Galectin-9 as a Potential Modulator of Lymphocyte Adhesion to Endothelium via Binding to Blood Group H Glycan

The recruitment of leukocytes from blood is one of the most important cellular processes in response to tissue damage and inflammation. This multi-step process includes rolling leukocytes and their adhesion to endothelial cells (EC), culminating in crossing the EC barrier to reach the inflamed tissue. Galectin-8 and galectin-9 expressed on the immune system cells are part of this process and can induce cell adhesion via binding to oligolactosamine glycans. Similarly, these galectins have an order of magnitude higher affinity towards glycans of the ABH blood group system, widely represented on ECs. However, the roles of gal-8 and gal-9 as mediators of adhesion to endothelial ABH antigens are practically unknown. In this work, we investigated whether H antigen–gal-9-mediated adhesion occurred between Jurkat cells (of lymphocytic origin and known to have gal-9) and EA.hy 926 cells (immortalized endothelial cells and known to have blood group H antigen). Baseline experiments showed that Jurkat cells adhered to EA.hy 926 cells; however when these EA.hy 926 cells were defucosylated (despite the unmasking of lactosamine chains), adherence was abolished. Restoration of fucosylation by insertion of synthetic glycolipids in the form of H (type 2) trisaccharide Fucα1-2Galβ1-4GlcNAc restored adhesion. The degree of lymphocyte adhesion to native and the “H-restored” (glycolipid-loaded) EA.hy 926 cells was comparable. If this gal-9/H (type 2) interaction is similar to processes that occur in vivo, this suggests that only the short (trisaccharide) H glycan on ECs is required.


Introduction
The recruitment of leukocytes to a site of inflammation results from a three-step adhesion cascade [1].In the first step, leukocytes circulating in blood start to roll on the endothelial surface; this process, known as rolling, is in part mediated by P-and E-selectins on endothelial cells (EC) and L-selectin on leukocytes reacting with specific oligoglycans.Rolling also requires the binding of CD44 [1][2][3][4] to the hyaluronic acid of EC.Both selectin-mediated and CD44-mediated rolling cause the activation of leukocytes.Activated leukocytes then express integrins of the β1 and β2 family, which bind to intercellular cell adhesion molecules ICAM-1-5 and VCAM-1, leading to the inhibition of leukocyte rolling and the facilitation of attachment to the endothelium.Adhesion is a key step of the cascade, since attached leukocytes cross the endothelial barrier and migrate to the site of inflammation [5,6].

Galectins and Galectin-Specific Antibodies
Recombinant human gal-8 and gal-9 were prepared as previously described [21] and purified by affinity chromatography on lactosylated Sepharose 4B.Polyclonal antibodies against these recombinant galectins were raised in rabbits.The IgG fraction was isolated by affinity chromatography using protein-A Sepharose 4B (Pharmacia, Freiburg, Germany) and checked for a lack of cross-reactivity against other lectin family members using Western blotting and ELISA.Cross-reactivity, if present, was removed by affinity chromatography on galectin-specific resins [22].
For studies of Jurkat cell adhesion to EA.hy 926 cells, polyclonal antibodies against endogenic gal-9 obtained from Antibody Verify Co (Las Vegas, CA, USA) were used.

Galectins and Galectin-Specific Antibodies
Recombinant human gal-8 and gal-9 were prepared as previously described [21] and purified by affinity chromatography on lactosylated Sepharose 4B.Polyclonal antibodies against these recombinant galectins were raised in rabbits.The IgG fraction was isolated by affinity chromatography using protein-A Sepharose 4B (Pharmacia, Freiburg, Germany) and checked for a lack of cross-reactivity against other lectin family members using Western blotting and ELISA.Cross-reactivity, if present, was removed by affinity chromatography on galectin-specific resins [22].
For studies of Jurkat cell adhesion to EA.hy 926 cells, polyclonal antibodies against endogenic gal-9 obtained from Antibody Verify Co (Las Vegas, CA, USA) were used.

