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Originally published In Press as doi:10.1074/jbc.M404456200 on June 2, 2004

J. Biol. Chem., Vol. 279, Issue 33, 35001-35008, August 13, 2004
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N-Acetylglucosamine 6-O-Sulfotransferase-1 Regulates Expression of L-Selectin Ligands and Lymphocyte Homing*

Kenji Uchimura{ddagger}§, Kenji Kadomatsu{ddagger}, Fathy M. El-Fasakhany{ddagger}, Mark S. Singer§, Mineko Izawa||, Reiji Kannagi||, Naoki Takeda**, Steven D. Rosen§, and Takashi Muramatsu{ddagger}{ddagger}{ddagger}

From the {ddagger}Department of Biochemistry, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan, the §Department of Anatomy, Program in Immunology, University of California, San Francisco, California 94143, the ||Program of Molecular Pathology, Aichi Cancer Center, Research Institute, Nagoya 464-8681, Japan, and the **Center for Animal Resources and Development, Kumamoto University, Kumamoto 860-0811, Japan

Received for publication, April 22, 2004 , and in revised form, June 1, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lymphocyte homing is initiated by the binding of L-selectin on lymphocytes to its ligands on high endothelial venules (HEV). Sialyl 6-sulfo Lewis X is a major capping group of L-selectin ligands. N-Acetylglucosamine (GlcNAc) 6-sulfation is essential for the ligand activity, and is catalyzed by GlcNAc 6-O-sulfotransferases (GlcNAc6STs) of which GlcNAc6ST-1 and GlcNAc6ST-2 are expressed in HEV. Here, we report that mice deficient in GlcNAc6ST-1 were impaired in the elaboration of sialyl 6-sulfo Lewis X in HEV and that an epitope of L-selectin ligands recognized by the MECA-79 anti-body was greatly reduced or abolished in the abluminal aspect of HEV. Lymphocyte homing to peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches was significantly reduced in GlcNAc6ST-1 null mice. These results demonstrate that GlcNAc6ST-1 is involved in lymphocyte homing in vivo, and indicate that GlcNAc6ST-1 and -2 play complementary roles. The importance of GlcNAc6ST-1 is particularly high-lighted by its involvement in lymphocyte homing to Peyer's patches where GlcNAc6ST-2 expression is undetectable.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lymphocytes circulate between the blood and lymphoid organs. Lymphocytes in the blood enter lymph nodes by crossing specialized vessels known as high endothelial venules (HEV)1 (1–3). Using L-selectin to bind to specific ligands on HEV, lymphocytes tether and roll along the luminal aspects of HEV, initiating a cascade of steps (2). Subsequently, rolling cells are activated by chemokines, arrested onto the HEV by integrin-mediated firm adhesion and finally transmigrate across the HEV (1, 2). L-selectin functions as a lectin-like homing receptor (4). Consistent with its C-type lectin domain in the N-terminal region (5), the HEV-expressed ligands for L-selectin identified so far are glycoproteins including Gly-CAM-1, CD34, and MAdCAM-1 (6–8). These molecules all contain O-sialomucin regions having reactivity with an adhesion-blocking monoclonal antibody called MECA-79 (9). MECA-79 staining is now regarded as a predictor of L-selectin reactivity (10).

Three modifications, i.e. sialylation (11), fucosylation (12), and sulfation (13) of L-selectin ligands are all required for optimal recognition by L-selectin. Furthermore, staining lymph nodes with specific monoclonal antibodies, G72 and G152, has identified a novel capping group known as sialyl 6-sulfo Lewis X (i.e. NeuAc{alpha}2,3Gal{beta}1,4[Fuc{alpha}1,3][SO4-6]GlcNAc) (Fig. 1) in human HEV (14). The presence of the structure in O-glycans of GlyCAM-1 (15) and human CD34 (16) has also been demonstrated. The requirement of sialyl 6-sulfo Lewis X for L-selectin binding has been established by the finding that G152 blocks the binding of lymphocytes to HEV with absolute dependence on GlcNAc-6-O-sulfation (14, 17). The importance of GlcNAc-6-O-sulfation is reinforced by the finding that the MECA-79 epitope requires this modification (17, 18) and that the full MECA-79 epitope consists of an extended core 1 O-glycan capped by Gal{beta}1,4(SO4-6)GlcNAc (Fig. 1) (19).



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  		FIG. 1.
L-selectin ligands on extended core 1 and core 2 branch structures. Sulfated O-linked chains are shown. The sulfation of Glc-NAc depicted in red is essential for recognition by L-selectin. Sialyl 6-sulfo Lewis X, a major capping group of L-selectin ligands, can cap both in core 2 and extended core 1 structures (open boxes). The epitope recognized by MECA-79 antibody is a component of the extended core 1 structure (shaded box) (19).

 
A great deal of effort has been devoted to the identification of the enzymes that catalyze sulfation at the C-6 position of Glc-NAc (i.e. GlcNAc 6-O-sulfotransferases (GlcNAc6STs)) (3, 20). The identification of enzymes involved in the synthesis of L-selectin ligands may contribute to the understanding and ultimately the treatment of inflammatory diseases. We previously cloned the first known GlcNAc6ST (GlcNAc6ST-1, also known as CHST2 and GST-2) in mouse and human (21, 22) based on sequence homology with chondroitin 6-sulfotransferase (23, 24). GlcNAc6ST-1 mRNA was shown to be expressed in the HEV of mouse lymph nodes (21). Another GlcNAc6ST, GlcNAc6ST-2 (originally named HEC-GlcNAc6ST or LSST and also known as CHST4 and GST-3), has been isolated (25, 26). GlcNAc6ST-2 is preferentially expressed in the HEV of lymph nodes (25, 26), whereas the expression of GlcNAc6ST-1 is broad (21). The cloned HEV-expressed sulfotransferases are capable of catalyzing the transfer of sulfate to C-6 of GlcNAc in recombinant L-selectin ligands (e.g. CD34, GlyCAM-1, and MAd-CAM-1) (17, 25–27). Transfection with cDNA encoding either of the two GlcNAc6STs (GlcNAc6ST-1 and GlcNAc6ST-2) contribute to the formation of sialyl 6-sulfo Lewis X and the MECA-79 epitope on the transfectants (17, 25, 26, 28). We further characterized their substrate specificities and enzymological properties employing protein A-fused GlcNAc6ST-1 and GlcNAc6ST-2 proteins. Both GlcNAc6ST-1 and GlcNAc6ST-2 can efficiently sulfate oligosaccharides based upon O-linked glycans (29), and thus both enzymes are implicated in the elaboration of L-selectin ligands in vitro.

