by F1 and F2 in vitro. A, numbers of rolling leukocytes from mouse spleens that had been pretreated with or without (None) MEL-14 (10 μg/ml) for 30 s and added to CHO/CD34/C1/C2/FucT-7/GlcNAc6ST-2 ...

Inhibition of Lymphocyte Homing by F1 and F2

To assess the functional effects of the newly generated mAbs in vivo, a lymphocyte homing assay was performed (Fig. 7). F1 and F2 nearly completely inhibited lymphocyte homing to PLNs and MLNs in WT mice (Fig. 7A). These mAbs also significantly inhibited lymphocyte homing to Peyer's patches (PPs). Consistent with these findings, the mAbs specifically stained HEVs in PLNs, MLNs, and PPs in WT mice, but not those in FucT-IV and FucT-IV/-VII DKO mice, in the immunofluorescence analysis (Fig. 7D). In addition, a modified Stamper-Woodruff cell-binding assay indicated that F1 and F2 significantly inhibited the binding of fluorescently labeled leukocytes to HEVs in PLN tissue sections, similar to the MECA-79 antibody (Fig. 7E). The binding observed in this assay was dependent on L-selectin because the anti-L-selectin mAb MEL-14 (22) completely blocked the cell binding. Thus, these results indicate that both F1 and F2 inhibited the interaction between L-selectin on leukocytes and its ligands on HEVs.

Previously, it was reported that lymphocyte homing was diminished by ~75% in GlcNAc6ST-1 and GlcNAc6ST-2 DKO mice and that the residual homing was probably mediated by unsulfated sLex, as suggested by our group and others (13, 23). To determine whether this is the case, the ability of F1 and F2 to effect the residual homing in the sulfotransferase DKO mice was examined (Fig. 7B). Both F1 and F2 nearly completely blocked lymphocyte homing to PLNs and MLNs, indicating that the previous hypothesis was correct. It was also reported previously that lymphocyte homing to PLNs in C1β3GnT and C2GnT-I DKO mice was only partially diminished (15). To confirm the importance of N-glycans in the residual lymphocyte homing in those mutant animals, a short term homing assay using WT mice and C1β3GnT and C2GnT-I DKO mice, which lack sulfated O-glycans, was performed. Both F1 and F2 nearly completely inhibited the residual lymphocyte homing to PLNs in the C1β3GnT and C2GnT-I DKO mice (Fig. 7C). These results suggest that the sLex- or 6-sulfo-sLex-containing N-glycans expressed in the HEVs mediate the residual lymphocyte homing in C1β3GnT and C2GnT-I DKO mice.

Inhibition of CHS Responses by F1 and F2

To further examine the effects of F1 and F2 in vivo, their effects on CHS responses were examined. In Schedule 1, in which mice were injected intravenously with F1 or F2 on three consecutive days during the sensitization phase, ear swelling and leukocyte infiltration were significantly blocked by the treatment with F1 or F2 (Fig. 8, A and B). Under the same protocol, mAb S2, which recognizes 6-sulfo-sialyl LacNAc and 6-sulfo-sLex, inhibited CHS responses, as described previously (11). ELISA indicated that 11.8 ± 4.07 μg/ml F1 and 0.07 ± 0.05 μg/ml F2 persisted in the sera on day 5 after separately intravenously injecting these mAbs according to Schedule 1 (Fig. 8E), suggesting that administration of the mAbs during the sensitization phase might affect not only hapten sensitization but also leukocyte infiltration in the elicitation phase. To examine the effects of the mAbs in the elicitation phase, the mAbs were injected intravenously into the hapten-sensitized mice 30 min before the second oxazolone challenge (Schedule 2). Interestingly, even under this protocol, both ear swelling and leukocyte infiltration were significantly blocked by the mAb treatment (Fig. 8, C and D), suggesting that sLex-dependent leukocyte infiltration in the elicitation phase is critical for CHS responses.

Inhibition of in Vivo Rolling of Leukocytes by F1 and F2 during the Elicitation Phase of CHS Responses

To determine if F1 and F2 actually inhibited leukocyte rolling in vivo, leukocyte rolling along the vein in the inflamed ear was analyzed by fluorescence intravital microscopy. As shown in Fig. 9A, two peaks of leukocyte rolling were observed, at 30 min and 14 h after elicitation. Both of these peaks of leukocyte rolling were significantly blocked by intravenously injecting F1 or F2 30 min prior to the elicitation (Fig. 9, B and C). RT-PCR analysis indicated that P-selectin was expressed at all time points examined, whereas E-selectin was expressed at 6 and 14 h after elicitation and faintly at 24 h (Fig. 9D), suggesting that P- and E-selectin differentially contribute to leukocyte rolling at different time points in CHS responses.