Enzymatic Defucosylation of EA.hy 926
Cells (1 × 10 6 cells/mL) were grown to confluence in medium using 24-well plates (Nunc, Roskilde, Denmark), washed three times with DMEM-F12-0.3%FCS, and treated with fucosidase (4 U/mL) at 37 • C in a humidified atmosphere of 5% CO 2 overnight.The next day, cells were detached with Versene solution (PBS containing 0.02% EDTA v/v) and washed three times with phosphate-buffered saline containing 0.2% BSA (PBA) using centrifugation at 450× g (Jouan, rotor T20, France) at 4 • C. The binding of labeled UEA I served as a control for defucosylation.Briefly, the cells (1 × 10 5 cells per well in 50 µL) were carefully resuspended in PBA and incubated with 50 µL of biotinylated UEA I in PBA (final concentration 20 µg/mL) for 30 min at 4 • C under gentle agitation on a shaker, followed by incubation with FITC-labeled streptavidin (1:50 dilution in PBA) for 20 min under the same conditions.The cells were washed three times with PBA and analyzed by flow cytometry.

Insertion of FSL-H (Type 2) into EA.hy 926 Cells
Cells (1 × 10 6 cells/mL) were grown to confluence in medium using 24-well plates (Nunc, Denmark), washed three times with DMEM-F12-0.3%FCS, treated with fucosidase as described above, washed three times with PBA, and incubated with FSL-H (type 2) (final concentration: 5 µM in PBA) at 37 • C in a humidified atmosphere of 5% CO 2 for 1 h.FCS was used in a low concentration (0.3% FCS in DMEM-F12 in medium), as high percentages of serum can interfere with glycolipid insertion into cell membranes [23,24].To analyze FSL-H (type 2) insertion, cells were detached with Versene solution, washed three times with PBA by centrifugation at 95× g and 4 • C for 3 min, and incubated with biotinylated UEA I for 30 min at 4 • C under gentle agitation on a shaker.Cells were then incubated with Str-FITC (1:50 dilution in PBA) for 20 min under the same conditions.The cells were washed three times with PBA and analyzed by flow cytometry.

Binding of Galectins to EA.hy 926 Cells
After the insertion of FSL-H (type 2), cells were detached with Versene solution and washed three times by centrifugation, as described above.Aliquots of the cell suspension (1 × 10 5 cells in 50 µL) were incubated with 50 µL of gal-8 or gal-9 (0.1 mg/mL) for 30 min at 4 • C under gentle agitation on a shaker.To remove unbound galectins, cells were carefully washed once using centrifugation under the previously described conditions [25].The measurement of galectin binding was determined with galectin-specific biotinylated antibodies (5 µg/mL in PBA) and Str-FITC (1:50 dilution in PBA) at 4 • C for 20 min.As a negative control, native galectin-free cells were incubated with the galectin-specific antibody.

Flow Cytometry
After the washing steps, cells were transferred into a tube and mixed with 2 mL of PBS.Flow cytometry was performed at room temperature using a FACScan instrument (Becton-Dickinson Co, Franklin Lakes, NJ, USA) equipped with the software FlowJo V10.5.3 or a FC500 cytofluorimeter (Beckman Coulter, Miami, FL, USA) equipped with the software Kaluza 1.3.Live cell populations were first gated using morphological parameters of forward light scatter (FSC) vs. sideward light scatter (SSC).Then, the logarithmic fluorescence intensity (FL-FITC) of the gated population was measured.The fluorescence (in some articles, this parameter is called Fluorescence increase) shown in the figures was calculated as [(F i /F 0 ) × 100] − 100, where F i is the geometric mean of the fluorescence intensities of cells after incubation with anti-galectin + second IgG-FITC or UEA I + Str-FITC, and F 0 is the geometric mean of the fluorescence intensities of cells stained only with IgG-FITC or Str-FITC.In parallel, the fluorescences of microspheres (in the dilution range recommended by the manufacturer) were measured.The values of the obtained fluorescence intensities were used to calculate the number of FSL-H (type 2) molecules per cell.