The question of whether GlcNAc6ST-1, GlcNAc6ST-2, or both are involved in the formation of L-selectin ligands has motivated efforts to produce mice deficient in the GlcNAc6ST genes. Genetic deletion of GlcNAc6ST-2 results in the almost complete loss of the MECA-79 epitope in the luminal aspect of peripheral lymph node (PLN)-HEV and a 50% reduction of L-selectin-mediated lymphocyte homing to PLN (30), thus establishing the importance of this enzyme for lymphocyte homing in vivo. However, it is clear that there are GlcNAc6ST-2-independent ligands for L-selectin (30, 31) in lymph nodes. Furthermore, homing to Peyer's patches (PP) is not decreased in GlcNAc6ST-2 null mice compared with wild-type mice (30). To determine whether GlcNAc6ST-1 is involved in the formation of the residual ligands observed in GlcNAc6ST-2 null mice, we have generated mice deficient in the GlcNAc6ST-1 gene (Gn6st-1, also called Chst2). Here, we report that GlcNAc6ST-1 contributes to the generation of L-selectin ligands in HEV of lymph nodes and PP.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Cloning of the Mouse GlcNAc6ST-1 Gene and Construction of the GlcNAc6ST-1 Targeting Vector—The GenBankTM mouse EST data base was BLAST screened using the sequence of the mouse GlcNAc6ST-1 cDNA (21). ESTs BM947059 [GenBank] , BM231531 [GenBank] , and W83672 [GenBank] were identified to encode the 5' termini (BM947059 [GenBank] and BE985866 [GenBank] ) and 3' termini (BM231531 [GenBank] , W83672 [GenBank] , AK048226 [GenBank] , and AK038805 [GenBank] ) of the cDNA. Using the cDNA as a probe, we screened a 129 SV/J mouse genomic library (Stratagene) to obtain mouse GlcNAc6ST-1 genomic DNA. Two positive overlapping clones termed 5-1 and 6-1 contained a 16-kb and a 13-kb DNA insert, respectively (Fig. 2A). The GlcNAc6ST-1 targeting vector was constructed from a basic targeting vector (32) and fragments of GlcNAc6ST-1 genomic DNA. To delete a 1.2-kb portion of a GlcNAc6ST-1 coding exon (Sau3AI-AccI sites in Fig. 2B), a 3.7-kb XbaI/Sau3AI fragment in the 5-1 clone insert and a 2.5-kb AccI fragment in the 6-1 clone were used as the 5'-arm and the 3'-arm, respectively (Fig. 2B).



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  		FIG. 2.
Targeting strategy for the mouse GlcNAc6ST-1 locus. A, the protein-coding region of GlcNAc6ST-1 is encoded in one exon. Boxes denote exons. Solid and open boxes represent protein-coding and untranslated regions, respectively. The isolated genomic clones, 5-1 and 6-1, are indicated. Arrows show the transcription initiation sites. The sequence data for the mouse GlcNAc6ST-1 gene are available from GenBankTM/EMBL/DDBJ under accession number AB125058 [GenBank] . B, the targeting vector was constructed to replace the 3'-PAPS binding region in the GlcNAc6ST-1 catalytic domain with the neomycin resistance cassette (neo). The diphtheria toxin fragment A gene (DTA) was used for the negative screening of recombinant ES cells. Restriction enzyme sites indicated are: E, EcoRI; Xb, XbaI; Su, Sau3AI; Ac, AccI; EV, EcoRV. Boxes denote exons. C, in homozygous mice, Gn6st-1 -/-, Southern blot analysis confirmed the homologous recombination. Southern blots were prepared using EcoRI-digested genomic DNA from the F2 progeny. The fragment of AccI/AccI (0.5-kb, shown in B) DNA was used as a probe. D, total RNAs from PLN, MLN, and PP of GlcNAc6ST-1 wild-type (+/+) and null (-/-) mice were used to synthesize cDNAs. Fragments corresponding to GlcNAc6ST-1 (491 bp) and {beta}-actin (476 bp) were amplified by PCR using the cDNA preparations as templates. No expression of GlcNAc6ST-1 transcripts was detected in -/- mice organs. Specific products were not observed when reverse transcriptase (RT) was omitted from the preparation of cDNA.

 
Generation of Targeted ES Cells and Mice—Aliquots of 17 µg of ScaI-linearized targeting vector DNA were electroporated into 1 x 107 D3 ES cells (33), and homologously recombined cells were selected as described previously (34). The production of chimeric mice, which are derived from the targeted ES clones, was carried out as described previously (34). Chimeric male mice were bred with C57BL/6J mice. Then, F1 progeny were mated to yield Gn6st-1 -/- mice. The null mice were backcrossed with C57BL/6J mice more than 6 times, and mated with each other. The resulting -/- and +/+ mice were used for analysis. The animals were maintained in a barrier facility. All animal studies were approved by the Institutional Review Boards of our institutions.

