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(Journal of Leukocyte Biology. 2001;69:565-574.)
© 2001 by Society for Leukocyte Biology

CHST1 and CHST2 sulfotransferase expression by vascular endothelial cells regulates shear-resistant leukocyte rolling via L-selectin

Xuan Li, LiLi Tu, Patricia G Murphy, Takafumi Kadono, Douglas A Steeber and Thomas F. Tedder

Department of Immunology, Duke University Medical Center, Durham, North Carolina

Correspondence: Thomas Tedder, Box 3010, Department of Immunology, Room 353 Jones Building, Research Drive, Duke University Medical Center, Durham, NC 27710. E-mail thomas.tedder{at}duke.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sulfation is an essential component of the selectin ligands, potentially mediated by members of a new family of carbohydrate sulfotransferases. In this study, we assessed the contributions of CHST1, CHST2, CHST3, and CHST4 in producing functional L-selectin ligands. Human umbilical vein endothelial cells predominantly expressed CHST1 and CHST2 transcripts with low levels of CHST3 mRNA, while cytokine activation up-regulated CHST2 expression and induced low-level CHST4 expression. A human umbilical vein endothelial cell line, EA.hy926, displayed functional L-selectin ligands that correlated with CHST1 and CHST2 expression in the absence of CHST4 expression. Increased CHST1 or CHST2 expression by a cell line expressing low-level L-selectin ligand activity during in vitro flow chamber assays increased rolling leukocyte numbers, reduced rolling velocities, and enhanced leukocyte rolling under higher shear stresses. These results suggest that CHST1 and CHST2 contribute to the generation of optimal L-selectin ligands in vascular endothelial cells at sites of inflammation.

Key Words: inflammation • vascular biology • leukocyte adhesion • cell trafficking • adhesion molecules


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocyte recruitment to sites of inflammation depends on interactions between adhesion molecules and the ligands expressed by leukocytes and vascular endothelial cells. An early event in leukocyte transmigration is leukocyte capture and rolling on vascular endothelial cells, which is mediated in part by L-selectin [1 ]. L-Selectin ligands are constitutively expressed by high endothelial venules (HEVs) [2 ] and are induced on vascular endothelium by inflammatory cytokines [3 4 5 6 7 ]. L-Selectin ligands are sialylated, fucosylated, and sulfated O-linked carbohydrates displayed in the proper context on a limited number of glycoproteins or proteoglycans. Although mucin ligands for L-selectin have been identified on HEVs [8 9 10 11 ], the vascular L-selectin ligands remain to be defined. L-Selectin ligand activity also requires fucosylation of appropriate carbohydrate determinants, with fucosyltransferase VII (FucT-VII) dominating during selectin ligand generation by HEV and vascular endothelial cells [12 13 14 ]. Cytokine activation of human umbilical vein endothelial cells (HUVECs) up-regulates FucT-VII and HECA-452 antigen expression and induces L-selectin ligand activity [14 ]. Enhanced FucT-VII expression in EA.hy926, an HUVEC cell line [15 ], generates L-selectin ligands that support leukocyte rolling and attachment [14 , 16 ]. Vascular L-selectin ligands induced by FucT-VII expression predominantly involve the subset of sialyl Lewisx (sLex) tetrasaccharides identified by the HECA-452 and 2H5 monoclonal antibodies (mAbs) [14 , 17 ]. The HECA-452 mAb abrogates L-selectin-dependent leukocyte rolling under physiological-flow conditions, although other anti-sLex mAbs such as CSLEX-1 do not [14 , 16 ].

L-Selectin ligands on HEVs characteristically bear a sulfate-dependent carbohydrate epitope defined by the MECA-79 mAb [18 19 20 21 22 23 ]. Others have proposed that sialyl 6-sLex and 6'-sulfo-sLex, sulfated derivatives of the sLex tetrasaccharide, are structural components for L-selectin ligand recognition [23 , 24 ]. However, vascular endothelial cells do not normally express the MECA-79 antigen [14 ]. Nonetheless, sulfation of L-selectin ligands on vascular endothelial cells is required because chlorate treatment of cytokine-activated HUVEC and FucT-VII cDNA-transfected EA.hy926 cells (926-FtVII cells) eliminates L-selectin-mediated leukocyte attachment [14 ]. Membrane-bound sulfotransferases potentially involved in the generation of selectin ligands have been identified recently [25 26 27 28 29 30 31 32 33 ]. This new family of membrane-bound enzymes currently includes CHST1 (chondroitin 6-sulfotransferase, keratan sulfate galactose-6-sulfotransferase, KSST), CHST2 (6-sul-T and N-acetylglucosamine-6-O-sulfotransferase), CHST3 (chrondroitin 6-sulfotransferase), and CHST4 (HEC-GlcNAc6ST). Among this family of enzymes, CHST4 is reported to be specifically expressed by HEVs, while CHST2 is highly expressed by mouse HEVs [27 , 29 , 33 ]. CHST1, CHST2, and CHST3 are expressed by a broad range of other tissues [25 , 26 , 28 , 30 31 32 , 34 , 35 ]. However, the expression of the CHSTs by vascular endothelial cells and their involvement in the biosynthesis of vascular L-selectin ligands remain unclear. Therefore, we assessed the role of CHSTs in generating vascular L-selectin ligands.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell lines
EA.hy926 cells were provided by C.-J. Edgell (University of North Carolina at Chapel Hill, NC). ECV-304 cells were from the American Type Culture Collection (ATCC; Manassas, VA). Both adherent cell lines were cultured in Dulbecco’s modified Eagle’s medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS) (Sigma, St. Louis, MO) and penicillin/streptomycin (Life Technologies). HeLa, SVEC4-10, HL-60, and COS-7 cells were obtained from ATCC and were cultured as recommended. Initial reports indicated that ECV-304 is a spontaneously transformed human endothelial cell line. More recent findings indicate that ECV-304 cells have a short-terminal repeat microsatellite fingerprint and marker chromosomes in common with the human bladder cancer line T-24 and are therefore a variant of T-24. Mouse pre-B-cell line 300.19 stably transfected with L-selectin (300-LAM) was described previously [16 , 36 , 37 ]. HUVECs were isolated and cultured as previously described [3 , 14 ]. In some cases, HUVECs were stimulated with 100 U/mL of recombinant human tumor necrosis factor (TNF)-{alpha} (Genzyme, Cambridge, MA) for 6 h.

