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Tenascin supports lymphocyte rolling.

Clark RA, Erickson HP, Springer TA - J. Cell Biol. (1997)

Bottom Line: Tenascin has been reported to have both adhesive and anti-adhesive effects in static assays.When compared to rolling of the same cell type on E-selectin, rolling on tenascin was found to be smoother at all shear stresses tested, suggesting that cells formed a larger number of bonds on the tenascin substrate than on the E-selectin substrate.When protein plating densities were adjusted to give similar profiles of cell detachment under increasing shears, the density of tenascin was 8.5-fold greater than that of E-selectin.

View Article: PubMed Central - PubMed

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

ABSTRACT
Tenascin is a large extracellular matrix molecule expressed at specific sites in the adult, including immune system tissues such as the bone marrow, thymus, spleen, and T cell areas of lymph nodes. Tenascin has been reported to have both adhesive and anti-adhesive effects in static assays. We report here that tenascin supports the tethering and rolling of lymphocytes and lymphoblastic cell lines under flow conditions. Binding was calcium dependent and was not inhibited by treatment of lymphocytes with O-glycoprotease or a panel of glycosidases including neuraminidase and heparitinase but was inhibited by treatment of cells with proteinase K. Binding was to the fibrinogen-like terminal domain of tenascin as determined by antibody blocking studies and binding to recombinant tenascin proteins. When compared to rolling of the same cell type on E-selectin, rolling on tenascin was found to be smoother at all shear stresses tested, suggesting that cells formed a larger number of bonds on the tenascin substrate than on the E-selectin substrate. When protein plating densities were adjusted to give similar profiles of cell detachment under increasing shears, the density of tenascin was 8.5-fold greater than that of E-selectin. Binding to tenascin was not dependent on any molecules previously identified as tenascin receptors and is likely to involve a novel tenascin receptor on lymphocytes. We postulate that the ability of tenascin to support lymphocyte rolling may reflect its ability to support cell migration and that this interaction may be used by lymphocytes migrating through secondary lymphoid organs.

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Localization of antibody binding sites with immunoblotting and inhibition of cell binding to tenascin. (a) Recognition of tenascin and recombinant tenascin proteins by antibodies. Proteins were spotted on nitrocellulose and detected with the indicated antibodies  and alkaline phosphatase–anti-Ig. Antibodies included normal rabbit serum (NRS); rabbit antibodies against intact tenascin (α-TN), recombinant TNfnA-D (α-TNfnA-D), recombinant TNfn1-5 (α-TNfn1-5), and a mixture of recombinant TNfn6-8 and TNfbg (TNfn68fbg); a negative IgG control mAb; and mAb M112, M139, M168, and M171 against tenascin. (b) Inhibition of SKW3 cell binding to  plastic-adsorbed tenascin by tenascin antibodies. Binding of SKW3 cells was measured before and after treatment of the spots of plasticimmobilized tenascin with polyclonal antisera (1:50), ascites (1:100), or purified mAb (50 μg/ml) for 10 min. Binding to control NRStreated spot was 250 cells and binding to IgG control-treated spot was 224 cells. Mean values of two experiments are shown, with the  range indicated by bars.
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Figure 4: Localization of antibody binding sites with immunoblotting and inhibition of cell binding to tenascin. (a) Recognition of tenascin and recombinant tenascin proteins by antibodies. Proteins were spotted on nitrocellulose and detected with the indicated antibodies and alkaline phosphatase–anti-Ig. Antibodies included normal rabbit serum (NRS); rabbit antibodies against intact tenascin (α-TN), recombinant TNfnA-D (α-TNfnA-D), recombinant TNfn1-5 (α-TNfn1-5), and a mixture of recombinant TNfn6-8 and TNfbg (TNfn68fbg); a negative IgG control mAb; and mAb M112, M139, M168, and M171 against tenascin. (b) Inhibition of SKW3 cell binding to plastic-adsorbed tenascin by tenascin antibodies. Binding of SKW3 cells was measured before and after treatment of the spots of plasticimmobilized tenascin with polyclonal antisera (1:50), ascites (1:100), or purified mAb (50 μg/ml) for 10 min. Binding to control NRStreated spot was 250 cells and binding to IgG control-treated spot was 224 cells. Mean values of two experiments are shown, with the range indicated by bars.

