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L-Selectin ligands that are O-glycoprotease resistant and distinct from MECA-79 antigen are sufficient for tethering and rolling of lymphocytes on human high endothelial venules.

Clark RA, Fuhlbrigge RC, Springer TA - J. Cell Biol. (1998)

Bottom Line: Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant.We conclude that a pool of O-glycoprotease-resistant sLex-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb.We postulate that these ligands may participate in lymphocyte binding to HEV.

View Article: PubMed Central - PubMed

Affiliation: The Center for Blood Research and Harvard Medical School, Department of Pathology, Boston, Massachusetts 02115, USA.

ABSTRACT
During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewisx (sLex)-containing carbohydrate ligands expressed on the surface of high endothelial venules (HEV). We have examined the expression of sLex on HEV using a panel of mAbs specific for sLex and sLex-related structures, and have examined the function of different sLex-bearing structures using an in vitro assay of lymphocyte rolling on HEV. We report that three sLex mAbs, 2F3, 2H5, and CSLEX-1, previously noted to bind with high affinity to glycolipid-linked sLex, vary in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked sLex on glycoproteins. Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast majority of MECA-79 staining, only partially reduced staining with the 2F3 and 2H5 mAbs. Using a novel rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of O-sialoglycoprotease-resistant molecules is present on HEV that is sufficient for attachment and rolling of lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only 30%, whereas the sLex mAb 2H5 blocks binding by approximately 60% and a combination of MECA-79 and 2H5 mAb blocks binding by 75%. We conclude that a pool of O-glycoprotease-resistant sLex-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We postulate that these ligands may participate in lymphocyte binding to HEV.

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Effect of MECA-79  mAb on binding of neuraminidase-treated PBMC under  flow conditions to HEV of  (a and b) untreated and (c  and d) O-sialoglycoprotease– treated tonsil sections. Binding to an individual HEV was  assessed before (a and c) and  (b and d) after incubation of  sections with MECA-79 mAb.  Flow parameters were identical to those used in Fig. 4.  The direction of flow was  from the bottom of the figure  to the top.
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Figure 6: Effect of MECA-79 mAb on binding of neuraminidase-treated PBMC under flow conditions to HEV of (a and b) untreated and (c and d) O-sialoglycoprotease– treated tonsil sections. Binding to an individual HEV was assessed before (a and c) and (b and d) after incubation of sections with MECA-79 mAb. Flow parameters were identical to those used in Fig. 4. The direction of flow was from the bottom of the figure to the top.

Mentions: PBMC were isolated as previously described (56). For experiments using desialylated cells, cells (7.5 × 106 in 150 μl of HBSS with 2 mM Ca2+, 0.1% BSA) were treated with 0.1 U/ml of neuraminidase for 40 min at 37°C. For antibody blocking experiments using DREG-56 mAb, cells (7.5 × 106 cells in 150 μl of HBSS with 2 mM Ca2+) were pre-incubated for 40 min on ice with a final concentration of 50 μg/ml blocking mAb. Freshly cut 9 μm sections of human tonsil identified as “highly reactive” (see above) were placed on 70 × 50 mm glass slides (Corning Glass Works, Corning, NY) pre-coated with 0.1 mg/ml poly-L-lysine (Sigma Chemical Co.), allowed to dry 1–3 h at room temperature, and then stored at 4°C for up to 2 h until use. A parallel plate flow chamber was attached directly to the glass slide such that the tissue section made up the floor of the chamber (19, 38). PBMC were introduced into the chamber under flow, and the tethering of cells to HEV at 25°C was observed and recorded using video microscopy. Flow experiments used the following shear stresses: cells were accumulated at 0.75 dyn/cm2 for 80 s, followed by rinsing with cell-free medium at 1.2, 1.7, 2.1, 3.0, 3.9, 4.8, 6.3, 7.8, 9.3, 10.8, and 12.3 dyn/cm2 for 10 s each. Binding was evaluated at the end of the 1.7 dyn/cm2 step and the higher shears were used to remove all adherent lymphocytes from the sections. In general, an individual section could be used for up to four consecutive evaluations of cell binding without diminution of lymphocyte attachment to HEV; after the fourth run, a progressive decrease in lymphocyte binding was usually observed. Individual sections were therefore used for only three to four runs. For Figs. 4–6, video images were captured using NIH Image software version 1.59 and unbound cells were darkened by comparing two consecutive frames and using the minimum value for each pixel. For antibody-blocking experiments using mAbs MECA-79, 2H5, and 2F3, cells were first bound to the HEV in the absence of mAb. The mAb (50 μg/ml of MECA-79 or culture supernatants of 2H5 or 2F3) was then infused into the flow chamber for 15 min at a rate of 0.02 ml/min. Binding of cells to the same HEV was then re-evaluated at the end of mAb infusion. For MECA-79 experiments, mAb at a final concentration of 50 μg/ml was added to the cell suspension before infusion into the flow chamber and wash medium was also supplemented with 50 μg/ml MECA-79.


L-Selectin ligands that are O-glycoprotease resistant and distinct from MECA-79 antigen are sufficient for tethering and rolling of lymphocytes on human high endothelial venules.