Antibodies and Flow Cytometry Analysis
The anti-L-selectin (LAM1-3) mAb was produced and purified as described elsewhere [36 , 38 ]. The HECA-452 (anti-CLA), CSLEX-1 (anti-sLex), and MECA-79 mAb-producing hybridomas were obtained from the ATCC. Reactivity of the MECA-79 mAb was verified by staining HEVs of mouse lymph node tissue sections using standard immunochemical techniques (unpublished results). The anti-PSGL-1 (PL-1; K. Moore, University of Oklahoma Health Science Center, Oklahoma City, OK), anti-CD57 (HNK-1; M. Cooper, University of Alabama in Birmingham, Birmingham, AL), anti-P-selectin (G1; B. Furie, Beth Israel Medical Center, Boston, MA), and anti-VAP1 (TK8-14; S. Jalkanen, University of Turku, Turku, Finland) mAbs were generous gifts. The mouse mAbs to human E-selectin (HAE-1f), vascular cell adhesion molecule 1 (VCAM-1 [HAE-2d]), and intercellular adhesion molecule 1 (ICAM-1 [HAE-4b]) were produced as previously described [3 ]. The anti-CD34 mAb (My10; Becton Dickinson, San Jose, CA), anti-mouse CD25 mAb (rat immunoglobulin M [IgM]), fluorescein isothiocyanate-conjugated goat anti-rat IgM (anti-rat Ig [H + L]), and anti-mouse IgM antibodies were purchased from Southern Biotechnology Associates (Birmingham, AL). All mAbs were used at predetermined optimal concentrations for indirect immunofluorescence staining. Staining and immunofluorescence analysis were performed as previously described with a FACScan flow cytometer (Becton Dickinson) [14 , 16 ].

CHST and FucT-VII cDNA expression vectors
A CHST1 cDNA clone [28 ] was amplified by polymerase chain reaction (PCR) using the 5'-GCT CTA GAC ATG CAA TGT TCC TGG AAG-3' and 5'-CCC TCG AGT CAC GAG AAG GGG CGG AAG-3' primer set. A CHST2 cDNA clone [28 ] was PCR amplified using the 5'-GCT CTA GAA TGA GCC GCA GCC CGC AGC GA-3' and 5'-GCG TCG AGA ACC CCT TTT AGA GAC GGG G-3' primer set. The PCR products were cloned into the BlueScript vector (Stratagene, La Jolla, CA) and sequenced. The CHST1 and CHST2 coding regions were excised using the XbaI and XhoI restriction enzymes and cloned into the pcDNA3.1(-) vector (Invitrogen, Carlsbad, CA) at the appropriate sites. An internal ribosome entry site (IRES)-ß-gal fusion gene (from Y. Zhuang, Duke University Medical Center, Durham, NC) was then inserted into the XhoI site downstream of the CHST1 or CHST2 coding region, resulting in new vector pcDNAIßST1 or pcDNAIßST2. A FucT-VII cDNA (from B. Weston, University of North Carolina at Chapel Hill, NC) was modified and used as described elsewhere [14 ].

Generation of cDNA-transfected cell lines
EA.hy926 cells transfected with FucT-VII cDNA (926-FtVII) were generated previously [14 ]. In this study, EA.hy926 and ECV-304 cells were transfected with either a FucT-VII cDNA expression vector or a pcDNAIßST vector or with both vectors together using the calcium phosphate method [3 , 14 ]. Transfected cells were selected in Dulbecco’s modified Eagle’s medium containing 10% FCS and G418 (0.5 mg/mL; Sigma). Surviving cells expressing the HECA-452 antigen or ß-gal were isolated by fluorescence-based cell sorting using a FACStar flow cytometer (Becton Dickinson). Cells transfected with both FucT-VII and CHST cDNAs were first sorted based on high levels of HECA-452 antigen expression. Subsequently, cells were sorted based on ß-gal expression using the fluorescein di-ß-D-galactopyranoside (FDG; Molecular Probes, Eugene, OR) ß-gal substrate as previously described [39 ]. Briefly, the cells (1 x 106) were resuspended in 50 µL of phosphate-buffered saline (PBS) at 37°C. FDG solution (50 µL; 2 mmol in 1% (v/v) dimethyl sulfoxide) at 37°C was added to the cell suspension and mixed quickly. After incubation at 37°C for 2.5 min, 10 v of ice-cold PBS containing 5% FCS were added to stop FDG loading. The cells were maintained on ice for 30–60 min until flow cytometry analysis or cell sorting. In additional experiments, cells transfected with both FucT-VII and CHST cDNAs were isolated first based on increased ß-gal activity, with subsequent isolation based on HECA-452 antigen expression. These cells were phenotypically and functionally identical to the comparable cells isolated as first described above (unpublished data).