Mentions: To determine the region of the tenascin molecule that binds to lymphocytes under flow, we used a panel of antibodies against tenascin. Rabbit polyclonal antibodies were prepared against recombinant tenascin fragments TNfn1-5, TNfnA-D, and TNfn6-8+TNfbg (Fig. 3). Monoclonal antibodies recognizing tenascin were produced by immunizing mice with preparations of human tonsillar stroma. mAbs were screened by immunohistochemistry, and those that recognized high endothelial venule (HEV) or ECM components of secondary lymphoid organs were further analyzed. A significant proportion of mAbs made in this way was found to recognize tenascin. The sites of antibody binding to tenascin were mapped by immunoblotting of recombinant tenascin proteins (Fig 4 a). Rabbit antibodies showed the expected specificity, and additionally, antibodies to TNfn6-8+TNfbg crossreacted weakly with TNfnA-D and TNfn1-5. mAbs M139 and M171 were specific for TNfn1-5, and mAb M112 reacted with TNfn1-5 and more weakly with TNfn6-8fbg and TNfbg. mAb M168 reacted with TNfn6-8fbg and TNfbg and not with TNfn6-8 and therefore appears specific for the fibrinogen-like terminal domain of tenascin. Antisera produced by immunizing rabbits with the TNfn1-5 and TNfnA-D tenascin proteins had little effect on the binding of SKW3 cells to intact tenascin under flow, while antisera raised against the TNfn6-8fbg recombinant tenascin protein completely blocked binding (Fig. 4 b). Antibodies M112, M139, and M171 to TNfn1-5 domains of tenascin did not significantly inhibit SKW3 cell binding. By contrast, mAb M168 to the terminal fbg domain of tenascin reduced binding by 99%. These studies suggest that the terminal fibrinogen-like domain of tenascin is responsible for binding to SKW3 cells.


Tenascin supports lymphocyte rolling.

Clark RA, Erickson HP, Springer TA - J. Cell Biol. (1997)

Localization of antibody binding sites with immunoblotting and inhibition of cell binding to tenascin. (a) Recognition of tenascin and recombinant tenascin proteins by antibodies. Proteins were spotted on nitrocellulose and detected with the indicated antibodies  and alkaline phosphatase–anti-Ig. Antibodies included normal rabbit serum (NRS); rabbit antibodies against intact tenascin (α-TN), recombinant TNfnA-D (α-TNfnA-D), recombinant TNfn1-5 (α-TNfn1-5), and a mixture of recombinant TNfn6-8 and TNfbg (TNfn68fbg); a negative IgG control mAb; and mAb M112, M139, M168, and M171 against tenascin. (b) Inhibition of SKW3 cell binding to  plastic-adsorbed tenascin by tenascin antibodies. Binding of SKW3 cells was measured before and after treatment of the spots of plasticimmobilized tenascin with polyclonal antisera (1:50), ascites (1:100), or purified mAb (50 μg/ml) for 10 min. Binding to control NRStreated spot was 250 cells and binding to IgG control-treated spot was 224 cells. Mean values of two experiments are shown, with the  range indicated by bars.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139881&req=5