Detection of FSL-H (Type 2) in the Cell Membrane
To visualize the membrane, DPH stock solution (8 mM) in dimethyl sulfoxide was prepared and added to the cells (final concentration: 4 µM).Cells were incubated with DPH at 4 • C for 1 h and then washed with PBS.The inserted FSL-H (type 2) was detected with biotinylated UEA I followed by Str-FITC, as described above.Microscopy mounting solution containing 2.4 g of Mowiol 4-88, 6 g of glycerol, 6 mL of water, and 12 mL of 0.2 MTris-HCl (pH 8.5) was placed on the microscope slide, followed by the cell suspension (10 µL).All images were obtained with a confocal microscope Nikon Eclipse TE-2000-E (Nikon, Minato City, Japan) and analyzed with ImageJ.At least ten randomly selected cells were analyzed in each experiment.

Cell Adhesion Assay
The adhesion of Jurkat cells to EA.hy 926 cells was evaluated using a confocal microscope (Nikon Eclipse TE-2000-E).Briefly, EA.hy 926 cells (1 × 10 6 cells/mL) were grown to confluence in DMEM-F12 medium, washed with DMEM-F12 medium containing 0.3% FCS, and treated with fucosidase, as described above.FSL-H constructs were then inserted into the EA.hy 926 cells, as described above, cells were washed with RPMI-0.3%FCS, and then Jurkat cells (1 × 10 6 cells/well) in the same media were added.The plate was incubated at 37 • C in a humidified atmosphere of 5% CO 2 overnight.The next day, non-adhered cells were removed by gentle washing with PBA.Jurkat cells adhered to the monolayer of EA.hy 926 were stained with endogenic anti-gal-9 and IgG-Alexa Fluor 594.Three confocal microscope images per well were taken, and adherent cells were counted using ImageJ software 1.53t.
To confirm involvement of gal-9 in adhesion inhibition of its binding to EA.hy 926 cells by anti-gal-9 antibodies and polyacrylamide conjugate H (type 2)-PAA was performed.Jurkat cells were incubated with antibodies against gal-9 or H (type 2)-PAA (100 µM by trisaccharide) for 60 min at 37 • C and then added to EA.hy 926 cells.

Statistical Analysis
Data represent means +/− standard deviations.The unpaired Student's t-test for statistical analysis of the results was used.

Binding of Galectins to Unmodified and Fucosidase-Treated EA.hy 926 Cells
To determine if gal-8 and gal-9 recognized cell surface-exposed H glycans, the binding of galectins to unmodified cells was compared against defucosylated cells (i.e., treated with fucosidase).Quantitative data were obtained using flow cytometry, measuring the fluorescence intensity's (MFI) geometric mean.The decreased binding of UEA I (a plant lectin recognizing the Fucα1-2Galβ1-4GlcNAc glycotope [19]) verified the level of defucosylation.(The fluorogram is presented in Supplementary Figure S1.) After treatment with fucosidase, gal-9 binding to EA.hy 926 cells significantly decreased (Figure 2B), while defucosylation did not affect gal-8 binding (Figure 2A).The concentrations of 7 µg/mL of gal-8 and 55 µg/mL of gal-9 were found to be required to reach 50% binding, indicating that gal-9 binds predominantly to H-glycans, while gal-8 utilizes other glycans to attach to cells.
Because only gal-9 could bind the H antigen of EA.hy 926, further experiments were conducted solely with gal-9.Because only gal-9 could bind the H antigen of EA.hy 926, further experiments were conducted solely with gal-9.