Physiologic shear flow assays
In vitro flow chamber assays were performed as previously described [16 ]. Monolayers of EA.hy926 cells, ECV-304 cells, and their cDNA-transfected counterparts were grown to confluence on 25-mm circular glass coverslips and mounted in a parallel-plate flow chamber. 300-LAM cells (106 cells/mL) in flow medium (PBS containing Ca2+-Mg2+ and 0.5% bovine serum albumin) were drawn through the chamber with a syringe pump (Harvard Apparatus, Natick, MA) at defined flow rates. Wall shear stress was varied at 1-min intervals by changing the flow rate through the flow chamber. For antibody-blocking experiments, 300-LAM cells (in flow medium) were incubated with the LAM1-3 mAb (10 µg/mL) at room temperature for 15 min before a fivefold dilution with flow medium. Rolling cells were observed using an inverted phase-contrast microscope (Olympus Corporation, Lake Success, NY) and videotaped using a charge-coupled device video camera (Hitachi Denshi, Ltd., Tokyo, Japan), a Sony SuperVHS video recorder (model SVO-9500MD; Sony Corp. of America, New York, NY), and an attached time-date generator (Microimage Video Systems Co., Bechtelsville, PA). Interacting cells (tethering and rolling) were quantified at each shear rate by analysis of videotapes in which 300-LAM cells interacting with the adherent cell monolayer were counted in 16 randomly chosen 0.16-mm2 fields near the center line of the flow chamber. For calculating velocities, the distance that individual rolling cells traveled between two time points was measured, converted into actual distance, and divided by the elapsed time.

RNA isolation and Northern blot analysis
Cytoplasmic RNA free of DNA contamination was isolated from each cell type using an RNeasy Mini Kit (Qiagen, Inc., Chatsworth, CA) according to the manufacturer’s instructions. RNA concentrations were determined by ultraviolet absorbance. RNA (30 µg/lane) was electrophoresed through 1% agarose-formaldehyde gels and transferred to nitrocellulose filters. CHST1, CHST2, and ß-actin probes were generated by labeling PCR-generated DNA fragments with [{alpha}-32P]dATP using the Prime-a-Gene® labeling system with random hexadeoxynucleotide primers (Promega, Madison, WI). After overnight hybridization at 65°C, the filters were washed once at room temperature for 10 min in 1x saline sodium citrate containing 0.1% sodium dodecyl sulfate and twice at 60°C for 20 min in 0.1x saline sodium citrate containing 0.1% sodium dodecyl sulfate. Hybridization was visualized using a Storm 680 Phosphorimager (Molecular Dynamics, Sunnyvale, CA).

PCR analysis of sulfotransferase expression
Semiquantitative reverse transcription (RT)-PCR analysis was performed using equal amounts of RNA (~2 µg) from each cell type for RT reactions as described elsewhere [35 , 40 ]. Briefly, cDNA over a range of dilutions (neat to 1:100) was PCR amplified using predetermined optimal conditions. PCR amplification of CHST1 cDNA used sense 5'-CAC GCG CAG CGG CTC CTC CTT CGT-3' and antisense 5'-GCC AGG TCC TCG TAG CGC ACC G-3' primers to generate a 713-bp fragment. CHST2 amplification used sense 5'-GGG CGC AAC CTC ACC ACG-3' and antisense 5'-CCA CGA AAG GCT TGG AGG AGG-3'—primers to generate a 690-bp fragment. CHST3 amplification used sense 5'-CAA CCA GCA GGG CAA CAT CT-3' and antisense 5'-CCC TAC GTG ACC CAG AAG G-3' primers to generate a 980-bp fragment. CHST4 amplification used sense 5'-GTG GTG GAG AAG GCC TGC CG-3' and antisense 5'-ACC CTC TTA GTG GAT TTG CT-3' primers to generate a 680-bp fragment. Primers for ß-actin were sense 5'-ATG TTT GAG ACC TTC AAC AC-3' and antisense 5'-CAC GTC ACA CTT CAT GAT GG-3', which generate a 495-bp fragment. Primers for hypoxanthine phosphoribosyltransferase (HPRT) were sense 5'-CCT GCT GGA TTA CAT CAA AGC AC-3' and antisense 5'-TCC AAC ACT TCG TGG GGT CCT-3', which generate a 300-bp fragment. As controls, RNA from each cell type equal to the amount of RNA used for cDNA generation was used in PCR reactions without RT. PCR signals were not detected in any of the control reactions, ruling out the possibility that the PCR products were generated from contaminating genomic DNA.

PCR conditions for the CHSTs were 94°C for 2 min, then 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 40 s. PCR conditions for ß-actin were 94°C for 2 min, then 25 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 30 s. PCR conditions for HPRT were 94°C for 2 min, then 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 20 s. PCR products were electrophoresed on agarose gels and visualized by ethidium bromide staining. Relative PCR product concentrations were assessed by scanning densitometry and analyzed using NIH Image software (version 1.60). The relative intensities of the HPRT bands generated from each cDNA dilution were used to generate dose-response curves for each cDNA aliquot. In each case, the intensity of PCR products generated at a 1:10 or 1:20 dilution of cDNA was in the midpoint of the linear range where band intensity was proportional to the amount of input cDNA. RNA isolated from HL-60 and COS cells served as positive controls for CHST1, CHST2, CHST3, and CHST4 amplification by PCR as previously described [35 ].

