A two-step adhesion cascade for T cell/endothelial cell interactions under flow conditions. D A Jones, … , C W Smith, L J Picker J Clin Invest. 1994;94(6):2443-2450. https://doi.org/10.1172/JCI117612. Research Article Neutrophil adherence to endothelial cells (ECs) under conditions of flow occurs in successive steps, including selectin- dependent primary adhesion and CD18-dependent secondary adhesion. We used a parallel-plate flow chamber to assess the steps in T cell adherence in vitro. On monolayers of L cells transfected with the EC adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1), or intercellular adhesion molecule-1 (ICAM-1), E-selectin was capable of mediating only primary adhesion, ICAM-1 was capable of mediating only secondary adhesion, and VCAM-1 was capable of mediating both primary and secondary adhesion. Studies using human umbilical vein EC monolayers stimulated for 24 h with IL-1 also revealed distinct primary and secondary steps in T cell adhesion under flow, and the secondary adhesion was inhibited > 90% by blocking both VCAM-1/alpha 4 beta 1 integrin and ICAM-1/CD18 integrin pathways. However, the primary adhesion under conditions of flow could not be attributed to any of the mechanisms known to support adhesion of leukocytes to ECs. Alone, this pathway was shown to mediate T cell rolling and was a necessary prerequisite for engagement of the two integrin pathways in this system. Thus, T cell adherence to 24-h IL-1-stimulated human umbilical vein ECs at venular wall shear stresses involves at least two successive steps, with clear molecular distinctions from the mechanisms accounting for neutrophil/EC adhesion. Find the latest version: https://jci.me/117612/pdf A Two-Step Adhesion Cascade for T Cell/Endothelial Cell Interactions under Flow Conditions David A. Jones, * Larry V. McIntire, * C. Wayne Smith, t and Louis J. Picker § *Cox Laboratory for Biomedical Engineering, Institute of Biosciences and Bioengineering, Department of Chemical Engineering, Rice University, Houston, Texas 77251-1892; tSperos P. Martel Laboratory of Leukocyte Biology, Department of Pediatrics, Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030; and 'Laboratory of Molecular Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9072 Abstract Introduction Neutrophil adherence to endothelial cells (ECs) under con- Detailed studies of acute inflammatory processes suggest that ditions of flow occurs in successive steps, including selectin- neutrophil/endothelial cell (EC)' interaction can be divided dependent primary adhesion and CD18-dependent second- into several successive steps. These steps include primary adhe- ary adhesion. We used a parallel-plate flow chamber to as- sion, in which free-flowing neutrophils bind and roll slowly sess the steps in T cell adherence in vitro. On monolayers along the surface of activated endothelium, secondary firm ad- of L cells transfected with the EC adhesion molecules E- hesion of the rolling cells, and subsequent transmigration. Dis- selectin, vascular cell adhesion molecule-1 (VCAM-1), or tinct families of adhesion molecules have been shown to partici- intercellular adhesion molecule-1 (ICAM-1), E-selectin was pate in the primary and secondary adhesive events for neutro- capable of mediating only primary adhesion, ICAM-1 was phils. Primary adhesion involves all three members of the capable of mediating only secondary adhesion, and VCAM- selectin family (1-5) and their oligosaccharide ligands, while 1 was capable of mediating both primary and secondary secondary adhesion involves 62 (CD18) integrins on neutro- adhesion. Studies using human umbilical vein EC mono- phils (1) and immunoglobulin gene superfamily counterrecep- layers stimulated for 24 h with IL-1 also revealed distinct tors on EC (primarily intercellular adhesion molecule-I primary and secondary steps in T cell adhesion under flow, [ICAM-1]) (2). Selectin-ligand interactions are activation in- and the secondary adhesion was inhibited > 90% by dependent, are rapid enough to bind free-flowing neutrophils, blocking both VCAM-1/a43, integrin and ICAM-1/CD18 and appear to break in such a way as to allow adherent leuko- integrin pathways. However, the primary adhesion under cytes to roll slowly along the endothelium (6, 7). In contrast, conditions of flow could not be attributed to any of the CD18 integrin-ligand interactions are activation dependent mechanisms known to support adhesion of leukocytes to (i.e., high avidity can be induced through a conformation ECs. Alone, this pathway was shown to mediate T cell rolling change), are not able to initiate adhesion under flow conditions, and was a necessary prerequisite for engagement of the two but can mediate highly stable adhesion under static conditions integrin pathways in this system. Thus, T cell adherence to or once binding is initiated by selectins (1, 4, 8, 9). 