In Vivo Analysis of Uropod Function during Physiological T Cell Trafficking Silvia F. Soriano, Miroslav Hons, Kathrin Schumann, Varsha Kumar, Timo J. Dennier, Ruth Lyck, Michael Sixt This information is current as and Jens V. Stein of September 26, 2021. J Immunol published online 27 July 2011 http://www.jimmunol.org/content/early/2011/07/27/jimmun ol.1100935 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2011/07/27/jimmunol.110093 Material 5.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 27, 2011, doi:10.4049/jimmunol.1100935 The Journal of Immunology In Vivo Analysis of Uropod Function during Physiological T Cell Trafficking Silvia F. Soriano,*,1 Miroslav Hons,*,1 Kathrin Schumann,† Varsha Kumar,* Timo J. Dennier,* Ruth Lyck,* Michael Sixt,†,‡ and Jens V. Stein* Migrating lymphocytes acquire a polarized phenotype with a leading and a trailing edge, or uropod. Although in vitro experiments in cell lines or activated primary cell cultures have established that Rho-p160 coiled-coil kinase (ROCK)-myosin II-mediated uropod contractility is required for integrin de-adhesion on two-dimensional surfaces and nuclear propulsion through narrow pores in three-dimensional matrices, less is known about the role of these two events during the recirculation of primary, nonacti- vated lymphocytes. Using pharmacological antagonists of ROCK and myosin II, we report that inhibition of uropod contractility blocked integrin-independent mouse T cell migration through narrow, but not large, pores in vitro. T cell crawling on chemokine- coated endothelial cells under shear was severely impaired by ROCK inhibition, whereas transendothelial migration was only Downloaded from reduced through endothelial cells with high, but not low, barrier properties. Using three-dimensional thick-tissue imaging and dynamic two-photon microscopy of T cell motility in lymphoid tissue, we demonstrated a significant role for uropod contractility in intraluminal crawling and transendothelial migration through lymph node, but not bone marrow, endothelial cells. Finally, we demonstrated that ICAM-1, but not anatomical constraints or integrin-independent interactions, reduced parenchymal motility of inhibitor-treated T cells within the dense lymphoid microenvironment, thus assigning context-dependent roles for uropod con- traction during lymphocyte recirculation. The Journal of Immunology, 2011, 187: 000–000. http://www.jimmunol.org/ he high diversity of the Ag-receptor repertoire and the along the luminal surface of HEV, which is covered with high resulting low frequency of Ag-specific T cells in an or- levels of the CCL21 chemokine (4). Binding of CCL21 to its re- T ganism pose an important logistic challenge, which is to ceptor CCR7 induces rapid (.1 s) conformational activation of quickly allow for efficient encounters between APCs and rare the LFA-1 integrin required for firm arrest of rolling lymphocytes, specific T cells close to potential microbial entry sites. To meet and it induces over the next tens of seconds to few minutes the this challenge, strategically positioned secondary lymphoid organs, acquisition of a polarized phenotype (5–7). Polarized lymphocytes including spleen, peripheral lymph nodes (PLNs), and bone then crawl in an ICAM-1–dependent manner along the HEV by guest on September 26, 2021 marrow (BM), have evolved, and these secondary lymphoid organs surface prior to transendothelial migration (TEM) through narrow serve as meeting points for tissue-borne APCs and continuously pores of the endothelial barrier and negotiate their passage passing lymphocytes (1–3). Recruitment of blood-borne lympho- through the cuff of basement membrane meshworks surrounding cytes in PLNs occurs in high endothelial venules (HEVs), spe- fibroblast reticular cells (FRCs) and occasional pericytes (8–12). cialized postcapillary venules characterized by their expression FRCs constitute the stromal backbone of the T cell area in the of peripheral node addressin (PNAd). PNAd serves as a ligand for PLN parenchyma and form a three-dimensional (3D) network, L-selectin expressed on naive lymphocytes, allowing cells to roll which T cells use as contact guidance cues during their scanning of APCs inside the tightly packed lymphoid microenvironment (13). This migration is mediated, in part, by ICAM-1 and