Statistical analysis
Data were expressed as means ± SE. The Student’s t-test was used to determine the significance of differences in sample population means.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CHST expression by vascular endothelial cells
Because sulfate is a critical component of vascular L-selectin ligands, we assessed CHST expression by vascular endothelial cells, L-selectin ligand-expressing lymphoblastoid cell lines, and COS cells. CHST expression by HUVECs, the EA.hy926 HUVEC cell line, the SVEC4-10 mouse endothelial cell line, ECV-304 cells, HeLa cells, and HL-60 cells was assessed by Northern blot analysis and semiquantitative RT-PCR. By Northern blot analysis, EA.hy926, ECV-304, and SVEC4-10 cells expressed higher levels of CHST1 mRNA than the other cell types (Fig. 1A ). EA.hy926 and HL-60 cells expressed the highest levels of CHST2 mRNA, although all cells expressed CHST2 transcripts (Fig. 1A) . CHST1 and CHST2 transcripts varied in size depending on the cell source, as previously described [28 ].



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  		Figure 1. Expression of CHST transcripts by vascular and nonvascular cells. For Northern blot analysis of CHST1 and CHST2 expression by cell lines, filters bearing ~30 µg of RNA from each cell type were hybridized with radiolabeled CHST1, CHST2, or ß-actin cDNA probes (A). Hybridization was visualized using a phosphorimager. Ribosomal RNA migration is shown as an indicator of size. To analyze CHST expression by resting and activated HUVECs, we performed semiquantitative RT-PCR amplification of total RNA prepared from equivalent numbers of HUVEC cells cultured in medium alone or containing TNF-{alpha} (100 U/ml) for 6 h (B). CHST expression by EA.hy926 and ECV-304 cells was analyzed as follows (C): for controls, HPRT (not shown) or ß-actin primers were used to amplify both mRNA (ß + RNA negative control) and its cDNA product (ß + cDNA positive control). Semiquantitative RT-PCR products were visualized by electrophoresis and ethidium bromide staining and represent results from four separate experiments. The migration of standard size markers is shown.

 
Expression of the CHSTs by resting and cytokine-activated HUVECs was too low to be detected by Northern blot analysis (data not shown) and was therefore assessed by RT-PCR analysis (Fig. 1B) . CHST1, CHST2, and a low level of CHST3 mRNAs were detected in resting HUVECs, whereas CHST4 transcripts were not amplified. TNF-{alpha} treatment enhanced CHST2 mRNA expression by 2.8-fold (n = 2) and induced expression of CHST4 mRNA, whereas CHST1 and CHST3 mRNA levels did not change significantly. In addition, EA.hy926 cells consistently expressed three- to fivefold-higher levels of CHST1 and CHST2 transcripts than did ECV-304 cells, whereas CHST3 mRNA expression was equivalent in both cell lines (n = 2; Fig. 1C ). CHST4 transcripts were not detected in either cell line. Thus, vascular endothelial cells may express multiple CHSTs, and inflammatory mediators may influence their transcription.

EA.hy926 and ECV-304 cells express functional L-selectin ligands
Functional L-selectin ligand expression by EA.hy926 and ECV-304 cells was quantified by assessing their ability to support rolling of 300.19 cells stably transfected with L-selectin cDNA (300-LAM) during in vitro flow chamber assays [16 ]. 300-LAM cells were used for these studies, because native 300.19 cells do not attach to or roll on EA.hy926 and ECV-304 cells or cytokine-activated endothelial cell monolayers [14 , 16 , 41 ]. Moreover, the use of 300.19 cells allows examination of L-selectin function in isolation since 300.19 cells do not express most of the known vascular adhesion molecules [42 , 43 ].

Neither EA.hy926 nor ECV-304 cells supported 300-LAM cell rolling (Fig. 2B ), consistent with their lack of HECA-452 antigen expression (Fig. 2A , Table 1 ). However, transfection of EA.hy926 and ECV-304 cells with FucT-VII cDNA (resulting in 926-FtVII and 304-FtVII cells, respectively) induced HECA-452 antigen expression (Fig. 2A , Table 1 ) and 300-LAM cell rolling (Fig. 2B) . The majority of 300-LAM cells in contact with 926-FtVII or 304-FtVII monolayers remained in contact for their entire transit across the field of view, and there was no stationary adhesion of 300-LAM cells to the cell monolayers. 300-LAM cells rolled at velocities significantly lower than the theoretical velocity of a lymphocyte not interacting with the endothelial cell surface (526 µm/s at 1.85 dynes/cm2) [44 ]. 926-FtVII cells supported leukocyte tethering and rolling over a broad range of shear stress levels (0.25–2.5 dynes/cm2), while 304-FtVII cells supported tethering and rolling only within a narrow range of shear stress levels (0.25–1.25 dynes/cm2). Similarly, 304-FtVII cells supported the rolling of significantly fewer 300-LAM cells when compared with 926-FtVII cells at the same shear stresses. 300-LAM cell rolling on 926-FtVII and 304-FtVII cells was completely abolished by blocking L-selectin function with the LAM1-3 mAb (Fig. 2B) . Therefore, 926-FtVII cells appeared more efficient than 304-FtVII cells in supporting transient interactions (tethering) between L-selectin and its ligands and in mediating sustained rolling. It is unlikely that this results from FucT-VII expression differences by 926-FtVII and 304-FtVII cells because both cell lines displayed similar HECA-452 antigen levels (Fig. 2A , Table 1 ). In addition, 926-FtVII and 304-FtVII cells were similar in their lack of expression of other reported ligands for L-selectin and other adhesion receptors (Table 1) . Therefore, the differential abilities of 926-FtVII and 304-FtVII cells to support L-selectin-mediated leukocyte rolling suggested a correlation between L-selectin ligand activity and endogenous cellular expression of CHST1 and CHST2 transcripts.