Figure 4: Localization of antibody binding sites with immunoblotting and inhibition of cell binding to tenascin. (a) Recognition of tenascin and recombinant tenascin proteins by antibodies. Proteins were spotted on nitrocellulose and detected with the indicated antibodies and alkaline phosphatase–anti-Ig. Antibodies included normal rabbit serum (NRS); rabbit antibodies against intact tenascin (α-TN), recombinant TNfnA-D (α-TNfnA-D), recombinant TNfn1-5 (α-TNfn1-5), and a mixture of recombinant TNfn6-8 and TNfbg (TNfn68fbg); a negative IgG control mAb; and mAb M112, M139, M168, and M171 against tenascin. (b) Inhibition of SKW3 cell binding to plastic-adsorbed tenascin by tenascin antibodies. Binding of SKW3 cells was measured before and after treatment of the spots of plasticimmobilized tenascin with polyclonal antisera (1:50), ascites (1:100), or purified mAb (50 μg/ml) for 10 min. Binding to control NRStreated spot was 250 cells and binding to IgG control-treated spot was 224 cells. Mean values of two experiments are shown, with the range indicated by bars.
Mentions: To determine the region of the tenascin molecule that binds to lymphocytes under flow, we used a panel of antibodies against tenascin. Rabbit polyclonal antibodies were prepared against recombinant tenascin fragments TNfn1-5, TNfnA-D, and TNfn6-8+TNfbg (Fig. 3). Monoclonal antibodies recognizing tenascin were produced by immunizing mice with preparations of human tonsillar stroma. mAbs were screened by immunohistochemistry, and those that recognized high endothelial venule (HEV) or ECM components of secondary lymphoid organs were further analyzed. A significant proportion of mAbs made in this way was found to recognize tenascin. The sites of antibody binding to tenascin were mapped by immunoblotting of recombinant tenascin proteins (Fig 4 a). Rabbit antibodies showed the expected specificity, and additionally, antibodies to TNfn6-8+TNfbg crossreacted weakly with TNfnA-D and TNfn1-5. mAbs M139 and M171 were specific for TNfn1-5, and mAb M112 reacted with TNfn1-5 and more weakly with TNfn6-8fbg and TNfbg. mAb M168 reacted with TNfn6-8fbg and TNfbg and not with TNfn6-8 and therefore appears specific for the fibrinogen-like terminal domain of tenascin. Antisera produced by immunizing rabbits with the TNfn1-5 and TNfnA-D tenascin proteins had little effect on the binding of SKW3 cells to intact tenascin under flow, while antisera raised against the TNfn6-8fbg recombinant tenascin protein completely blocked binding (Fig. 4 b). Antibodies M112, M139, and M171 to TNfn1-5 domains of tenascin did not significantly inhibit SKW3 cell binding. By contrast, mAb M168 to the terminal fbg domain of tenascin reduced binding by 99%. These studies suggest that the terminal fibrinogen-like domain of tenascin is responsible for binding to SKW3 cells.

Bottom Line: Tenascin has been reported to have both adhesive and anti-adhesive effects in static assays.When compared to rolling of the same cell type on E-selectin, rolling on tenascin was found to be smoother at all shear stresses tested, suggesting that cells formed a larger number of bonds on the tenascin substrate than on the E-selectin substrate.When protein plating densities were adjusted to give similar profiles of cell detachment under increasing shears, the density of tenascin was 8.5-fold greater than that of E-selectin.

View Article: PubMed Central - PubMed

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

ABSTRACT
Tenascin is a large extracellular matrix molecule expressed at specific sites in the adult, including immune system tissues such as the bone marrow, thymus, spleen, and T cell areas of lymph nodes. Tenascin has been reported to have both adhesive and anti-adhesive effects in static assays. We report here that tenascin supports the tethering and rolling of lymphocytes and lymphoblastic cell lines under flow conditions. Binding was calcium dependent and was not inhibited by treatment of lymphocytes with O-glycoprotease or a panel of glycosidases including neuraminidase and heparitinase but was inhibited by treatment of cells with proteinase K. Binding was to the fibrinogen-like terminal domain of tenascin as determined by antibody blocking studies and binding to recombinant tenascin proteins. When compared to rolling of the same cell type on E-selectin, rolling on tenascin was found to be smoother at all shear stresses tested, suggesting that cells formed a larger number of bonds on the tenascin substrate than on the E-selectin substrate. When protein plating densities were adjusted to give similar profiles of cell detachment under increasing shears, the density of tenascin was 8.5-fold greater than that of E-selectin. Binding to tenascin was not dependent on any molecules previously identified as tenascin receptors and is likely to involve a novel tenascin receptor on lymphocytes. We postulate that the ability of tenascin to support lymphocyte rolling may reflect its ability to support cell migration and that this interaction may be used by lymphocytes migrating through secondary lymphoid organs.

Show MeSH
Related in: MedlinePlus