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.
The integrin LFA-1 mediates activation-dependent leukocyte adhesion. The beta subunit cytoplasmic domain has been demonstrated previously to modulate the adhesiveness of LFA-1. To investigate whether the alpha subunit cytoplasmic domain is also involved in the regulation of LFA-1-adhesive function, we stably expressed cytoplasmic domain truncated forms of the alpha subunit in a Jurkat mutant (Jurkat-beta2.7) deficient in the endogenous LFA-1 alpha subunit and in K562 cells. Clones expressing similar levels of cell surface LFA-1 were tested for their ability to bind to immobilized ICAM-1. Truncation of the alpha subunit cytoplasmic domain before, but not after, the conserved GFFKR sequence motif resulted in constitutive ICAM-1 binding of both Jurkat-beta2.7 and K562 transfectants. However, truncation after the GFFKR motif reduced sensitivity to stimulation by PMA or stimulatory Abs. Internal deletion of the GFFKR motif, or point mutations of the Gly (G), the two Phe (F), or the Arg (R) in the GFFKR motif to Ala (A) rendered LFA-1 constitutively active. Mutation of the Lys (K) did not affect LFA-1 adhesion to ICAM-1. These findings indicate that the GFFKR motif maintains the low adhesive state of LFA-1, possibly by restraining the receptor conformation. We further demonstrate that the alpha subunit cytoplasmic domain and the conserved GFFKR motif are also required for efficient formation of LFA-1 alphabeta heterodimers.