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  		Figure 2. EA.hy926 and ECV-304 cells expressed functional L-selectin ligands after transfection with FucT-VII cDNA. HECA-452 antigen expression by 926, 304, 926-FtVII, and 304-FtVII cells was assessed by indirect immunofluorescence staining with flow cytometry analysis (A). Solid lines indicate HECA-452 mAb staining, while isotype-matched control mAb staining is indicated by dashed lines. Flow cytometry histograms are shown on a four-decade log scale. To examine the tethering and rolling of 300-LAM cells on monolayers of EA.hy926, ECV-304, 926-FtVII and 304-FtVII cells, flow was initiated in these in vitro parallel-plate flow chamber assays at 3.0 dynes/cm2, and shear stress was reduced as indicated at 1-min intervals by changing the flow rate through the flow chamber (B). 300-LAM cells preincubated with either medium or the LAM1-3 mAb (10 µg/mL) were diluted to 106 cells/mL and perfused through the flow chamber. Values represent means ± SE of results from three experiments. In each experiment, the mean number of 300-LAM cells interacting with the adherent cell monolayer was counted in 16 randomly chosen fields at each shear stress.

 

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  		Table 1. Adhesion Molecule Expression by EA.hy926 and ECV-304 Cell Lines

 
We have previously shown that reductions in sulfotransferase activity by treatment of 926-FtVII cells with sodium chlorate for 24 h also significantly reduces L-selectin-dependent binding by 74–84% in nonstatic binding assays [14 ]. In addition, treatment of TNF-activated HUVECs with sodium chlorate for 24 h significantly reduces lymphocyte attachment by 31–40%, whereas blocking lymphocyte attachment through L-selectin reduces attachment by 65–75% in nonstatic binding assays [14 ]. We therefore assessed whether this requirement for sulfotransferase activity could be visualized during in vitro flow chamber assays. However, culturing 926-FtVII and 304-FtVII cells with sodium chlorate for 24 h as previously described [14 ] did not decrease the number of rolling leukocytes (data not shown). The disparity between results obtained using the nonstatic binding assay and the in vitro flow chamber assay are best explained by our observations that the nonstatic binding assay is more sensitive to alterations in receptor density. Therefore, because the reduction in sulfation is likely incomplete after sodium chlorate treatment, sufficient ligand function may remain to sustain normal L-selectin engagement and rolling.

Effects of CHST2 overexpression by 926-FtVII cells
Because CHST2 expression was found to be up-regulated in HUVECs after TNF-{alpha} treatment, the relationship between sulfotransferase expression levels and L-selectin ligand activity was assessed by increasing CHST2 expression in EA.hy926 cells. To monitor sulfotransferase transcript levels in transfected cells, bicistronic CHST2 expression constructs were generated by placing an IRES element and a ß-gal gene downstream of CHST2 cDNA in the pcDNA3.1(-) vector (Fig. 3A ). Thereby, ß-gal levels correlated linearly with CHST transcription levels because ß-gal translation was initiated via an IRES site within CHST transcripts. EA.hy926 cells were cotransfected with a CHST2/ß-gal-encoding vector and a conventional FucT-VII cDNA expression vector. Repeated sorting of cDNA-transfected EA.hy926 cells resulted in the isolation of EA.hy926 and 926-FtVII cells with elevated ß-gal activity that were termed 926-CHST2 and 926-FtVII-CHST2 cells, respectively (Fig. 3B) . On average, ß-gal activity in 926-CHST2 cells was 20-fold higher (n = 2) than in EA.hy926 cells, while 926-FtVII-CHST2 cells expressed fourfold higher ß-gal activity (n = 3). By contrast, repeated sorting of mock transfected EA.hy926 cells for high ß-gal activity did not select for EA.hy926 cells with ß-gal activity higher than the levels endogenous to parental EA.hy926 cells (data not shown). Enhanced CHST2 expression by EA.hy926 cells was further verified by semiquantitative RT-PCR amplification of cDNA from each transfected cell line. 926-CHST2 and 926-FtVII-CHST2 cells expressed four- to fivefold higher levels of CHST2 transcripts than parental EA.hy926 cells (n = 4; Fig. 3C ). HECA-452 or CSLEX1 antigen expression levels were not influenced by enhanced CHST2 expression or FDG-based cell sorting (Fig. 3 ; data not shown).