Southern Blot Analysis—Southern blot analysis was performed as described previously (21) for DNA samples digested with EcoRI. The membrane was hybridized with a 0.5-kb AccI fragment corresponding to the genomic DNA downstream of the 3'-arm of the targeting vector (Fig. 2B). The homologously recombined DNA gave a 4.2-kb band, whereas the wild-type DNA gave an 8.8-kb band (Fig. 2C).

Polymerase Chain Reaction—Aliquots of ~0.5 µg of DNA were mixed with 20 µl of 1x Taq DNA polymerase buffer containing 0.2 mM of each dNTP, 1.5 mM MgCl2, 10 pmol of each primer, 2.5% (v/v) dimethyl sulfoxide, and 1 unit of Taq DNA polymerase (Invitrogen). PCR amplification was carried out at 94 °C for 3 min, with 35 cycles of 94 °C for 0.5 min, 57 °C for 1 min, and 72 °C for 1 min. To screen for homologously recombined DNA, GlcNAc6ST-1 primers were used: 5'-AAGCCTACAGGTGGTGCGAA-3' (GST2KO-F) and 5'-CAGGACTGTTAACCCGCTCA-3' (GST2KO-R). The PCR products were then separated by electrophoresis on 1% agarose gels and visualized with ethidium bromide. The wild-type allele gave a 0.5-kb band, whereas the mutated allele gave no band. Neo primers were also used: 5'-CAGCGTCTTGTCATTGGCGA-3' (Neo3) and 5'-GCTCTTCGTCCAGATCATCC-3' (Neo4). The wild-type allele gave no band, whereas the mutated allele gave a 0.6-kb band.

Reverse Transcription-PCR—Total RNA was extracted from PLN, mesenteric lymph nodes (MLN), and PP of +/+ and -/- mice using RNAZol (Tel-Test Inc., Friendswood, TX). Random hexamers and SuperScriptTM II reverse transcriptase (Invitrogen) were used to synthesize double-stranded cDNA. PCRs were performed with the cDNA and the following primers sets for GlcNAc6ST-1 (5' primer, GST2KO-F; 3' primer, GST2KO-R) and {beta}-actin (5' primer, 5'-CTTCTACAATGAGCTGCGTGTGG-3'; 3' primer, 5'-TGATGACCTGGCCGTCAGGCA-3').

Analysis of Blood and Lymphoid Organs—Peripheral blood was obtained from 12-week-old Gn6st-1 +/+ and -/- mice by cardiac puncture and placed in Microtainer tubes containing EDTA (BD Biosciences). A complete hematological profile was obtained using a Hemavet 850 automated hematological analyzer (CDC Technologies Inc., Oxford, CT). Freshly isolated lymphoid organs of 12-week-old Gn6st-1 +/+ and -/- mice were place in a cold PBS and teased with two 25-gauge needles to prepare a uniform single cell suspension. The prepared suspension was rinsed with PBS. Trypan blue negative cells were counted using a hemocytometer.

Immunofluorescence Staining—Mouse (9- to 12-week-old) tissues were embedded in the Tissue-Tek® O.C.T. compound (Sakura Finetek U.S.A., Inc., Torrance, CA) and frozen in 2-methylbutane cooled in liquid nitrogen. Cryostat sections (10 µm thick) were prepared on poly-L-lysine-coated glass slides, fixed in ice-cold acetone for 5 min, and then allowed to dry for 30 min. Sections were incubated with blocking solution (PBS containing 3% bovine serum albumin and 5% normal mouse serum) for 30 min at room temperature. Subsequently, the sections were reacted with MECA-79 (rat IgM; 1.0 µg/ml) (30) for 1 h at room temperature. The sections were washed in PBS and then incubated with Cy3-conjugated goat anti-rat IgM (1.5 µg/ml, Jackson ImmunoResearch Laboratories, Inc.) for 30 min at room temperature. After washing in PBS, sections were counterstained with Harris' hematoxylin (Sigma) and mounted in FluorSaverTM reagent (Calbiochem). All antibodies were diluted in the blocking solution described above. The digital images were recorded with a digital camera system (Axio-Cam, Carl Zeiss, Inc.) equipped with a Nikon OPTIPHOT-inverted microscope and an Epi-Fluorescence Equipment EF-D Mercury Set (Nikon).

To distinguish the MECA-79 epitope in the abluminal aspects of HEV from the epitope in the luminal aspects, a two-step in vivo/ex vivo staining approach was employed (35). Unlabeled MECA-79 (50 µg/mouse in 100 µl of PBS) was infused into the tail veins of mice and allowed to circulate for 30 min. Mice were killed and cryostat sections were prepared as described above. The MECA-79 epitope in the abluminal aspect of HEV was detected using biotinylated MECA-79 (1.0 µg/ml) (30) and Cy2-conjugated streptavidin (1.8 µg/ml, Jackson ImmunoResearch Laboratories, Inc.).

Immunohistochemical Analysis with AG223 Antibody—The AG223 monoclonal antibody (murine IgM) was produced using 6-sulfo Lewis X ceramide as the immunogen (14). On enzyme-linked immunosorbent assay, AG223 antibody reacted with 6-sulfo Lewis X ceramide, and to a lesser extent, with 6,6'-disulfo Lewis X ceramide. The AG223 antibody was not reactive to Lewis X ceramide, 6'-sulfo Lewis X ceramide, or 3'-sulfo Lewis X ceramide (21, 36). Cryostat sections of mouse lymph nodes were prepared as above. The tissue sections were immunostained with biotinylated AG223 antibody after digestion with 0.02 units/ml neuraminidase (from Arthrobacter ureafaciens, Nakarai Tesque, Kyoto Japan) in PBS (pH 7.4) at 37 °C for 30 min. The avidin-biotin complex was used with a Vectastain® ABC kit (Vector Laboratories, Burlingame, CA). The reaction was visualized with 3,3'-diaminobenzidine tetrahydrochloride followed by counterstaining with 1% methyl green (14).