Clark RA, Fuhlbrigge RC, Springer TA - J. Cell Biol. (1998)

Effect of MECA-79  mAb on binding of neuraminidase-treated PBMC under  flow conditions to HEV of  (a and b) untreated and (c  and d) O-sialoglycoprotease– treated tonsil sections. Binding to an individual HEV was  assessed before (a and c) and  (b and d) after incubation of  sections with MECA-79 mAb.  Flow parameters were identical to those used in Fig. 4.  The direction of flow was  from the bottom of the figure  to the top.
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Related In: Results  -  Collection

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Figure 6: Effect of MECA-79 mAb on binding of neuraminidase-treated PBMC under flow conditions to HEV of (a and b) untreated and (c and d) O-sialoglycoprotease– treated tonsil sections. Binding to an individual HEV was assessed before (a and c) and (b and d) after incubation of sections with MECA-79 mAb. Flow parameters were identical to those used in Fig. 4. The direction of flow was from the bottom of the figure to the top.
Mentions: PBMC were isolated as previously described (56). For experiments using desialylated cells, cells (7.5 × 106 in 150 μl of HBSS with 2 mM Ca2+, 0.1% BSA) were treated with 0.1 U/ml of neuraminidase for 40 min at 37°C. For antibody blocking experiments using DREG-56 mAb, cells (7.5 × 106 cells in 150 μl of HBSS with 2 mM Ca2+) were pre-incubated for 40 min on ice with a final concentration of 50 μg/ml blocking mAb. Freshly cut 9 μm sections of human tonsil identified as “highly reactive” (see above) were placed on 70 × 50 mm glass slides (Corning Glass Works, Corning, NY) pre-coated with 0.1 mg/ml poly-L-lysine (Sigma Chemical Co.), allowed to dry 1–3 h at room temperature, and then stored at 4°C for up to 2 h until use. A parallel plate flow chamber was attached directly to the glass slide such that the tissue section made up the floor of the chamber (19, 38). PBMC were introduced into the chamber under flow, and the tethering of cells to HEV at 25°C was observed and recorded using video microscopy. Flow experiments used the following shear stresses: cells were accumulated at 0.75 dyn/cm2 for 80 s, followed by rinsing with cell-free medium at 1.2, 1.7, 2.1, 3.0, 3.9, 4.8, 6.3, 7.8, 9.3, 10.8, and 12.3 dyn/cm2 for 10 s each. Binding was evaluated at the end of the 1.7 dyn/cm2 step and the higher shears were used to remove all adherent lymphocytes from the sections. In general, an individual section could be used for up to four consecutive evaluations of cell binding without diminution of lymphocyte attachment to HEV; after the fourth run, a progressive decrease in lymphocyte binding was usually observed. Individual sections were therefore used for only three to four runs. For Figs. 4–6, video images were captured using NIH Image software version 1.59 and unbound cells were darkened by comparing two consecutive frames and using the minimum value for each pixel. For antibody-blocking experiments using mAbs MECA-79, 2H5, and 2F3, cells were first bound to the HEV in the absence of mAb. The mAb (50 μg/ml of MECA-79 or culture supernatants of 2H5 or 2F3) was then infused into the flow chamber for 15 min at a rate of 0.02 ml/min. Binding of cells to the same HEV was then re-evaluated at the end of mAb infusion. For MECA-79 experiments, mAb at a final concentration of 50 μg/ml was added to the cell suspension before infusion into the flow chamber and wash medium was also supplemented with 50 μg/ml MECA-79.

Bottom Line: Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant.We conclude that a pool of O-glycoprotease-resistant sLex-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb.We postulate that these ligands may participate in lymphocyte binding to HEV.

View Article: PubMed Central - PubMed

Affiliation: The Center for Blood Research and Harvard Medical School, Department of Pathology, Boston, Massachusetts 02115, USA.

ABSTRACT
During the process of lymphocyte recirculation, lymphocytes bind via L-selectin to sulfated sialyl-Lewisx (sLex)-containing carbohydrate ligands expressed on the surface of high endothelial venules (HEV). We have examined the expression of sLex on HEV using a panel of mAbs specific for sLex and sLex-related structures, and have examined the function of different sLex-bearing structures using an in vitro assay of lymphocyte rolling on HEV. We report that three sLex mAbs, 2F3, 2H5, and CSLEX-1, previously noted to bind with high affinity to glycolipid-linked sLex, vary in their ability to stain HEV in different lymphoid tissues and bind differentially to O-linked versus N-linked sLex on glycoproteins. Treatment of tissue sections with neuraminidase abolished staining with all three mAbs but slightly increased staining with MECA-79, a mAb to a sulfation-dependent HEV-associated carbohydrate determinant. Treatment of tissue sections with O-sialoglycoprotease under conditions that removed the vast majority of MECA-79 staining, only partially reduced staining with the 2F3 and 2H5 mAbs. Using a novel rolling assay in which cells bind under flow to HEV of frozen tissue sections, we demonstrate that a pool of O-sialoglycoprotease-resistant molecules is present on HEV that is sufficient for attachment and rolling of lymphocytes via L-selectin. This interaction is not inhibited by the mAb MECA-79. Furthermore, MECA-79 mAb blocks binding to untreated sections by only 30%, whereas the sLex mAb 2H5 blocks binding by approximately 60% and a combination of MECA-79 and 2H5 mAb blocks binding by 75%. We conclude that a pool of O-glycoprotease-resistant sLex-like L-selectin ligands exist on human HEV that is distinct from the mucin-associated moieties recognized by MECA-79 mAb. We postulate that these ligands may participate in lymphocyte binding to HEV.

Show MeSH
Related in: MedlinePlus