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  		Figure 3. Enhanced CHST2 expression by transfected EA.hy926 cells. The structure of the bicistronic CHST expression vector that encodes ß-galactosidase (ß-gal) via an IRES element was determined (A). Abbreviations: IRES, internal ribosome entry site; ß-gal, bacterial ß-galactosidase gene; BGA pA, bovine growth hormone gene polyadenylation signal sequence; SV40 Ori, simian virus 40 origin of replication; Neor, neomycin-resistant gene; SV40 pA, simian virus 40 polyadenylation signal sequence; Ori, ColE1 origin of replication; Ampr, ampicillin-resistant gene; Pcmv, cytomegalovirus promoter sequence. HECA-452 antigen and ß-gal expression by cDNA-transfected cells was also determined (B). The indicated cell lines (above columns) were assessed for HECA-452 antigen expression (solid lines) by indirect immunofluorescence staining or were loaded with FDG (solid lines) to assess ß-gal expression levels, with subsequent flow cytometry analysis. Dashed histograms represent cell staining with an isotype-matched control mAb or the fluorescence intensity of cells not loaded with FDG. Results represent those obtained in three separate experiments and are shown on a four-decade log scale. To determine CHST2 mRNA expression, semiquantitative RT-PCR analysis was carried out with serial dilutions (as indicated) of cDNA prepared from RNA of the indicated cell lines (C). PCR products were visualized by electrophoresis with ethidium bromide staining. Results represent those from at least two separate experiments.

 
The effect of enhanced CHST2 expression by EA.hy926 cells on L-selectin ligand expression was assessed under physiologic flow using in vitro flow chamber assays. Rolling of 300-LAM cells on EA.hy926 transfectants was assessed at shear stresses from 0.25 to 3.0 dynes/cm2 (Fig. 4A ). EA.hy926 cells transfected with CHST2 alone did not support rolling of 300-LAM cells, whereas both 926-FtVII and 926-FtVII-CHST2 cell monolayers supported L-selectin-mediated rolling at shear stresses of 0.25–2.5 dynes/cm2. Overexpression of CHST2 by 926-FtVII cells did not increase the number of 300-LAM cells rolling across the 926-FtVII cell monolayers (Fig. 4A) . Similarly, the velocity of 100 randomly chosen 300-LAM cells rolling on EA.hy926 transfectants at 1.0 dyne/cm2 was not affected by enhanced CHST2 expression (Fig. 4B) . Therefore, it appears that endogenous CHST expression by EA.hy926 cells is sufficient for the optimal generation of functional L-selectin ligands. Alternatively, CHST2 does not contribute significantly to the generation of L-selectin ligands on vascular endothelial cells.



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  		Figure 4. Effect of enhanced CHST2 expression by EA.hy926 cells on leukocyte rolling. Rolling of 300-LAM cells on monolayers of cDNA-transfected EA.hy926 cells during in vitro flow chamber assays was assessed as in Fig. 2B (A). Values represent means ± SE of results from three experiments. Velocities of rolling leukocytes were determined (B). Velocity measurements are from one of three experiments with similar results. Each symbol represents the velocity of an individual 300-LAM cell plotted in rank order at 1 dyne/cm2 of shear stress.

 
CHST overexpression by ECV-304 cells augmented L-selectin ligand activity
The above results obtained using EA.hy926 cells suggested that high levels of endogenous CHST expression may preclude the use of EA.hy926 cells for examining the function of any single CHST by overexpression. However, since endogenous CHST1 and CHST2 mRNA expression levels were three- to fivefold lower in ECV-304 cells than EA.hy926 cells, the effect of enhanced CHST expression on L-selectin ligand function was assessed using ECV-304 cells. Combinations of single- and double-transfected cell lines were generated as described above for EA.hy926 cells, producing 304-CHST2, 304-FtVII-CHST2, 304-CHST1, and 304-FtVII-CHST1 cells. The ß-gal activity of CHST cDNA-transfected ECV-304 cells was 13- to 18-fold higher than in parental cells (n = 3; Fig. 5A ). Enhanced CHST expression was further verified by semiquantitative RT-PCR analysis of transcripts isolated from each cell line. 304-CHST1, 304-CHST2, 304-FtVII-CHST1, and 304-FtVII-CHST2 cells expressed CHST1 or CHST2 transcripts 10- to 20-fold higher than parental ECV-304 cells (Fig. 5B) . At these levels of expression, 304-FtVII-CHST2 cells expressed CHST2 transcripts at levels threefold higher than those found in parental EA.hy926 cells and at levels equivalent to those expressed by 926-FtVII-CHST2 cells (data not shown). Similarly, 304-FtVII-CHST1 cells expressed CHST1 transcripts at levels threefold higher than those found in parental EA.hy926 cells (data not shown). Enhanced CHST1 or CHST2 expression did not significantly alter HECA-452, CSLEX1, or MECA-79 antigen expression levels (Fig. 5A , Table 1 ). Similarly, E-selectin, P-selectin, CD34, PCLP-1, MAdCAM-1, PSGL-1, ICAM-1, VAP-1, and HNK-1 antigen expression was unaltered in any of the ECV-304 transfectants (Table 1) .



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  		Figure 5. Enhanced CHST1 and CHST2 expression by ECV-304 cells. HECA-452, CSLEX1, and MECA-79 antigen and ß-gal expression by cDNA-transfected ECV-304 cells are shown (A). Each cell line was assessed for cell surface antigen expression (solid lines) by indirect immunofluorescence staining or was loaded with FDG to assess ß-gal expression levels, with subsequent flow cytometry analysis. Dashed histograms represent cell staining with an isotype-matched control mAb or the fluorescence intensity of cells not loaded with FDG. Results represent three separate experiments and are shown on a four-decade log scale. CHST1 and CHST2 mRNA expression by ECV-304 transfectants was determined by semiquantitative RT-PCR analysis performed as described for Fig. 3C (B). PCR amplification of mRNA samples is shown as a negative control. Results represent two separate experiments.