CCL21 † *Theodor Kocher Institute, University of Bern, 3012 Bern, Switzerland; Hof- presumably presented on the FRC surface, before T cells exit PLNs schneider Group Leukocyte Migration, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany; and ‡Institute of Science and Technology Austria, 3400 Klos- through efferent lymphatic vessels after an average dwelling time of terneuburg, Austria a few hours (14–16). CXCL12 and a4 integrins are likely candi- 1S.F.S. and M.H. contributed equally to this study. dates for inducing firm adhesion and guiding naive lymphocytes Received for publication April 6, 2011. Accepted for publication June 24, 2011. through BM sinusoids into BM parenchyme (17). This work was supported by a Marie Curie Excellence grant (MEXT-CT-2005- The chemokine-driven rapid transition of spherical blood-borne 025405) from the European Union and Swiss National Foundation Grants 31-120640 lymphocytes to fully polarized cells with a leading edge and a and CRSII3-125447 (to J.V.S.). uropod is orchestrated by the concerted activation of small Address correspondence and reprint requests to Dr. Jens V. Stein, Theodor Kocher GTPases of the Ras and Rho family (7, 18). Although Rac1 and Institute, University of Bern, Freiestrasse 1, 3012 Bern, Switzerland. E-mail address: [email protected] Rac2 direct T cell protrusion at the leading edge (19), the for- The online version of this article contains supplemental material. mation of the uropod depends on the activity of Rho, and, at least in part, of its downstream effector Rho-p160 coiled-coil kinase Abbreviations used in this article: BM, bone marrow; CMAC, 7-amino-4-chlorome- thylcoumarin; CMFDA, 5-chloromethyfluorescein diacetate; CMTMR, (5-(and-6)- (ROCK) (5, 20). Nonmuscle myosin IIA also localizes to the (((4-chloromethyl)benzoyl)amino) tetramethylrhodamine); 2D, two-dimensional; 3D, uropod, where it promotes actin–myosin contractions downstream three-dimensional; 3-DIF, 3D immunofluorescence; FRC, fibroblast reticular cell; HEV, high endothelial venule; PFA, paraformaldehyde; PLN, peripheral lymph node; of ROCK-dependent phosphorylation of the myosin L chain, ei- 2PM, two-photon microscopy; pMBMEC, primary mouse brain microvascular endo- ther directly or indirectly by inactivating MLC phosphatase (21, thelial cell; PNAd, peripheral node addressin; ROCK, Rho-p160 coiled-coil kinase; 22). In vitro experiments support an important role for the Rho– TEM, transendothelial migration; WT, wild type. ROCK–myosin signaling axis and uropod contraction during Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 leukocyte migration in at least three processes. First, in two- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100935 2 IN VIVO FUNCTION OF ROCK IN T CELLS dimensional (2D) systems, Rho–ROCK activity limits the adhe- Reagents sion interface, probably by promoting cortical tension. Therefore, Y27632 and blebbistatin were purchased from Calbiochem (San Diego, upon blocking ROCK activity using the pharmacological inhibi- CA). CCL21 and CXCL12 were provided by PeproTech (London, U.K.), tor Y27632 or myosin II ATPase activity with blebbistatin, cells and CXCL13 was from R&D Systems (Minneapolis, MN). CFSE, 5- spread more, even on nonadhesive substrates, thereby limiting chloromethyfluorescein diacetate (CMFDA; CellTracker Green), 7-amino- migration speed (23–27). Second, myosin contractility is required 4-chloromethylcoumarin (CMAC; CellTracker Blue), and (5-(and-6)-(((4- chloromethyl)benzoyl)amino) tetramethylrhodamine) (CMTMR; Cell- for LFA-1 de-adhesion during migration on ICAM-1–coated 2D Tracker Orange) were purchased from Molecular Probes (Eugene, OR). surfaces. Upon cell treatment with Y27632 or blebbistatin, leu- kocytes remain attached with their uropod, restraining net cell Lymphocyte-polarization assay displacement promoted by the probing leading edge (28–30). Fi- T cells were treated with DMSO (wild type [WT]), Y27632 (20 mM), or nally, uropod contractility is required for integrin-independent blebbistatin (30 mM) for 1 h; stimulated in suspension with 100 nM dendritic cell squeezing through 3D meshworks. This is mainly CCL21 final concentration; and immediately plated on fibronectin-coated due to the trapping of the relatively large nucleus in narrow pores; chamber slides for incubation at 37˚C.