In Vitro Adherence Assay—Axillary and brachial lymph nodes of 9- to 12-week-old GlcNAc6ST-1 +/+ and -/- mice were dissected and frozen in Tissue-Tek® O.C.T. compound. The assay was performed as described previously (37). Cryostat sections (10 µm thick) were adhered onto 3-well epoxy-coated slides (Carlson Scientific, Peotone, IL) and air dried for 30 min. Then, sections were fixed in 1% paraformaldehyde in 0.1 M sodium cacodylate, pH 7.3, for 20 min on ice. Sections were overlaid with 100 µl/well of a suspension (8 x 106/ml) of L-selectin-expressing lymphoma cells (i.e. 38C13) (9) in RPMI 1640 containing 25 mM HEPES and 1% bovine serum albumin. The slides were rotated (80 rpm) using a gyratory shaker. In some cases, L-selectin-expressing 38C13 cells were treated with MEL-14 antibody (rat IgG2a; 10 µg/ml) for 20 min before being added to sections. The number of cells bound per HEV was quantitated as described previously (37). MEL-14 antibody generated from hybridoma cells (American Type Culture Collection) was purified on a column of recombinant Protein G-Sepharose 4B (Zymed Laboratories Inc., South San Francisco, CA).

In Vivo Homing Assay—Lymphocyte homing in vivo was carried out based on published procedures (12, 30). Mesenteric lymphocytes from 5- to 10-week-old ICR mice were isolated and then labeled with 5 µM 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes). After being washed with PBS, the cells (1.7 x 107 in 100 µl of PBS) were injected into the tail veins of age-matched (12-week-old) Gn6st-1 +/+ and -/- mice. One hour after injection, the animals were sacrificed and secondary lymphoid organs were dissected. Lymphocytes in these organs were teased out with two 25-gauge needles. The fractional content of CMFDA-positive cells in lymphocyte suspensions of these organs was determined by flow cytometry (FACScan, BD Biosciences). For each suspension, the data on 500,000 cells were analyzed and acquired with CellQuestTM software (BD Biosciences). The data were normalized for each animal (fractional value of CMFDA-positive cells divided by the mean of the fractional values for all of the wild-type mice within the experiment). This normalization procedure allowed pooling of data from four separate experiments. To determine the contribution of L-selectin to lymphocyte homing in Gn6st-1 +/+ and -/- mice, we employed L-selectin null mice (38). Splenocytes from L-selectin null mice and wild-type mice were isolated and then labeled with CMFDA and CMRA (Molecular Probes) dyes, respectively. The cells were mixed in an equal number (3.4 x 107 cells total in 200 µl of PBS for an animal) and injected intravenously into either Gn6st-1 +/+ or -/- mice. One hour after injection, fractional contents of CMFDA-positive and CMRA-positive cells were determined in each recipient organ by flow cytometry as described previously (30).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Genomic Organization of the Mouse GlcNAc6ST-1 Gene— The full-length sequence of mouse GlcNAc6ST-1 cDNA with the 5'- and 3'-untranslated regions (7862 bp) was determined by assembling the sequence of EST clones (GenBankTM accession numbers BM947059 [GenBank] , BE985866 [GenBank] , BM231531 [GenBank] , W83672 [GenBank] , AK048226 [GenBank] , and AK038805 [GenBank] ) that are related to mouse GlcNAc6ST-1 cDNA cloned previously (21). Two mouse GlcNAc6ST-1 genomic clones (termed 5-1 and 6-1) were isolated from a 129SV/J mouse genomic library (Stratagene) using mouse GlcNAc6ST-1 cDNA as a probe. Sequence comparison between the inserted genomic DNA and GlcNAc6ST-1 cDNA indicated that the open reading frame of GlcNAc6ST-1 is encoded in one exon (Exon II, Fig. 2A), whereas the 5'-untranslated region appeared to be encoded by two exons (Exon I and Exon II, Fig. 2A) divided by a 103-bp intron (Fig. 2A). The intron features canonical mammalian GT-AG splice sites (39). Thus, the coding sequence of the mouse GlcNAc6ST-1 gene is split into 2 exons and spans 8.0 kb.

Gene Targeting of Mouse GlcNAc6ST-1—We designed a targeting construct to delete a part of the catalytic region in Exon II (Fig. 2B). The deletion resulted in loss of the whole 3'-PAPS binding domain of the sulfotransferase (40), so that any resulting translated protein would not have the ability to catalyze sulfation. Standard gene knockout technology was used to produce mice with the deletion at the GlcNAc6ST-1, Gn6st-1 -/-. Homologous recombination occurred in 2 of 60 ES clones examined. One chimeric mouse transmitted the mutation to the germ line. By intercrossing F1 Gn6st-1 heterozygous (+/-) mice, Gn6st-1 -/- mice were born in the predicted Mendelian ratio. There were no apparent differences between Gn6st-1 wild-type +/+ and -/- mice in gross morphology. The null mice reproduced normally. Southern blot analysis confirmed that the GlcNAc6ST-1 gene was deleted in the knockout mice (Fig. 2C). Reverse transcriptase-PCR analysis performed on lymph node RNA with primers corresponding to the targeted regions confirmed the absence of GlcNAc6ST-1 mRNA in -/- mice (Fig. 2D).

Leukocyte Distribution and Histology of Lymphoid Organs in GlcNAc6ST-1 Null Mice—Numbers of leukocytes, neutrophils, lymphocytes, and monocytes in the blood were unaltered in Gn6st-1 -/- mice as compared with +/+ mice (Fig. 3A). Lymphocyte numbers in spleen, thymus, axillary and brachial lymph nodes (PLN), MLN, and PP of +/+ and -/- mice were determined. Although no significant differences were observed in the organs between +/+ and -/- mice, the number of lymphocytes in -/- MLN and PP showed a tendency to be reduced (Fig. 3C).