 
The ability of enhanced CHST1 or CHST2 expression by ECV-304 monolayers to affect 300-LAM cell rolling was assessed using in vitro flow chamber assays. 304-FtVII-CHST1 and 304-FtVII-CHST2 cells supported rolling of 300-LAM cells at shear stresses up to 1.75 dynes/cm2, while 304-FtVII cells supported L-selectin-mediated rolling at shear stresses only up to 1.25 dynes/cm2 (Fig. 6A B ). In addition, the number of 300-LAM cells tethering and rolling on 304-FtVII monolayers was significantly increased (two- to fourfold) by enhanced CHST1 or CHST2 expression, which also reduced the average velocity of 100 randomly selected cells rolling on 304-FtVII transfectants at 1 dyne/cm2 (Fig. 6C ; 127 µm/s for 304-FtVII, 92 µm/s for 304-FtVII-CHST2, and 104 µm/s for 304-FtVII-CHST1 monolayers; P < 0.01). Specifically, enhanced CHST expression reduced the frequency of 300-LAM cells that were rolling at >100 µm/s on 304-FtVII cells. In all cases, 300-LAM cell rolling was blocked completely by the LAM1-3 mAb. Therefore, enhanced CHST1 or CHST2 expression by cell monolayers significantly altered the dynamics of L-selectin-dependent leukocyte rolling.



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  		Figure 6. Effect of enhanced CHST1 and CHST2 expression by ECV-304 cells on leukocyte rolling. Rolling of 300-LAM cells on ECV-304 cell monolayers during in vitro flow chamber assays was assessed as in Fig. 2B (A, B). Values represent means ± SE of results from three experiments. Asterisks indicate that 300-LAM cell rolling on 304-FtVII-CHST1 or 304-FtVII-CHST2 monolayers was significantly different from rolling on 304-FtVII monolayers (P < 0.001). Velocities of leukocytes rolling on ECV-304 cell monolayers were determined (C). Each symbol represents the velocity of an individual 300-LAM cell plotted in rank order at 1 dyne/cm2 of shear stress. These results represent three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results of this study demonstrate that (1) HUVECs and an HUVEC-derived cell line constitutively express CHST1, CHST2, and CHST3 transcripts (Fig. 1) ; (2) cytokine activation of HUVECs up-regulated CHST2 mRNA levels and induced low-level CHST4 mRNA expression (Fig. 1B) ; (3) 926-FtVII and 304-FtVII cells displayed different levels of L-selectin ligand activities that correlated with their endogenous CHST expression levels (Fig. 1 and 2) ; (4) augmenting CHST1 or CHST2 mRNA expression in 304-FtVII cells significantly enhanced their L-selectin ligand activity during in vitro flow chamber assays (Fig. 6) . Specifically, augmenting CHST1 or CHST2 expression in 304-FtVII cells increased the number of rolling 300-LAM cells, reduced overall 300-LAM cell rolling velocities, and significantly enhanced their ability to support 300-LAM cell rolling under higher shear stress (Fig. 6) . Thus, collectively these data indicate that CHST1, CHST2, and CHST3 expression by vascular endothelial cells may contribute to the sulfation of vascular L-selectin ligands. Moreover, optimal sulfation of L-selectin ligands may have a marked effect on the attachment efficiency, overall velocity, and binding strength of rolling interactions, all of which would significantly enhance leukocyte interactions with endothelial cells at sites of inflammation.

926-FtVII cells constitutively expressed CHST1, CHST2, and CHST3 transcripts and supported leukocyte rolling via L-selectin (Fig. 1 and 2) . It is surprising that further increases in CHST2 expression by 926-FtVII cells had little effect on leukocyte rolling via L-selectin (Fig. 3 and 4) . This result may be explained by their relatively high expression of endogenous CHST2 as well as CHST1 mRNA (Fig. 1) , which may already reach appropriate levels for optimal L-selectin ligand synthesis (Fig. 4) . Consistent with this, EA.hy926 cells constitutively expressed CHST1 and CHST2 transcripts at levels significantly higher than those found in TNF-activated HUVECs. Moreover, 304-FtVII-CHST2 cells expressed CHST2 transcripts at levels threefold higher than those found in parental EA.hy926 cells and at levels equivalent to those expressed by 926-FtVII-CHST2 cells, which may explain why 300-LAM cell rolling on 304-FtVII-CHST2 cells was equivalent to 300-LAM cell rolling on 926-FtVII and 926-FtVII-CHST2 cells (Fig. 4 and 6B) . Because each CHST protein has enzymatic activity, small changes in CHST transcription levels may be functionally significant. Thereby, increased CHST2 expression by cytokine-activated HUVECs may be physiologically relevant (Fig. 1) . Cytokine-induced expression of CHST4 mRNA by HUVECs suggests that CHST4 may also contribute to L-selectin ligand generation (Fig. 1B) . However, the current study demonstrates that CHST4 expression was not required for the generation of functional L-selectin ligands in EA.hy926 or ECV-304 cells. Thus, functional redundancy or overlapping specificity of the CHSTs may promote optimal L-selectin ligand generation.