Binding of Gal-9 to Defucosylated EA.hy 926 Cells after Their "Refucosylation" with FSL-H (Type 2)
FSL-H (type 2) (Figure 3, Supplementary Figure S1) was inserted in defucosylated cells, and UEA I binding to these defucosylated cells increased four-fold following FSL-H (type 2) insertion (see in Supplementary Figure S1), indicating a significant degree of modification.Comparing the fluorescence of FSL-H (type 2)-modified cells with Immuno-Brite fluorosphere-calibrating particles, it was calculated that the number of FSL-H (type 2) molecules inserted per cell was ~10 5 (after 1 h of incubation).
The binding of gal-9 to FSL-H (type 2)-modified cells was determined, and fluorograms are presented in Supplementary Figure S2.The insertion of FSL-H (type 2) into defucosylated cells resulted in a three-fold increase in gal-9 and at a level that was comparable to unmodified cells (Figure 3).The low signal seen in the defucosylated cells indicated incomplete defucosylation.FSL-H (type 2) (Figure 3 and Supplementary Figure S1) was inserted in defucosylated cells, and UEA I binding to these defucosylated cells increased four-fold following FSL-H (type 2) insertion (see in Supplementary Figure S1), indicating a significant degree of modification.Comparing the fluorescence of FSL-H (type 2)-modified cells with Immuno-Brite fluorosphere-calibrating particles, it was calculated that the number of FSL-H (type 2) molecules inserted per cell was ~10 5 (after 1 h of incubation).Because only gal-9 could bind the H antigen of EA.hy 926, further experiments were conducted solely with gal-9.

Binding of Gal-9 to Defucosylated EA.hy 926 Cells after Their "Refucosylation" with FSL-H (Type 2)
FSL-H (type 2) (Figure 3, Supplementary Figure S1) was inserted in defucosylated cells, and UEA I binding to these defucosylated cells increased four-fold following FSL-H (type 2) insertion (see in Supplementary Figure S1), indicating a significant degree of modification.Comparing the fluorescence of FSL-H (type 2)-modified cells with Immuno-Brite fluorosphere-calibrating particles, it was calculated that the number of FSL-H (type 2) molecules inserted per cell was ~10 5

(after 1 h of incubation).
The binding of gal-9 to FSL-H (type 2)-modified cells was determined, and fluorograms are presented in Supplementary Figure S2.The insertion of FSL-H (type 2) into defucosylated cells resulted in a three-fold increase in gal-9 and at a level that was comparable to unmodified cells (Figure 3).The low signal seen in the defucosylated cells indicated incomplete defucosylation.The binding of gal-9 to FSL-H (type 2)-modified cells was determined, and fluorograms are presented in Supplementary Figure S2.The insertion of FSL-H (type 2) into defucosylated cells resulted in a three-fold increase in gal-9 and at a level that was comparable to unmodified cells (Figure 3).The low signal seen in the defucosylated cells indicated incomplete defucosylation.

Localization of Inserted FSL-H (Type 2)
The FSL-H (type 2) distribution in the EA.hy 926 cell membrane was assessed using confocal microscopy.For membrane tracing, the fluorescent dye DPH (1,6-diphenylhexatriene), which has no polar head and is known to accumulate and fluoresce only in a hydrophobic environment [26], was used.FSL-H (type 2) stained with UEA I/Str-FITC was detected near the DPH staining zone; its distribution was similar to that of H glycan in unmodified cells (Figure 4).The fucosidase-treated cells showed poor staining with UEA I, which reflected, as mentioned above (see in Supplementary Figure S1), the expected incomplete defucosylation.These results showed that FSL-H (type 2) was able to restore H reactivity to a level similar to that seen on unmodified cells, and that defucosylated cells only had trace levels of UEA I-reactive H glycan.
Figure 3. Binding of gal-9 to EAhy 926 cells modified with FSL-H (type 2), as determined by flow cytometry.Cells were treated with fucosidase and incubated with FSL-H (type 2) at 37 °C for 1 h.After insertion and washing, cells were incubated with gal-9 and stained with anti-gal-9, as described in Material and Methods.Data represent the means of fluorescence +/− standard deviations (n = 3); *** p < 0.001.