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  		FIG. 3.
Distribution of leukocytes in blood and lymphoid organs of GlcNAc6ST-1 null mice. A, leukocytes in the blood of GlcNAc6ST-1 wild-type (+/+) and null (-/-) mice were differentially counted. An automated count was performed using a Hemavet 850 automated hematological analyzer. WBC, PMN, LY, and MO denote total white blood cell, neutrophil, lymphocyte, and monocyte counts, respectively. Six mice of each genotype were compared. B and C, lymphocytes recovered from each organ were quantitated manually using a hemocytometer. Six mice of each genotype were compared. Results are presented as mean ± S.E. Statistical analysis was performed using Student's t test. The numbers of lymphocytes obtained in -/- MLN and PP were reduced to 75 and 57% of wild-type levels, respectively, although the trends are not statistically significant (MLN, p = 0.17; PP, p = 0.23).

 
Altered Expression Pattern of MECA-79 Epitope in HEV of GlcNAc6ST-1 Null Mice—HEV, which are characterized by plump endothelial lining cells, were present in lymph nodes and PP of null -/- mice as in the case of wild-type mice (Fig. 4, A–C). Because we have previously shown the ability of GlcNAc6ST-1 to form functional L-selectin ligands in vitro (17, 29, 41), we employed MECA-79 to determine whether the expression of L-selectin ligands was perturbed in HEV of null -/- mice. Because the MECA-79 antibody recognizes a critical GlcNAc-6-sulfate modification on L-selectin ligands and has been widely used to detect the ligands (10, 19), we examined MECA-79 staining of the HEV in null mice. As shown in Fig. 4, A and B, a pericellular pattern of staining was observed in PLN- and MLN-HEV of GlcNAc6ST-1 null mice as in wild-type mice. The expression level of the other HEV-expressed GlcNAc 6-sulfotransferase, namely, GlcNAc6ST-2, was not altered in PLN- and MLN-HEV of -/- mice as revealed with an anti-HEC-GlcNAc6ST (GlcNAc6ST-2) antibody (data not shown). PP-HEV lack the expression of GlcNAc6ST-2 (35) and demonstrate only abluminal expression of the MECA-79 epitope (9). We wanted to know how the absence of GlcNAc6ST-1 would affect the MECA-79 epitope at this location. Indeed, we found that the abluminal MECA-79 epitope in HEV of Gn6st-1 PP was completely abolished (arrows in Fig. 4C). To establish that the expression of other adhesion molecules was not affected in GlcNAc6ST-1 null HEV, we stained MLN and PP for MAd-CAM-1 and found an identical pattern of HEV staining in +/+ and -/- mice (data not shown).



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  		FIG. 4.
Staining for the MECA-79 epitope in GlcNAc6ST-1 null lymphoid organs. Cryostat sections of PLN (A), MLN (B), and PP (C) from GlcNAc6ST-1 +/+ and -/- mice were stained with MECA-79 (Cy3, red) antibody. Sections were counterstained with hematoxylin. Pericellular MECA-79 staining was observed in HEV of the PLN and MLN in -/- mice (A and B). Abluminal MECA-79 staining was observed in PP-HEV of +/+ mice, whereas the signal in the -/- PP-HEV was totally abolished (C). Arrows denote HEV. Bars represent 25 µm.

 
Our observations in PP prompted us to determine whether the expression level of the abluminal MECA-79 epitope in PLN- and MLN-HEV was impaired in null mice. We employed a combined in vivo/ex vivo staining technique to discriminate luminal from abluminal epitopes. Unlabeled MECA-79 was infused into the tail veins of mice. The infused MECA-79 was allowed to bind to its epitopes in the blood-exposed regions of the HEV in vivo. Cryostat sections of the PLN and MLN of the injected mice were prepared and stained with biotinylated MECA-79 and Cy2-conjugated streptavidin to detect the abluminal epitope that was not accessible to infused MECA-79. As shown in Fig. 5D, the PLN-HEV of -/- mice exhibited very limited abluminal MECA-79 staining relative to that in wild-type mice, and MLN-HEV of -/- mice showed almost no staining (Fig. 5H). Thus, we have established that GlcNAc6ST-1 is involved in synthesis of the abluminal MECA-79 epitopes in the HEV of PLN, MLN, and PP.



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  		FIG. 5.
Staining for the abluminal MECA-79 epitope in the HEV. GlcNAc6ST-1 +/+ and -/- mice were intravenously injected with unlabeled MECA-79 antibody. The antibody infused was allowed to circulate for 30 min. Cryostat sections of PLN (A–D) and MLN (E–H) were prepared from the injected Gn6st-1 +/+ (A, B, E, and F) and -/- mice (C, D, G, and H). The MECA-79 epitope in the abluminal aspect (i.e. blood-unexposed region) of HEV was detected using biotinylated MECA-79 antibody followed by Cy2-conjugated streptavidin (green) (B, D, F, and H). Sections were counterstained with hematoxylin (A, C, E, and G). In PLN-HEV of -/- mice, the abluminal staining signal was slightly weakened (D) as compared with that of +/+ mice (B). Notably, the abluminal MECA-79 epitope in MLN-HEV of -/- mice was significantly reduced (white arrows in H) as compared with that of +/+ mice (F). Bars represent 50 µm.