Expression of CHST1 and CHST2 by vascular endothelial cells is consistent with our current understanding of the biochemical nature of L-selectin ligands on HEV. The four known sulfotransferases expressed by vascular endothelial cells demonstrate overlapping abilities to catalyze sulfation of chondroitin, keratan sulfate, and sialyl lactosamine oligosaccharides [25 26 27 , 29 30 31 32 33 34 , 45 ]. CHST1 exhibits galactose-6-sulfotransferase activity, while CHST2 and CHST4 display GlcNAc-6-sulfotransferase activity [29 , 30 ]. These activities correlate with the two major sulfated carbohydrate groups found on putative L-selectin ligands: 6-sulfo sLex and 6'-sulfo sLex [22 , 23 ]. Since increased expression of either CHST1 or CHST2 by 304-FtVII cells enhanced L-selectin ligand activity, these results are consistent with both 6-sulfo sLex and 6'-sulfo sLex groups as functional components of vascular L-selectin-ligands. The biochemical specificity of vascular L-selectin ligand sulfation awaits further characterization and identification of the vascular L-selectin ligands. Identification of the specific proteins mediating L-selectin-dependent binding in HUVECs and endothelial cell lines is required because a large number of endothelial cell proteins are sulfated. This information will also allow a direct assessment of how the CHSTs regulate ligand interactions.

The current study extends a recent report showing that CHST2 and FucT-VII cotransfection of ECV-304 cells induces L-selectin ligand expression as detected in a nonstatic binding assay [17 ]. However, our study assessed the effect of either enhanced CHST1 or CHST2 expression on leukocyte rolling and attachment via L-selectin under physiological flow conditions. Nonetheless, significant differences in results were observed between the two studies. Differences in sensitivity of the assay systems may explain why 304-FtVII cells supported sustained leukocyte rolling in the current studies (Fig. 2B) , although L-selectin-mediated attachment to 304-FtVII cells was not detected in the nonstatic binding assays used in the earlier studies unless the cells were also transfected with CHST2 cDNA [17 ]. In addition, ECV-304 cells transfected with CHST2 and FucT-VII cDNAs were reported to express the MECA-79 antigen in the previous study [17 ]. However, none of the 304-CHST1, 304-CHST2, 304-FtVII-CHST1, or 304-FtVII-CHST2 cells expressed detectable MECA-79 antigen in the current studies (Fig. 5 , Table 1 ). Similarly, HUVECs, EA.hy926 cells, COS cells, neutrophils, KG-1a cells, and HL-60 cells express FucT-VII and endogenous CHSTs, yet do not display detectable MECA-79 antigen [14 , 35 ]. In addition, Chinese hamster ovary (CHO) cells transfected with CHST4 or mouse CHST2 cDNAs do not express the MECA-79 epitope [33 ]. It remains possible that MECA-79 antigen expression requires a high threshold of sulfation that was not achieved in our cDNA-transfected cells or those of others [14 , 33 , 35 ]. Nonetheless, numerous studies have shown that MECA-79 antigen expression is neither sufficient nor required for vascular L-selectin ligand activity [12 , 14 , 46 ].

The current study of vascular L-selectin ligand sulfation complements recent studies focused on HEV-associated L-selectin ligands. Transfection of CHST1 and CHST4 cDNAs into CHO cells expressing CD34 and FucT-VII cDNAs generates ligands bound by L-selectin–IgM fusion proteins [29 ]. In this system, CHST1 and CHST4 synergize to enhance binding of this chimeric fusion protein [29 ]. Similarly, recombinant GlyCAM-1–IgG chimeras produced in COS cells overexpressing CHST1, CHST2, or CHST4 support increased L-selectin–dependent rolling of lymphocytes and Jurkat T cells in parallel-plate flow chambers by three- to sixfold [47 ]. In this system, COS cell transfection with a CHST1 cDNA generated optimal L-selectin ligands, although additive effects were not observed when the COS cells were cotransfected with combinations of CHST cDNAs [47 ]. In a separate study, CHST4 expression caused CHO cells transfected with CD34 and FucT-VII cDNAs to roll at slower velocities in parallel-plate flow chambers coated with an L-selectin–IgG fusion protein [33 ]. It was surprising that CHST2 expression by CHO cells transfected with CD34 and FucT-VII cDNAs did not affect rolling velocities in this assay system. These and other data led the authors of that study to propose that CHST4 is the only sulfotransferase relative to CHST1, CHST2, and CHST3 that generates L-selectin ligands such as 6-sulfo sLex in core 2-branched oligosaccharides attached to CD34 [33 ]. However, the results described above and in the current study, in combination with the broad expression patterns of the CHSTs, suggest that CHST1, CHST2, and CHST3 may also contribute to the generation of physiologic L-selectin ligands.

In summary, these results suggest that endothelial cells can use CHST1 and CHST2 for L-selectin ligand synthesis. Multiple CHSTs may be capable of generating functional ligands and overlapping enzymatic activities may be necessary for optimal ligand generation. Appropriate sialylation and fucosylation are also critical for the generation of HECA-452 mAb-defined sLex determinants that are essential components of vascular and leukocyte L-selectin ligands [14 , 35 ]. Increased CHST expression may synergize with increased FucT-VII mRNA expression in activated vascular endothelial cells [14 , 17 ] to generate optimal L-selectin ligands at sites of inflammation. In addition, functional vascular L-selectin ligands may require special protein scaffolds [14 , 37 ]. Therefore, regulation of sulfation, fucosylation, sialylation, and transcription of appropriate acceptor molecules provides critical points for modifying L-selectin ligand function. Each of these enzymatic pathways represents a potentially important target for therapeutic intervention.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH grants CA54464 and CA81776. We thank D. Patel, Y. Zhuang, F. W. Luscinskas, J. Ross, J. Poe, C.-J. Edgell, K. Moore, M. Cooper, B. Furie, S. Jalkanen, and B. Weston for reagents and help with these experiments.

Received August 21, 2000; revised November 6, 2000; accepted November 25, 2000.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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