Discussion

We previously developed an efficient method for generating anti-carbohydrate mAbs by immunizing mice deficient in glycan-synthesizing enzyme with transfectants that overexpress the glycan epitopes formed by the enzyme (11). In the present study, we examined whether this method is applicable to the generation of other anti-carbohydrate mAbs. To this end, FucT-IV- and FucT-VII-deficient mice were immunized with 6-sulfo-sLex-expressing transfectants, and novel anti-sLex mAbs, termed F1 and F2, were generated. Before immunization, Cmah cDNA was transiently transfected into 6-sulfo-sLex-expressing cells to modify the terminal sialic acid. This strategy successfully generated mAbs that were reactive with mouse tissues rich in Neu5Gc (4).

Glycan array analysis indicated that both F1 and F2 were highly specific for the sLex and 6-sulfo-sLex structures. Both of the mAbs bound well to sLex on the GlcNAcβ1–2Man present in N-glycans (Glycan 535). However, F2 bound only weakly to sLex on core 2-branched O-glycans (Glycan 330), and F1 failed to bind to this glycan, suggesting that the core 1 branch of the O-glycan chain may interfere with the accessibility of terminal sLex on the core 2 branch to these mAbs. We speculate that both F1 and F2 should make contact with the sLex structure on the core 2 branch of an O-glycan from such an angle that the core 1 branch might sterically interfere with the access. The reason why only F2 could bind to this glycan is probably that F2 has higher affinity toward sLex compared with F1. Indeed, F2 bound sLex more avidly than F1 did in our glycan array analysis. Previously, it was reported that PSGL-1-derived N-terminal sulfoglycopeptide modified with sLex-containing core 2 O-glycans could support interaction with P-selectin (24, 25). Because both F1 and F2 significantly blocked P-selectin-mediated leukocyte rolling (Fig. 6), F1 and F2 probably bound to sLex-containing glycans on PSGL-1 near its N terminus. We hypothesize that sLex-containing glycans other than those on core 2-branched structures might serve as the binding site for F1.

Previous studies indicated that lymphocyte homing was partially diminished in GlcNAc6ST-1 and GlcNAc6ST-2 DKO mice (13, 23). Although those reports suggested that the unsulfated sLex structure was involved in the residual homing in the sulfotransferase DKO mice, a definitive conclusion could not be reached because of the lack of mAbs reactive with mouse sLex. Our present study now clearly shows that the residual homing to PLNs in these mice is nearly completely dependent on unsulfated sLex (Fig. 7). In addition, it has also been reported that lymphocyte homing to PLNs was only partially diminished, by 55%, in C1β3GnT and C2GnT-I DKO mice in a short term homing assay (15). Further studies in that work showed the importance of N-glycans in lymphocyte homing, although it was not clear whether sLex or its related structure on N-glycans is involved in the residual lymphocyte homing. In the present study, by using specific mAbs, we provided evidence that the residual homing observed in C1β3GnT and C2GnT-I DKO mice is most likely mediated by N-glycans modified with sLex or 6-sulfo-sLex structures (Fig. 7). Because the majority of lymphocyte homing to the PLNs in C1β3GnT and C2GnT-I DKO mice was blocked by anti-sulfated glycan mAb S2 in our previous studies (11) and by mAb CL41 with similar carbohydrate binding specificity (26), it is likely that N-glycans modified with 6-sulfo-sLex play a major role in the residual lymphocyte homing to PLN in these mutant mice.

Both F1 and F2 were reactive with PP HEVs and significantly inhibited lymphocyte homing to PPs (Fig. 7). These results are consistent with a previous report using FucT-IV and FucT-VII DKO mice, in which short term lymphocyte homing to PP was significantly inhibited (9). In contrast, GlcNAc6ST-1 and GlcNAc6ST-2 DKO mice did not show any reduction of lymphocyte homing to PPs (13, 23). Furthermore, mAb S2 did not inhibit lymphocyte homing to PPs, although this mAb was reactive with PP HEVs (11). Therefore, these findings collectively suggest that unsulfated sLex structure plays an important role in lymphocyte homing to PPs. The residual homing to PP in the presence of F1 or F2 could be due to α4β7 integrin and MAdCAM-1 interaction (27) or CD22-mediated homing of B cells (28).