 
Decreased Expression of Sialyl 6-Sulfo Lewis X in HEV of GlcNAc6ST-1 Null Mice—The sialyl 6-sulfo Lewis X structure (i.e. Sia{alpha}2,3Gal{beta}1,4[Fuc{alpha}1,3]GlcNAc) is a sialylated, fucosylated, and sulfated oligosaccharide. The sulfation modification occurs at the C-6 position of GlcNAc. We have previously established that sialyl 6-sulfo Lewis X is a major capping structure of carbohydrate L-selectin ligands in human lymph nodes as revealed with a specific monoclonal antibody, G152 (14). We therefore wished to know whether the expression level of sialyl 6-sulfo Lewis X was altered in the HEV of GlcNAc6ST-1 null mice. The G152 antibody does not stain mouse tissues probably as a result of differences in acylation of sialic acids between human and mouse.2 Therefore, we employed an anti-6-sulfo Lewis X antibody, AG223 (21, 36), in combination with sialidase treatment to determine the presence of sialyl 6-sulfo Lewis X in mouse lymph nodes. There was no reactivity of this antibody on the HEV of either Gn6st-1 +/+ or -/- mice without sialidase pretreatment (data not shown). As shown in Fig. 6, A and C, sialidasetreated GlcNAc6ST-1 +/+ PLN and MLN showed strong reactivity with AG223 in both the luminal and abluminal aspect of HEV. Sialidase-treated +/+ PP showed strong reactivity with AG223 largely in the abluminal aspect of HEV (Fig. 6E). The AG223 staining signals were markedly altered in Gn6st-1 -/- PLN, MLN, and PP. In sialidase-treated -/- PLN, the AG223 staining signals were barely detectable on abluminal HEV, whereas the signals were reduced but persisted in the luminal aspect (Fig. 6B). In sialidase-treated -/- MLN, the AG223 staining signals were greatly reduced in both the luminal and abluminal aspects of HEV (Fig. 6D). AG223 staining was totally abolished in HEV of sialidasetreated -/- PP (Fig. 6F). We conclude that GlcNAc6ST-1 participates in the formation of sialyl 6-sulfo Lewis X in mouse PLN-, MLN-, and PP-HEV. In addition, the highly significant reduction of the AG223 epitope in PLN- and MLN-HEV of GlcNAc6ST-1 null mice, which still express GlcNAc6ST-2, implies that AG223 would prefer an epitope produced by GlcNAc6ST-1 to that produced by GlcNAc6ST-2.



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  		FIG. 6.
Evaluation of expression level of sialyl 6-sulfo Lewis X in HEV of GlcNAc6ST-1 null lymph nodes. Cryostat sections of PLN (A and B), MLN (C and D), and PP (E and F) from Gn6st-1 +/+ (A, C, and E) and -/- mice (B, D, and F) were preincubated with sialidase and stained with AG223 monoclonal antibody, which recognizes the 6-sulfo Lewis X structure. The sections were counterstained with 1% methyl green. No staining signal was observed in sections that had not been treated with sialidase (data not shown). Sialyl 6-sulfo Lewis X is present in both the luminal and abluminal aspects of HEV in +/+ PLN (A), MLN (C), and PP (E). Arrowheads in B indicate the luminal staining pattern in the PLN-HEV of -/- mice. Arrows in D and F denote the reduced luminal and abluminal AG223 staining signals in the MLN-HEV (D) and PP-HEV (F) of -/- mice. Bars represent 50 µm.

 
Diminished Lymphocyte Homing to Lymph Nodes in GlcNAc6ST-1 Null Mice—Reduced MECA-79 and AG223 staining signals seen in PLN, MLN, and PP of GlcNAc6ST-1 null mice led us to assess the functional significance of GlcNAc6ST-1. We therefore employed assays of in vitro lymphocyte adherence and in vivo homing assays. Lymphocytes bind to HEV in cryostat sections of lymph nodes under shear (11, 42, 43). To determine whether the reduced level of these epitopes affects the L-selectin-dependent cell binding, we performed an adherence assay with 38C13 cells, an L-selectin-bearing mouse lymphoma cell line (9, 42). The binding of 38C13 cells to PLN-HEV of GlcNAc6ST-1 null mice was decreased to 86% (p = 0.032) of +/+ mice (Fig. 7A). The binding of cells to PLN-HEV of Gn6st-1 +/+ and -/- mice was completely inhibited by an anti-mouse L-selectin antibody (i.e. MEL-14) (Fig. 7A). Consistent with a previous report (30), the PLN-HEV of GlcNAc6ST-2 null mice showed greatly reduced binding of these cells (Fig. 7A).



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  		FIG. 7.
In vitro adhesion and in vivo homing of lymphocytes in GlcNAc6ST-1 null mice. A, cryostat sections of the PLN from wild-type, GlcNAc6ST-1 null, and GlcNAc6ST-2 null mice (30) were overlaid with mouse 38C13 cells with or without MEL-14. After incubation for 30 min under shear, cells that had attached to HEV in the sections were counted. The mean number of bound cells per HEV was determined. The error bars indicate S.E. derived from the analysis of three independent sections per treatment. Statistical analysis was carried out using Student's t test. *, p < 0.05; **, p < 0.0001. B, mouse mesenteric lymphocytes labeled with a fluorescent dye (CMFDA; Molecular Probes) were injected into the tail veins of age-matched Gn6st-1 +/+ or -/- mice. After 1 h, CMFDA-positive lymphocytes in the PLN, MLN, PP, and spleen of each mouse were analyzed by flow cytometry. The number of positive cells was determined as a percentage of total lymphocytes in each organ. Homing in the null animals is shown as a fraction of that obtained in the wild-type animals. Data were pooled from four separate experiments. Eleven +/+ mice and 10 -/- mice were used. Error bars denote S.E. Statistical analysis was performed using Student's t test. *, p < 0.05. The result observed in the spleen was not significantly different between +/+ and -/- mice.

