Five Tumor Necrosis Factor-inducible Cell Adhesion Mechanisms on the Surface of Mouse Endothelioma Cells Mediate the Binding of Leukocytes

We have distinguished five TNF-ot-inducible cell adhesion mechanisms on microvasculature-derived endothelioma cells of the mouse which mediate the binding of different types of leukocytes. Three of these mechanisms could be identified as the mouse homologs of ICAM-1, VCAM-1, and E-selectin, of which the latter was defined by the novel mAb 21KC10. The fourth TNF-ot-inducible cell adhesion mechanism was blocked by antibodies specific for mouse P-selectin. We have recently shown that TNF-ot stimulates the synthesis of P-selectin in mouse endothelioma cells (A. Weller, S. Isenmann, D. Vestweber. 1992. J. Biol. Chem. 267:15176-15183). Here we show that this stimulation leads to maximal cell surface expression levels within 4 h after stimulation while the same endothelioma cells are also able to upregulate P-selectin at the cell surface within minutes after stimulation with PMA. Both effects are additive. The fifth TNF-induced cell adhesion mechanism is defined by mediating the binding to the mouse monocyte/macrophage cell line J774. This adhesion mechanism is not inhibited by antibodies against any of the other four CAMs; it functions well at 7°C (in contrast to ICAM-1 and VCAM-1) and it is as active after 16 h of TNF induction as after 4 h (in contrast to Eand P-selectin). Furthermore, this new adhesion mechanism only functions on two of three endothelioma cell lines and is undetectable on the third, although ICAM-1, VCAM-1, E-selectin, and P-selectin could be demonstrated to function well on this cell line. Thus, in addition to the three known TNF-inducible CAMs, ICAM-1, VCAM-1, and E-selectin, also P-selectin and a fifth, as yet molecularly undefined cell adhesion mechanism, are TNF inducible at the cell surface of mouse endothelioma cells. M IGRATION across the blood vessel wall is a prerequisite for leukocytes to mediate host defense mechanisms at sites of bacterial invasion or tissue damage. The central step in this migration process is the adhesion of leukocytes to activated endothelial cells lining the venules of such inflamed tissues. Cytokines and other inflammatory mediators induce cell adhesion molecules (CAMs) ~ on the surface of activated endothelial cells which allow leukocytes to recognize sites of inflammation and to bind to the blood vessel wall (Osborn, 1990). This adhesion process has been proposed to be based on a cascade of molecular interactions (Butcher, 1991; Shimizu et al., 1992) and is mediated by multiple pairs of CAMs. The selectins (Bevilacqua et al., 1991) are at present the best candidates for CAMs which function very early in the cell binding process and may even mediate the initial cell contact. Two of them, Eand P-selectin, are expressed on endothelial cells (Bevilacqua et al., 1989; Geng et al., 1. Abbreviations used in this paper: CAM, cell adhesion molecule; HUVEC, human umbilical vein endothelial cell; PCR, polymerase chain reaction; PMN, polymorphonuclear granulocytes. 1990), the third, L-selectin, is expressed on all leukocytes (Siegelman et al., 1989; Camerini et al., 1989). Antibody blocking studies have demonstrated, in vivo, that L-selectin is involved in "leukocyte-rolling" on the blood vessel wall which describes the initial, transient interactions of leukocytes with the surface of endothelial cells (von Andrian et al., 1991). This could also be the function of P-selectin which is stored in intracellular granula and is the most rapidly inducible endothelial CAM. It is transported to the cell surface within minutes upon stimulation by pre-inflammatory mediators, such as thrombin and histamine (Geng et al., 1990). For this selectin, binding to polymorphonuclear granulocytes (PMNs, neutrophils) in vitro could be demonstrated under shearing forces resembling those in flowing blood, while another endothelial CAM, ICAM-1, could only support PMN binding under static incubation conditions (Lawrence and Springer, 1991). In agreement with this, antibodies against CD18 (Mac-i) did not inhibit leukocyte rolling (von Andrian et al., 1991). Based on these in vitro and in vivo data, the selectins have been proposed to function as "rolling receptors" while ICAM-1 is probably essential for © The Rockefeller University Press, 0021-9525/93/05/655/10 $2.00 The Journal of Cell Biology, Volume 121, Number 3, May 1993 655-664 655 on N ovem er 7, 2017 jcb.rress.org D ow nladed fom leukocyte binding at a later stage in the cell adhesion process. In addition to mediating different molecular steps in the adhesion process, a variety of endothelial leukocyte receptors is necessary to allow the fine regulation of the extravasation of different categories of leukocytes. Eand P-selectin support the binding of PMNs and monocytes (Larsen et al., 1989; Geng et al., 1990; Bevilacqua et al., 1987) and also of certain subsets of lymphocytes (Picker et al., 1991; Shimizu et al., 1991; Damle et al., 1992). ICAM-1 binds to a larger repertoire of leukocytes including PMNs, monocytes, and lymphocytes (Kishimoto et al., 1989). VCAM-1 which is, like ICAM-1, also a member of the immunoglobulin super-gene family, mediates the binding of lymphocytes and monocytes, but not of PMNs to endothelial cells (Osborn et al., 1989). All these endothelial CAMs are inducible at the surface of endothelial cells partly by different mechanisms and with different kinetics. On cultured human umbilical vein endothelial cells (HUVECs), the synthesis of ICAM-1, VCAM-1, and E-selectin was found to be stimulated by TNF-c~ and IL1/$ (Osborn, 1990). In addition, -y-IFN was shown to stimulate the expression of ICAM-1 (Pober et al., 1986) and IL-4 was found to induce the expression of VCAM-1 (Thornhill et al., 1990). The kinetics of expression differs: E-selectin is rapidly induced with maximal expression levels at the cell surface 3-4 h after stimulation by TNF-ct and is down regulated within the next 20 h (Bevilacqua et al., 1987). In contrast, cell surface expression of ICAM-1 and VCAM-1 reaches maximal levels 4-6 h after TNF-a induction and these levels are maintained for more than 48 h (Dustin and Springer, 1991; Osborn, 1990). For P-selectin, only the thrombin and histamine induced transport regulation, which acts within minutes, has been described on HUVECs. In this report we have used various mouse endothelioma cell lines derived from microvasculature of different tissues (Williams et al., 1988) in order to search for TNF-inducible endothelial leukocyte adhesion molecules. We show, using cell adhesion assays, that in addition to ICAM-1, VCAM-1, and E-selectin, two more leukocyte adhesion mechanisms are TNF inducible at the surface of endothelial cells. We found recently that the synthesis of P-selectin, similar to that of E-selectin, is TNF inducible in mouse endothelioma cells (Weller et al., 1992). Here we report, that this TNFinduction leads to functionally detectable P-selectin at the cell surface and demonstrate that mouse endothelioma cells regulate the transport to the cell surface as well as the synthesis of P-selectin. The fifth TNF-inducible cell adhesion mechanism which we found on mouse endothelioma cells, mediates the binding of the mouse monocyte/macrophage cell line J774 and is not identical with any of the other four TNF-inducible CAMs. Materials and Methods