Localization of Inserted FSL-H (Type 2)
The FSL-H (type 2) distribution in the EA.hy 926 cell membrane was assessed using confocal microscopy.For membrane tracing, the fluorescent dye DPH (1,6-diphenylhexatriene), which has no polar head and is known to accumulate and fluoresce only in a hydrophobic environment [26], was used.FSL-H (type 2) stained with UEA I/Str-FITC was detected near the DPH staining zone; its distribution was similar to that of H glycan in unmodified cells (Figure 4).The fucosidase-treated cells showed poor staining with UEA I, which reflected, as mentioned above (see in Supplementary Figure S1), the expected incomplete defucosylation.These results showed that FSL-H (type 2) was able to restore H reactivity to a level similar to that seen on unmodified cells, and that defucosylated cells only had trace levels of UEA I-reactive H glycan.

Gal-9 Promotes Adhesion of Jurkat Cells to EA.hy 926 Cells
The adhesion of Jurkat cells to EA.hy 926 cells was assessed.Jurkat cells were added to a monolayer of EA.hy 926 cells, with variable degrees of FSL-H modification.The adhesion of Jurkat cells (as a measure of galectin-9 on the cell surface) to unmodified cells, fucosidase-treated cells, and FSL-H (type 2) fucosylation-restored cells were compared.Using the number of Jurkat cells attached to unmodified EA.hy 926 cells as 100%, calculations revealed that only 4% of Jurkat cells attached to defucosylated EA.hy 926 cells (Figure 5A,B,D), whereas 50% of Jurkat cells attached to defucosylated EA.hy 926 cells after the insertion of FSL-H (type 2) (Figure 5C,D).

Gal-9 Promotes Adhesion of Jurkat Cells to EA.hy 926 Cells
The adhesion of Jurkat cells to EA.hy 926 cells was assessed.Jurkat cells were added to a monolayer of EA.hy 926 cells, with variable degrees of FSL-H modification.The adhesion of Jurkat cells (as a measure of galectin-9 on the cell surface) to unmodified cells, fucosidasetreated cells, and FSL-H (type 2) fucosylation-restored cells were compared.Using the number of Jurkat cells attached to unmodified EA.hy 926 cells as 100%, calculations revealed that only 4% of Jurkat cells attached to defucosylated EA.hy 926 cells (Figure 5A,B,D), whereas 50% of Jurkat cells attached to defucosylated EA.hy 926 cells after the insertion of FSL-H (type 2) (Figure 5C,D).
To confirm that gal-9 mediated the adhesion of Jurkat to EA.hy 926 cells, we evaluated adhesion assay in the presence of anti-gal-9 antibodies or H (type 2)-PAA [27].(Previously, we used a similar assay for the study of galectin specificity [27,28].)Both reagents inhibited Jurkat cell adhesion (Figure 6).We explained the weak inhibition in the case of anti-gal-9 with the fact that only a small population of these polyclonal antibodies were directed to the carbohydrate recognition domain of gal-9.To confirm that gal-9 mediated the adhesion of Jurkat to EA.hy 926 cells, we evaluated adhesion assay in the presence of anti-gal-9 antibodies or H (type 2)-PAA [27].(Previously, we used a similar assay for the study of galectin specificity [27,28].)Both reagents inhibited Jurkat cell adhesion (Figure 6).We explained the weak inhibition in the case of anti-gal-9 with the fact that only a small population of these polyclonal antibodies were directed to the carbohydrate recognition domain of gal-9.To confirm that gal-9 mediated the adhesion of Jurkat to EA.hy 926 cells, we evaluated adhesion assay in the presence of anti-gal-9 antibodies or H (type 2)-PAA [27].(Previously, we used a similar assay for the study of galectin specificity [27,28].)Both reagents inhibited Jurkat cell adhesion (Figure 6).We explained the weak inhibition in the case of anti-gal-9 with the fact that only a small population of these polyclonal antibodies were directed to the carbohydrate recognition domain of gal-9.