 
Because the interaction between L-selectin and its ligands on HEV is essential for lymphocyte homing to secondary lymphoid organs (12, 30, 44), we performed short term homing assays in GlcNAc6ST-1 null mice. We injected fluorescently labeled mouse mesenteric lymphocytes into the tail veins of either Gn6st-1 +/+ or -/- mice. After 1 h, the number of labeled cells in each lymphoid organ was determined by flow cytometry as the ratio of fluorescent lymphocytes to total number of resident lymphocytes (30). A normal level of homing to spleen was observed in the -/- mice (Fig. 7B). Lymphocyte homing to PLN and MLN of -/- mice was diminished to 78 (p = 0.016) and 71% (p = 0.011) of +/+ levels, respectively (Fig. 7B). Moreover, homing to PP of -/- mice was reduced to 67% (p = 0.049) of the +/+ level. We verified the L-selectin contribution to homing in GlcNAc6ST-1 null mice by employing L-selectin null lymphocytes in homing assays. As shown in Table I, the remaining homing to PLN, MLN, and PP in GlcNAc6ST-1 null mice retained the similar quantitative dependence on L-selectin as in wild-type recipients.


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  		TABLE I
Relative homing of L-selectin -/- versus L-selectin +/+ lymphocytes to lymphoid organs of GlcNAc6ST-1 wild-type and null mice Splenocytes isolated from L-selectin +/+ and L-selectin -/- mice were fluorescently labeled with CMRA and CMFDA dyes (Molecular Probes), respectively. The labeled cells were mixed in an equal number and injected intravenously into either 7 GlcNAc6ST-1 wild-type or 7 null mice. Fractional contents of CMRA-positive and CMFDA-positive cells in each recipient organ were determined by flow cytometry. The ratio of the two fractions yielded the relative homing, which corresponds to the fraction of homing that was L-selectin independent. Means of the ratios ± S.E. are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
L-selectin is the major factor regulating the initial step of lymphocyte homing. The contribution of L-selectin-dependent homing to the homing overall is >95% in PLN, >85% in MLN, and about 50% in PP (30, 44). GlcNAc6ST-2, which is preferentially expressed in HEV (25, 26), has been established to participate in L-selectin-dependent lymphocyte homing (30). In mice deficient in GlcNAc6ST-2, lymphocyte homing to PLN and MLN is reduced to 50 and 69% compared with wild-type mice, and homing to PP is unchanged (30). Whereas most of luminal MECA-79 reactivity was eliminated in PLN and MLN of the null mice, considerable abluminal MECA-79 reactivity remained. In PP of the deficient mice, all of the abluminal staining persisted. These results raised many questions; the most important issue of which was the identity of the GlcNAc6ST involved in the formation of residual L-selectin ligands in the deficient mice.

GlcNAc6ST-1 null mice showed a significant decrease in lymphocyte homing to PLN (78% of wild-type) and MLN (71% of wild-type). Applying an in vitro adhesion assay to PLN of null mice, we found a decrease in binding to HEV, consistent with the homing results. A complete inhibition of cell adhesion to either +/+ or -/- HEV was revealed using an anti-L-selectin antibody. This result supports the conclusion that the homing defect in GlcNAc6ST-1 null mice is because of the defect of interaction between L-selectin and its ligands. Because GlcNAc6ST-1 is not involved in synthesis of heparan sulfate or chondroitin sulfates (21), it is not likely that GlcNAc6ST-1 deficiency affects lymphocyte homing by effects on glycosaminoglycans, which present and stabilize chemokines. Taken together, we conclude that GlcNAc6ST-1 is involved in lymphocyte homing to lymph nodes by contributing to the synthesis of L-selectin ligands in HEV. Apparently, GlcNAc6ST-1 and GlcNAc6ST-2 play complementary roles. It is highly likely that GlcNAc6ST-1 is one of the GlcNAc6ST responsible for the formation of L-selectin ligands in lymph nodes of GlcNAc6ST-2 null mice.

We also found that in GlcNAc6ST-1 null mice, homing to PP was reduced to 67% of that in wild-type mice. Furthermore, we observed the disappearance of epitopes associated with L-selectin ligands from PP of GlcNAc6ST-1 null mice (see below). Because homing to PP is unchanged in GlcNAc6ST-2 null mice (30), GlcNAc6ST-1 is the first GlcNAc6ST identified as an enzyme regulating homing to PP.

We analyzed the distribution of L-selectin ligands in GlcNAc6ST-1 null mice using two antibodies, MECA-79 and AG223. MECA-79 antibody has been widely used to detect L-selectin ligands in lymph node HEV (10). The complex of MECA-79-reactive molecules is called "peripheral nodes addressin," i.e. PNAd. The full epitope of MECA-79 was recently identified as an extended core 1 O-glycan that is indifferent to sialylation or fucosylation, but shows an absolute requirement for GlcNAc-6-sulfation (19) (Fig. 1). Using a two-step in vivo/ex vivo staining approach, we found reduced abluminal MECA-79 reactivity in both PLN and MLN of GlcNAc6ST-1 null mice with the most pronounced effect on the latter. Furthermore, the abluminal epitope observed in wild-type PP (9) was eliminated in GlcNAc6ST-1 null PP. In view of the results on GlcNAc6ST-2 null mice (30, 31), we conclude that both GlcNAc6ST-1 and GlcNAc6ST-2 contribute to the synthesis of the abluminal MECA-79 epitope in PLN and MLN, and it is most likely that only GlcNAc6ST-1 is involved in formation of the abluminal epitope in PP (Table II). This latter finding is consistent with the absence of GlcNAc6ST-2 protein in PP HEV (35). The function of the abluminal L-selectin ligand is not presently known. In recent studies, it has been proposed that L-selectin may be involved in postextravasation migration events in tissues (45, 46). Further analysis of GlcNAc6ST-1 null mice will help to clarify the role of the abluminal L-selectin ligand, especially in PP.


