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RhoA Is Involved in LFA-1 Extension Triggered by CXCL12 but Not in a Novel Outside-In LFA-1 Activation Facilitated by CXCL9¹

Ronit Pasvolsky^*, Valentin Grabovsky^*, Cinzia Giagulli, Ziv Shulman^*, Revital Shamri^*, Sara W. Feigelson^*, Carlo Laudanna and Ronen Alon^2,*

^* 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

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 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 Universitáe 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{at}weizmann.ac.il

³ Abbreviations used in this paper: GPCR, G protein-coupled receptor; HEV, high endothelial venule; DAG, diacylglycerol; PIP₂, 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.