Discussion
The interaction of β1 and β2 integrins of leukocytes with intercellular adhesion molecules of the ICAM and VCAM families [5,6] on activated EC mediated the firm adhesion of leukocytes to the endothelium.Other adhesion mechanisms of this type were also known, including leukocyte CD44 reacting with endothelial hyaluronic acid [2,3,29].There was also data on galectin-mediated adhesion of leukocytes, which recognize oligolactosamines on the endothelium [7] and other ligands, including glycans of the ABH blood group system (which are widely represented on EC glycolipids and glycoproteins) [13,14,30].However, there was no information about galectin involvement in ABHmediated adhesion to endothelium, although the affinity of tandem-repeat-type galectins to ABH glycans was reported [31][32][33].It was an order of magnitude higher than oligolactosamines [27,34].The extracellular gal-8 and gal-9 were present in the glycocalyx of leukocytes [7].As mentioned above, tandem-repeat-type galectins had two homologous carbohydrate recognition domains (CRD), namely N-CRD and C-CRD [34].N-CRD exhibited a higher affinity for oligolactosamines [27], while C-CRD "targeted" ABH antigens.In this work, we examined whether gal-8 and gal-9 were able to mediate the adhesion of lymphocytes to EC.To achieve this, we used a model system, namely, the interaction of galectin-positive Jurkat cells (T-lymphocyte origin [35] with EA.hy 926 endothelial cells (i) bearing natural H antigen [13], (ii) being artificially depleted of H antigen by fucosidase treatment, and (iii) defucosylated then refucosylated with only H (type 2) antigen (using synthetic FSL glycolipid)).Due to the absence of ABO glycosyltransferases in EA.hy 926 cells, only the involvement of H (type 2) was examined.Additionally, the lack of gal-8 and gal-9 in the glycocalyx of EA.hy 926 cells (unpublished observations) made these cells particularly suitable for this study.It should be noted that although defucosylation was incomplete, the inability of glycosidases to fully deplete glycan-substrates on viable cells was well established [36].However, despite the residual levels of H glycan remaining, fucosidase-treated cells showed high levels of experimental differentiation from untreated cells.The depletion of fucose residues affected the binding of exogenic gal-9 but not gal-8, indicating that fucose was not critical for gal-8 binding.It is known that the affinity of gal-8 to H glycan was an order of magnitude lower than that of gal-9 [27], and the most potent fucose-containing glycan for gal-8 interaction was blood group A tetrasaccharide, which was absent on EA.hy 926 cells.We believe that on EA.hy 926 cells, gal-8 bound to oligolactosamines, the amount of which increased significantly due to the defucosylation of native glycans.On the contrary, H glycans (type 1 and type 2) were the most potent ligands for gal-9, and their affinities were found to be much stronger compared to oligolactosamines and α2-3-sialylated glycans [27,28].
Fucosidase treatment disrupted H glycotopes and a large range of other fucose-related histo-blood group antigens.Therefore, to confirm the observations that the loss of gal-9 activity in defucosylated cells was due to H glycans, we restored fucosylation in cells with a defined blood group H (type 2) antigen.This was performed by using a synthetic glycolipid FSL-H (type 2) and the well-established principles and methodologies of Kode Technology [37].As a result of refucosylation with H (type 2), the ability of gal-9 to bind EA.hy 926 cells was restored Thus, the presence of the trisaccharide H (type 2) was sufficient for the interaction of Jurkat cell gal-9 to EA.hy 926 cells, suggesting that gal-9 of leukocytes was potentially able to mediate or participate in the adhesion via binding to H glycan on EC.Furthermore, this could be extrapolated to suggest that EC cells of different ABO blood types, where the amount of H antigen is much more significant in blood group O than in A and B phenotypes, could have some impact on their immunological response(s).

Figure 2 .
Figure2.Effect of fucosidase treatment on EA.hy 926 cell-binding with gal-8 (A) and gal-9 (B), as determined by flow cytometry.Cells were treated with fucosidase, incubated with galectins, and stained with anti-galectin, followed by IgG-FITC, as described in Material and Methods.Results shown include fluorograms (the log of fluorescence intensity FL-1, x-axis, was plotted against the cell number, y-axis), and the number given for the black curve represents the percentage of cells reactive with the lectins.Cells stained with only IgG-FITC were used as a negative (dashed line) control.