Interestingly, F1 and F2 blocked CHS responses not only when administered in the sensitization phase but also when administered in the elicitation phase in mice (Fig. 8). Because F1 and F2 nearly completely abrogated lymphocyte homing to the PLNs, where immune responses occur, these results suggest that F1 and F2 inhibited CHS responses at least in part by blocking L-selectin-dependent lymphocyte homing to the draining lymph nodes during the sensitization phase. However, because a small amount of F1 (11.8 ± 4.07 μg/ml) and F2 (0.07 ± 0.05 μg/ml) persisted in the sera on day 5 after intravenous injection, we think it likely that mAbs administered during the sensitization phase might have blocked not only L-selectin-dependent hapten sensitization but also P- and E-selectin-dependent leukocyte infiltration in the elicitation phase. In accordance with this possibility, F1 and F2 more efficiently inhibited CHS responses than S2 that selectively inhibits L-selectin-dependent lymphocyte homing to PLNs (11) under Schedule 1. Administration of F1 and F2 to the hapten-sensitized mice before the second hapten challenge (Schedule 2) clearly indicated that blocking leukocyte infiltration in the ear during the elicitation phase was effective in blocking the CHS responses. We also provided evidence that F1 and F2 indeed block leukocyte rolling in the inflamed ear in vivo, as evidenced by intravital fluorescence microscopy (Fig. 9). These findings suggest the interesting clinical implication that blocking acute leukocyte infiltration could be an effective way to block allergic reactions, even in allergen-sensitized individuals.

Because both F1 and F2 bound not only mouse but also human tissues and leukocytes, these mAbs will be useful for assessing the expression of sLex in pathophysiological situations in both humans and mice. HEV-like vessels are induced at various sites of chronic inflammation. For example, MECA-79-reactive HEV-like vessels are induced in human gastric mucosa infected with Helicobacter pylori (29), in ulcerative colitis (30), and in rheumatoid arthritis (31). Because F1 and F2 strongly block selectin-mediated leukocyte trafficking, those mAbs will be useful for examining the roles of sLex and its related structures at sites of chronic inflammation using animal models.

In conclusion, we have developed novel anti-sLex mAbs reactive with both human and mouse tissues. Because these mAbs block leukocyte trafficking mediated by all three selectins in vivo and can be used in immunohistochemical as well as flow cytometric studies, future studies using these mAbs will advance our understanding of the role of sLex in health and disease.

Supplementary Material

Supplemental Data:

Acknowledgments

We thank Drs. Yoko Ishihara (Kurume University, School of Medicine) and Toshio Nishikawa (Tokyo Women's Medical University) for providing the human tonsil tissue sections. We also thank Koichiro Tsuboi and Hiroaki Matsuura (University of Shizuoka, School of Pharmaceutical Sciences) for technical support and Drs. Richard D. Cummings, David F. Smith, and Jamie Heimburg-Molinaro (Emory University School of Medicine) at the Consortium for Functional Glycomics (supported by National Institutes of Health Grant U54GM62116) for performing the glycan array analysis and for providing the FucT-IV and FucT-VII DKO mice.

*This work was supported in part by Grants-in-Aid for Scientific Research 21390023 and 24390018, Category B, from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; by the MEXT-supported program of the Strategic Research Foundation at Private Universities, 2014–2018 (Grant S1411019); and by the Institute for Fermentation (Osaka, Japan). The authors declare that they have no conflicts of interest with the contents of this article.

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThis article contains supplemental Table S1.

3R. Matsumura and H. Kawashima, unpublished observation.

2The abbreviations used are:

sLex
sialyl Lewis X
HEV
high endothelial venule
PLN
peripheral lymph node
Neu5Gc
N-glycolylneuraminic acid
Neu5Ac
N-acetylneuraminic acid
FucT
fucosyltransferase
DKO
doubly deficient
GlcNAc6ST
N-acetylglucosamine-6-O-sulfotransferase
LacNAc
N-acetyllactosamine
Cmah
CMP-Neu5Ac hydroxylase
CHS
contact hypersensitivity
C1β3GnT
core 1 β1,3-N-acetylglucosaminyltransferase
C2GnT-I
core 2 β1,6-N-acetylglucosaminyltransferase-I
PAA
polyacrylamide
MLN
mesenteric lymph node
PP
Peyer's patch
PSGL-1
P-selectin glycoprotein ligand 1
CFSE
carboxyfluorescein diacetate succinimidyl ester.

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