24-h IL-1-stimulated human umbilical vein ECs at venular Fewer details are known about the interactions between lym- wall shear stresses involves at least two successive steps, phocytes and EC during extravasation at sites of inflammation. with clear molecular distinctions from the mechanisms ac- The selectin and CD18 integrin adhesion pathways, as well as counting for neutrophil/EC adhesion. (J. Clin. Invest. 1994. other pathways not used by neutrophils, including the a401 94:2443-2450.) Key words: cell adhesion molecules * vascu- integrin (VLA-4) and a4f37 integrin pathways (which preferen- - * lar endothelium * lymphocytes interleukin-1 monoclonal tially bind to the EC ligands vascular cell adhesion molecule- antibodies 1 [VCAM-1] and mucosal addressin cell adhesion molecule-I [ MAdCAM- 1 ], respectively), have been implicated in lympho- cyte-EC recognition (10, 11). However, most studies of the Address correspondence to David A. Jones, Cox Laboratory for Biomed- adhesive functions of these pathways have used assays which ical Engineering, Institute of Biosciences and Bioengineering, Rice Uni- do not discriminate the relative participation of these pathways versity, Houston, TX 77251-1892. in primary and secondary adhesion. It is therefore still not clear Received for publication 29 March 1994 and in revised form 5 August 1994. if lymphocyte-EC adhesive interactions under flow are analo- gous to the multistep process described for neutrophils, and if 1. Abbreviations used in this paper: CLA, cutaneous lymphocyte anti- so, which receptors contribute at each stage. gen; D-PBS, Dulbecco's phosphate-buffered saline; EC, endothelial cell; This study addresses these issues by examining the behavior HUVEC, human umbilical vein EC; ICAM-1, intercellular adhesion of purified peripheral blood T cells in static and flow adhesion molecule-i; L-ELAM, murine L cells expressing human E-selectin; L- assays using two model systems: (a) murine L cell monolayers ICAM-l; ICAM-1-transfected L cells; L-VCAM-1, L cells transfected transfected with a single human receptor (E-selectin, ICAM-1, with the seven-domain form of VCAM-1; MAdCAM-l, mucosal ad- or VCAM-1) to examine adhesion mediated by single adhesion dressin cell adhesion molecule-i; VCAM- 1, vascular cell adhesion mol- pathways; and (b) cultured human umbilical vein EC (HU- ecule-1. VEC) monolayers stimulated with cytokines to explore possible J. Clin. Invest. cooperative interactions between the various adhesion molecule © The American Society for Clinical Investigation, Inc. pairs in a physiologically relevant cell type. Monoclonal anti- 0021-9738/94/12/2443/08 $2.00 body (mAb) blocking was used to confirm the specificity of Volume 94, December 1994, 2443-2450 interaction using the transfected cells and to analyze the contri- T Cell Adhesion under Flow 2443 butions of particular adhesion pathways with the HUVECs. Our endothelial cell adhesion molecule-I (CD31). CL2 (anti-E-selectin, results confirm that T cell interactions with 24-h IL-I -stimu- CD62E), DREG56 (anti-L-selectin, CD62L), PJ-18 (binds an irrele- lated HUVEC monolayers involve distinct primary and second- vant endothelial cell epitope), Hermes-3 (anti-CD44), MECA-79, and ary adhesion steps and use both ICAM-l/CD18 integrin and HECA-452 (anti-CLA) have been described previously (14-17). VCAM-I/a431 integrin interactions. However, we were unable In static and flow adhesion assays, L cell and HUVEC monolayers to define the adhesion molecules that account for primary adhe- were treated with mAbs for 30 min at 37°C before use. T cells were treated with mAbs for 30 min at room temperature before use. Experi- sion of T cells to activated EC monolayers and suggest that this ments were also run in which T cells were treated with mAbs at 37 or event may be independent of the three known members of the 4°C instead of at room temperature to check for possible activating or selectin family. inhibitory side effects of the mAb treatments, and no differences in binding were observed. In most experiments, cells were pretreated with Methods antibodies before use in the flow system, since preliminary experiments showed identical results for pretreatment alone and pretreatment with Isolation of T lymphocytes. Human peripheral blood T cells were ob- inclusion of mAb in the flow buffer. tained by centrifugation of whole blood through Ficoll-Hypaque (Histo- Adhesion assays. Physiological flow conditions were produced in paque 1077; Sigma Chemical Co., St. Louis, MO) and negative selection vitro using a flow chamber with parallel-plate geometry as described using a T cell enrichment column (R&D Systems, Inc., Minneapolis, previously (2). The chamber produces a well-defined laminar flow over MN). Preparations were > 95% CD3+ and > 99% viable and were monolayers grown in 35-mm tissue culture dishes and was used for labeled when necessary using the fluorescent DNA-binding dye Hoescht both static and flow adhesion assays. 33342 (bis-benzamide; Sigma Chemical Co.; reference 12). Chymo- In the static assays, a suspension of 5 x 106 fluorescently labeled trypsin treatment to remove surface L-selectin was performed as de- T cells/ml in 37°C Dulbecco's phosphate-buffered saline (D-PBS) was scribed elsewhere (13).