Figure 2 .
Figure2.Effect of fucosidase treatment on EA.hy 926 cell-binding with gal-8 (A) and gal-9 (B), as determined by flow cytometry.Cells were treated with fucosidase, incubated with galectins, and stained with anti-galectin, followed by IgG-FITC, as described in Material and Methods.Results shown include fluorograms (the log of fluorescence intensity FL-1, x-axis, was plotted against the cell number, y-axis), and the number given for the black curve represents the percentage of cells reactive with the lectins.Cells stained with only IgG-FITC were used as a negative (dashed line) control.

Figure 2 .
Figure2.Effect of fucosidase treatment on EA.hy 926 cell-binding with gal-8 (A) and gal-9 (B), as determined by flow cytometry.Cells were treated with fucosidase, incubated with galectins, and stained with anti-galectin, followed by IgG-FITC, as described in Material and Methods.Results shown include fluorograms (the log of fluorescence intensity FL-1, x-axis, was plotted against the cell number, y-axis), and the number given for the black curve represents the percentage of cells reactive with the lectins.Cells stained with only IgG-FITC were used as a negative (dashed line) control.

Figure 3 .
Figure 3. Binding of gal-9 to EAhy 926 cells modified with FSL-H (type 2), as determined by flow cytometry.Cells were treated with fucosidase and incubated with FSL-H (type 2) at 37 • C for 1 h.After insertion and washing, cells were incubated with gal-9 and stained with anti-gal-9, as described in Material and Methods.Data represent the means of fluorescence +/− standard deviations (n = 3); *** p < 0.001.

Figure 4 .
Figure 4. Localization of FSL-H (type 2) in the EA.hy 926 cell membrane, as determined by confocal microscopy.FSL-H (type 2) was inserted in the defucosylated cells, as described in Material and Methods; it was washed with PBS, and the membrane was stained with DPH (4 μM, blue) for 1 h at 4 °C.FSL-H was visualized (in green) with biotinylated UEA I/Str-FITC.The white bar inset at the top right in each image corresponds to 5 μm.

Figure 4 .
Figure 4. Localization of FSL-H (type 2) in the EA.hy 926 cell membrane, as determined by confocal microscopy.FSL-H (type 2) was inserted in the defucosylated cells, as described in Material and Methods; it was washed with PBS, and the membrane was stained with DPH (4 µM, blue) for 1 h at 4 • C. FSL-H was visualized (in green) with biotinylated UEA I/Str-FITC.The white bar inset at the top right in each image corresponds to 5 µm.

Figure 6 .
Figure 6.Adhesion of Jurkat cells to EA.hy 926 cells in the presence of inhibitors, shown with confocal microscopy data.Jurkat cells were incubated with anti-gal-9 (10 μg/mL) or H (type 2)-PAA (100 μM) for 1 h at 37 °C, washed to remove the unbound inhibitor, and added to the EA.hy 926

Figure 6 .
Figure 6.Adhesion of Jurkat cells to EA.hy 926 cells in the presence of inhibitors, shown with confocal microscopy data.Jurkat cells were incubated with anti-gal-9 (10 μg/mL) or H (type 2)-PAA (100 μM) for 1 h at 37 °C, washed to remove the unbound inhibitor, and added to the EA.hy 926

Figure 6 .
Figure 6.Adhesion of Jurkat cells to EA.hy 926 cells in the presence of inhibitors, shown with confocal microscopy data.Jurkat cells were incubated with anti-gal-9 (10 µg/mL) or H (type 2)-PAA (100 µM) for 1 h at 37 • C, washed to remove the unbound inhibitor, and added to the EA.hy 926 cells.The adhesion cell assay was performed, as described in Material and Methods.To visualize the attached Jurkat cells, DAPI reagent (4 ,6-diamidino-2-phenylindole) was used.Adherent cells were counted in each well in triplicate.Data represent percentage of Jurkat cells bound to EA.hy 926 cells +/− standard deviations (n = 3); ** p < 0.01, * p < 0.1.