RhoA Is Involved in LFA-1 Extension Triggered by CXCL12 but Not in a Novel Outside-In LFA-1 Activation Facilitated by CXCL9 1 Ronit Pasvolsky,* Valentin Grabovsky,* Cinzia Giagulli, † Ziv Shulman,* Revital Shamri,* Sara W. Feigelson,* Carlo Laudanna, † and Ronen Alon 2 * Chemokines presented on endothelial tissues instantaneously trigger LFA-1-mediated arrest on ICAM-1 via rapid inside-out and outside-in (ligand-driven) LFA-1 activation. The GTPase RhoA was previously implicated in CCL21-triggered LFA-1 affinity triggering in murine T lymphocytes and in LFA-1-dependent adhesion strengthening to ICAM-1 on Peyer’s patch high endothelial venules stabilized over periods of at least 10 s. In this study, we show that a specific RhoA 23/40 effector region is vital for the initial LFA-1-dependent adhesions of lymphocytes on high endothelial venules lasting 1–3 s. Blocking the RhoA 23/40 region in human T lymphocytes in vitro also impaired the subsecond CXCL12-triggered LFA-1-mediated T cell arrest on ICAM-1 by eliminating the rapid induction of an extended LFA-1 conformational state. However, the inflammatory chemokine CXCL9 triggered robust LFA-1-mediated T lymphocyte adhesion to ICAM-1 at subsecond contacts independently of the RhoA 23/40 region. CXCL9 did not induce conformational changes in the LFA-1 ectodomain, suggesting that particular chemokines can activate LFA-1 through outside-in post ligand binding stabilization changes. Like CXCL9, the potent diacylglycerol-dependent protein kinase C agonist PMA was found to trigger LFA-1 adhesiveness to ICAM-1 also without inducing integrin extension or an a priori clustering and independently of the RhoA 23/40 region. Our results collectively suggest that the 23/40 region of RhoA regulates chemokine- induced inside-out LFA-1 extension before ligand binding, but is not required for a variety of chemokine and non-chemokine signals that rapidly strengthen LFA-1-ICAM-1 bonds without an a priori induction of high-affinity extended LFA-1 conformations. The Journal of Immunology, 2008, 180: 2815–2823. T he arrest of tethered or rolling leukocytes on target endo- thelium is nearly exclusively mediated by members of the integrin superfamily and their endothelial Ig superfamily ligands (1). Activating cytokines are key players in this arrest, because integrins do not mediate firm shear-resistant adhesions unless activated in situ at the leukocyte-endothelial interface. Key checkpoints in integrin activation on many types of leukocytes include the rapid modulation of integrin affinity and valency to endothelial ligands (2– 4). In situ activation of the  integrin het- erodimer is rapidly triggered by the binding of specialized endo- thelial chemokines to G protein-coupled receptors (GPCRs) 3 (5– 9). In vitro studies suggest that lymphocyte GPCRs such as CXCR4, CCR7 and CXCR3, when occupied by surface-presented rather than soluble ligands, can transmit a series of cytoplasmic signals that result in LFA-1 activation (8, 10). This signaling oc- curs within subseconds through Gi heterotrimeric G protein acti- vation. A critical step in integrin activation is the almost instanta- neous stabilization of an extended conformational state that must be rapidly coupled to the ligand-induced activation of the integrin headpiece (10, 11). Many of the downstream Gi targets suggested to mediate this initial integrin activation step are distinct from those participating in rapid integrin activation at lymphocyte-endothelial contacts sub- jected to shear forces as they are involved in more prolonged in- tegrin mobility, clustering, and cell-spreading events (12). Only few Gi targets triggered by endothelial-displayed chemokines have so far been implicated in the initial, rapid integrin activation events of lymphocytes. Among them, the small GTPase RhoA was shown to be involved in rapid LFA-1 activation by the CCL21 and CXCL12 chemokines in lymphocytes (13). RhoA-mediated LFA-1 activation was argued to involve two modalities, the first, activation of a high-affinity LFA-1 state capable of binding soluble ICAM-1 (14), and the second, triggering of LFA-1 lateral mobility. Using Trojan peptides with sequences corresponding to distinct effector regions of RhoA, each able to block RhoA-dependent sig- naling in a domain-selective manner, it was shown that RhoA- triggered LFA-1 affinity and lymphocyte adhesion strengthening on ICAM-1 involves the 23/40 effector domain of this GTPase (13). A distinct RhoA effector region, encompassing residues 92/ 119, was shown to regulate LFA-1 mobility and was argued to augment firm lymphocyte adhesions to low-density ICAM-1 (13). Nevertheless, the contribution of each RhoA modality to the very *Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; and † Department of Pathology, Division of General Pathology, School of Medicine and Center for Biomedical Computing, University of Verona, Verona, Italy Received for publication September 17, 2007. Accepted for publication December 14, 2007. 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 study was supported by the Israel Science Foundation, the Minerva Foundation of Germany, by MAIN, the 6th Program of the European Community for Migration and Inflammation, and by Associazione Italiana per la Ricerca sul Cancro 2006 and fondo 40% Ministero Universita ´e Ricerca 2006. R.A. is the Incumbent of The Linda Jacobs Chair in Immune and Stem Cell Research. 2 Address correspondence and reprint requests to Dr. Ronen Alon, Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address: ronen.alon@weizmann.ac.il 3 Abbreviations used in this paper: GPCR, G protein-coupled receptor; HEV, high endo- thelial venule; DAG, diacylglycerol; PIP 2 , phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; Bis, bisindolomaleimide I; CMFDA, 5-chloromethylfluorescein diacetate; CMTMR, 5- and 6-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; IDAS, I domain allosteric site; PP, Peyer’s patch; MFI, mean fluorescence intensity. Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 The Journal of Immunology www.jimmunol.org