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  		TABLE II
Contribution of GlcNAc6ST-1 and GlcNAc6ST-2 (35) to the synthesis of two L-selectin ligand determinants in mouse secondary lymphoid organs

 
A major capping structure of L-selectin ligands has been identified to be sialyl 6-sulfo Lewis X, in which sialylation, fucosylation, and GlcNAc-6-sulfation occur at N-acetyllactosamine (14, 15). In GlcNAc6ST-1 null lymph nodes, the amount of luminal sialyl 6-sulfo Lewis X was decreased, whereas there was no detectable effect on luminal staining by MECA-79. Although MECA-79 recognizes sialyl 6-sulfo Lewis X on core 1-extended O-glycans, it does not recognize this determinant on core 2-branched O-glycans (19) (Fig. 1). Therefore, it is conceivable that the reduction of the luminal sialyl 6-sulfo Lewis X in GlcNAc6ST-1 null lymph nodes was because of the reduction of the structure on core 2-branched glycans (Fig. 1). This point is important in considering the nature of L-selectin ligands remaining in GlcNAc6ST-2 null mice. In these mice, the reactivity to MECA-79 in the luminal aspect of PLN- and MLN-HEV is almost completely abolished. Therefore, the remaining L-selectin ligand is likely to be unreactive with MECA-79 (31). Consequently, we propose that a portion of the L-selectin ligand remaining in GlcNAc6ST-2 null mice is on core 2 glycans, the sulfation of which is mediated by GlcNAc6ST-1. Consistent with the possibility, de Graffenried and Bertozzi (28) found that GlcNAc6ST-1 preferably modifies the capping group on the core 2 branch rather than that on core 1-extended glycans. Furthermore, Hiraoka et al. (47) observed that mice deficient in both GlcNAc6ST-2 and C2GlcNAcT exhibit a greater reduction in lymphocyte homing than GlcNAc6ST-2 single knockout mice. The ability of GlcNAc6ST-1 to form the MECA-79 epitope in the abluminal aspect of HEV suggests that it also contributes to some extent to the formation of the epitope in the luminal aspect. However, this contribution appears to be less than that of GlcNAc6ST-2 (Table II).

The L-selectin ligands in lymphocyte homing to PP are MECA-79-unreactive because the pattern of MECA-79 staining in HEV of PP is abluminal and injection of the antibody does not inhibit short term homing (9). It is likely that sialyl 6-sulfo Lewis X on core 2 glycans plays a role as an L-selectin ligand in PP. Consistent with this proposal, we found that sialyl 6-sulfo Lewis X exists on the luminal as well as abluminal aspects of HEV in mouse PP (Fig. 6E). The reduction in lymphocyte homing to PP observed in GlcNAc6ST-1 null mice is consistent with the loss of the luminal sialyl 6-sulfo Lewis X in the null PP. However, L-selectin ligand activity remains in PP-HEV of GlcNAc6ST-1 null mice (~38% of total homing, Table I), indicating the existence of additional ligands.

We also noticed that the luminal expression of sialyl 6-sulfo Lewis X in HEV of PLN and MLN was greatly reduced in GlcNAc6ST-1 null mice, whereas the reduction in lymphocyte homing and 38C13 cell adhesion was much less. It is conceivable that AG223 prefers 6-sulfo Lewis X on a core 2 branch, which could explain the above discrepancy. It is also possible that a large portion of the sialyl 6-sulfo Lewis X epitope in PLN and MLN is on structures that are not functional as L-selectin ligands (26); such structures might include N-linked oligosaccharides and sparsely distributed O-glycans.

Future efforts to analyze mice that are doubly deficient in GlcNAc6ST-1 and GlcNAc6ST-2 genes will provide essential information on overall the contribution of GlcNAc-6-sulfation to L-selectin-mediated homing. Furthermore, the mice could be utilized to investigate mechanisms of inflammation-associated lymphoid neogenesis in which MECA-79-positive vessels are prominent (35, 48).


   FOOTNOTES
 
* This work was supported in part by a grant-in-aid for Priority Area (14082202) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Mizutani Foundation for Glycoscience (to T. M.), and National Institutes of Health Grants GM57411 and R37GM23547 (to S. D. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Research Fellow of the Japan Society for the Promotion of Science. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumaicho, Showa-ku, Nagoya 466-8550, Japan. Tel.: 81-52-744-2-59; Fax: 81-52-744-2065; E-mail: tmurama{at}med.nagoya-u.ac.jp.

1 The abbreviations used are: HEV, high endothelial venules; GlcNAc, N-acetylglucosamine; GlcNAc6ST, N-acetylglucosamine-6-O-sulfotransferase; PAPS, adenosine 3'-phosphate 5'-phosphosulfate; Lewis X, Gal{beta}1–4(Fuc{alpha}1–3)GlcNAc; 6-sulfo Lewis X, Gal{beta}1–4(Fuc{alpha}1–3)(SO4-6)GlcNAc; sialyl 6-sulfo Lewis X, NeuAc{alpha}2–3Gal{beta}1–4(Fuc{alpha}1–3)(SO4-6)GlcNAc; core 1, Gal{beta}1–3GalNAc; core 2, GlcNAc{beta}1–6(Gal{beta}1–3)GalNAc; PLN, peripheral lymph nodes; MLN, mesenteric lymph nodes; PP, Peyer's patches; PBS, phosphate-buffered saline; CMFDA, 5-chloromethylfluorescein diacetate; Gn6st-1, GlcNAc6ST-1 gene, also called Chst-2. Back

2 M. Izawa and R. Kannagi, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Durwin Tsay and Hideki Muramatsu for excellent technical support, and Drs. Annemieke van Zante and Annette Bistrup for helpful comments and discussion. One of us (F. M. E.) thanks Tanta University, Tanta, Egypt, for a leave of absence.



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 RESULTS
 DISCUSSION
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