Signaling Through Integrin LFA-1 Leads to Filamentous Actin Polymerization and Remodeling, Resulting in Enhanced T Cell Adhesion 1 Joanna C. Porter,* Madelon Bracke,* Andrew Smith,* Derek Davies, † and Nancy Hogg 2 * The integrins can activate signaling pathways, but the final downstream outcome of these pathways is often unclear. This study analyzes the consequences of signaling events initiated by the interaction of the leukocyte integrin LFA-1 with its ligand, dimeric ICAM-1. We show that the active form of LFA-1 regulates its own function on primary human T cells by directing the remodeling of the F-actin cytoskeleton to strengthen T cell adhesion to ICAM-1. Confocal microscopy revealed that both F-actin bundling and overall levels of F-actin are increased in the ICAM-1-adhering T cells. This increase in F-actin levels and change in F-actin distribution was quantitated for large numbers of T cells using the technique of laser scanning cytometry and was found to be significant. The study went on to show that clustering of conformationally altered LFA-1 is essential for the changes in F-actin, and a model is proposed in which clustered, high-avidity T cell LFA-1, interacting with multivalent ICAM-1, causes LFA-1 signaling, which results in F-actin polymerization and higher-order F-actin bundling. The findings demonstrate that LFA-1 acts not only as an adhesion receptor but also as a signaling receptor by actively initiating the F-actin reorganization that is essential for many T cell-dependent processes. The Journal of Immunology, 2002, 168: 6330 – 6335. I n the fibroblast, integrins are found in focal adhesions con- nected to prominent bundles of filamentous actin (F-actin), 3 known as stress fibers. This association with cytoskeletal structures is brought about by both integrin cross-linking and li- gand binding (1, 2). The clustering of fibroblast integrin  5  1 also leads to an accumulation of cytoskeletal proteins and 20 signal transduction molecules (1, 3). The interaction of integrins with the cytoskeleton is dynamic, depending on both integrin signaling and the state of the cytoskeletal organization within the cell (reviewed in Ref. 4). Much less is known about the association in leukocytes of in- tegrins with the cytoskeleton (5). Leukocytes are key migratory cells that relay information to other cells, and their correct func- tioning depends on successful cell:cell and cell:matrix contacts. For example, the leukocyte integrin, LFA-1, participates in the guided movement of leukocytes from the bloodstream across the vasculature toward the site of injury. LFA-1 also has a key role in the initiation of an immune response by providing adhesion strengthening at the immunological synapse where T cells make contact with APCs (6). The integrins on leukocytes, unlike those on fibroblasts, are constitutively inactive but receive activating stimuli through signaling from other cell surface receptors. Such receptor-mediated signaling not only will cause clustering of in- tegrins like LFA-1 (7) but will also activate other intracellular signaling pathways. This has made it difficult to distinguish such signals, some of which are necessary for integrin activation, from those that might emanate from the integrin itself upon ligand binding. In this study we have directly activated LFA-1 by manipulating the extracellular cation environment and have therefore bypassed the usual requirement for an intracellular integrin-activating event. This has allowed the signaling events initiated by integrin ligand binding to be analyzed in isolation from other intracellular signal- ing pathways (8, 9). The key finding is that the clustering of con- formationally altered LFA-1 can independently signal the forma- tion of an F-actin filament network, which is essential for T cell adhesion. Materials and Methods Abs, reagents, and cells mAb 38 (LFA-1 subunit, function blocking) was prepared in this labo- ratory. Other Abs were mAb G25.2 (LFA-1 subunit, non-function block- ing; BD Biosciences, Oxford, U.K.), mAb UCHT1 (CD3; (donated by Dr. P. Beverley, Jenner Vaccine Institute, Compton, U.K.), and rabbit anti- mouse-IgG (DAKO, Cambridge, U.K.). The human dimeric five-domain ICAM-1Fc chimeric protein was produced by previously described meth- ods (9). Labels 2',7'-bis-(carboxyethyl)-5(6')-carboxyfluorescein (Calbio- chem, Nottingham, U.K.), Alexa Fluor 488-, and TRITC-phalloidin were from Cambridge Biosciences (Cambridge, U.K.). Cytoskeletal inhibitors used were cytochalasin D (Sigma-Aldrich, Cambridge, Dorset, U.K.), la- trunculin A (from Dr. R Treisman, Cancer Research U.K., London, U.K.), and jasplakinolide (Cambridge Biosciences). Primary 10- to 14-day-cul- tured T lymphoblasts were prepared as previously described (9). Flow cytometry and assessment of soluble dICAM-1 binding T cells were washed in assay buffer (20 mM HEPES, 140 mM NaCl, 2 mg/ml glucose (pH 7.4)) and 2  10 5 T cells in 50 l assay buffer/0.1% BSA/1–5 mM Mg 2+ /1 mM EGTA were added to flexiwell plate wells (Dynex Technologies, Ashford, U.K.) containing 50 l of 2 M dimeric *Leukocyte Adhesion Laboratory and † Fluorescence Activated Cell Sorter Labora- tory, Cancer Research U.K. London Research Institute, London, United Kingdom Received for publication February 19, 2002. Accepted for publication April 3, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Cancer Research U.K. London Research Labora- tories. J.C.P. has been supported by a Medical Research Council Clinical Training Fellowship and M.B. is supported by a European Molecular Biology Organization Fellowship. 2 Address correspondence and reprint requests to Dr. Nancy Hogg, Leukocyte Ad- hesion Laboratory, Cancer Research U.K. London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, U.K. E-mail address: nancy.hogg@cancer.org.uk 3 Abbreviations used in this paper: F-actin, filamentous actin; dICAM, dimeric ICAM; PLL, poly-L-lysine. The Journal of Immunology Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00