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Lymphocytes Perform Reverse Adhesive Haptotaxis Mediated By © 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs242883. doi:10.1242/jcs.242883 RESEARCH ARTICLE Lymphocytes perform reverse adhesive haptotaxis mediated by LFA-1 integrins Xuan Luo1, Valentine Seveau de Noray1, Laurene Aoun1, Martine Biarnes-Pelicot1, Pierre-Olivier Strale2, Vincent Studer3, Marie-Pierre Valignat1 and Olivier Theodoly1,* ABSTRACT ligands ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 Cell guidance by anchored molecules, or haptotaxis, is crucial in (vascular cell adhesion molecule-1). ICAM-1 is particularly development, immunology and cancer. Adhesive haptotaxis, or important for transmigration in nearby portals (Park et al., 2010; guidance by adhesion molecules, is well established for Sumagin and Sarelius, 2010), suggesting that integrins might guide mesenchymal cells such as fibroblasts, whereas its existence leukocytes towards and through extravasation sites. After remains unreported for amoeboid cells that require less or no diapedesis, leukocytes migrate in three-dimensional environments adhesion in order to migrate. We show that, in vitro, amoeboid of tissues and organs, where integrin-mediated adhesion was shown human T lymphocytes develop adhesive haptotaxis mediated by to be dispensable for motility (Lämmermann et al., 2008; Park et al., densities of integrin ligands expressed by high endothelial venules. 2010). Nevertheless, integrins remain crucial in vivo for efficient Moreover, lymphocytes orient towards increasing adhesion with VLA- homing, and the microenvironment of inflamed tissues is known to enhance motility in an integrin-dependent manner (Hons et al., 4 integrins (also known as integrin α4β1), like all mesenchymal cells, but towards decreasing adhesion with LFA-1 integrins (also known as 2018; Lämmermann et al., 2013; Overstreet et al., 2013). An important role of integrins has been further established in the integrin αLβ4), which has not previously been observed. This counterintuitive ‘reverse haptotaxis’ cannot be explained by existing context of tumors, like the chronic lymphocytic leukemia (CLL), α β mechanisms of mesenchymal haptotaxis involving either competitive where VLA-4 (also known as integrin 4 1) expression on CLL anchoring of cell edges under tension or differential integrin-activated cells had prognostic impact by favoring B cell adhesion and growth of lamellipodia, because they both favor orientation towards proliferation on stromal cells (Burger et al., 2009; Gattei et al., increasing adhesion. The mechanisms and functions of amoeboid 2008). Furthermore, the traffic of lymphocytes in lymph nodes is adhesive haptotaxis remain unclear; however, multidirectional orchestrated by specialized stromal cells expressing integrin ligands integrin-mediated haptotaxis might operate around transmigration and chemokines, and it has been hypothesized that integrins ports on endothelia, stromal cells in lymph nodes, and inflamed tissue participate in guiding lymphocytes on the stromal network where integrin ligands are spatially modulated. (Bajénoff et al., 2006). Altogether, guided migration by integrins is potentially relevant in several steps of leukocyte survey functions; KEY WORDS: Haptotaxis, Cell guidance, Amoeboid migration, however, there is no direct evidence yet that integrins can guide Lymphocyte, Integrins, LFA-1 leukocytes in environments that present modulations in adhesion ligand density. The first goal of this work was therefore to INTRODUCTION investigate the existence and conditions of emergence of integrin- An efficient immune response requires the rapid recruitment of mediated guidance in amoeboid cells such as leukocytes. circulating leukocytes from the blood system to lymph nodes and Directed motion is a hallmark of the immune response, and inflamed tissues. Leukocytes arrest on the endothelium of blood several guiding cues are well characterised. Leukocytes are sensitive vessels (the cellular monolayer covering the lumen) then migrate to mechanical cues such as blood flow (mechanotaxis) (Dominguez spontaneously with a rapid ‘crawling’ mode and eventually cross et al., 2015; Gorina et al., 2014; Valignat et al., 2013, 2014), to the endothelium – an event called diapedesis. On the walls of blood soluble biochemical cues including bacterial fragments and vessels, leukocytes crawl distances of several tens or hundreds of chemokines (chemotaxis) (Liu et al., 2012; Malawista et al., micrometers, proposedly in search for optimal diapedesis sites 2000; Malet-Engra et al., 2015; Massena and Phillipson, 2012; (Massena and Phillipson, 2012; Sumagin and Sarelius, 2010; Poznansky et al., 2000; Tharp et al., 2006), and to anchored Sumagin et al., 2010) called ‘transmigratory cups’ (Carman and signaling molecules such as the chemokines CCL21 and IL8 Springer, 2004) or ‘portals’ (Sumagin and Sarelius, 2010). These (haptotaxis) (Canton, 2008; Roy et al., 2017; Schwarz et al., 2017; sites are composed of endothelial cells with vertical microvilli-like van Gils et al., 2013; Weber et al., 2013). Directed motion guided by projections enriched in adhesion molecules, notably the integrin adhesion molecules is documented for mesenchymal cells, such as cancer cells, fibroblasts, muscle cells and endothelial cells (King 1LAI, Laboratory adhesion and inflammation, Aix Marseille University, CNRS, et al., 2016; MacNearney et al., 2016; Oudin et al., 2016; Wen et al., INSERM, F-13-009 Marseille, France. 2Alvéole, F-75013 Paris, France. 3 University 2015; Wu et al., 2012), as well as for Schwann cells (Motta et al., of Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297, F-33000 Bordeaux, France. 2019) and neurons (Aznavoorian et al., 1990; Brandley and Schnaar, 1989; Carter, 1965, 1967; Klominek et al., 1993; *Author for correspondence ([email protected]) Mccarthy and Furcht, 1984; O’Connor et al., 1990; Smith et al., O.T., 0000-0001-8787-3709 2004; Thibault et al., 2007), but it has not been demonstrated yet for amoeboid cells in general or for leukocytes in particular. For clarity, Handling Editor: Arnoud Sonnenberg hereafter we refer to any guidance phenotype triggered by anchored Received 12 December 2019; Accepted 13 July 2020 molecules as ‘haptotaxis’, and to the guidance phenotypes Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs242883. doi:10.1242/jcs.242883 specifically triggered by adhesion molecules as ‘adhesive haptotaxis’ phenotype, and a novel mechanism remains to be haptotaxis’. For mesenchymal migration, adhesive haptotaxis was deciphered. initially explained by a tug-of-war in the cell adherence zone; the areas with lower grip destabilizing spontaneously under traction in RESULTS favor of areas with a higher grip (Caballero et al., 2015; Carter, Protein printing yields adherent substrates with modulated 1965, 1967; O’Connor et al., 1990). Mesenchymal cells are indeed densities of integrin ligands characterized by strong cell–substrate adhesion mediated by mature In order to pattern substrates with controlled amounts of adhesion focal adhesions and by strong traction forces transmitted to the molecules, we used a set-up of light-induced molecular adsorption, substrate by contractile actin stress fibers. In contrast, amoeboid or LIMA (Strale et al., 2016), which optically coupled a 375 nm UV cells migrate at high speed with weak adhesion and low traction light source with a digital micro-mirror device (DMD) on an forces transferred to the substrate (Lämmermann et al., 2008; Paluch inverted microscope (Fig. 1A). A specific patterning protocol of et al., 2016; Ricoult et al., 2015; Smith et al., 2007). Leukocytes can ‘subtractive printing’ was developed to engineer substrates with even swim without interaction with a substrate (Aoun et al., 2019 modulated and integrin-specific adherence for lymphocytes. Glass preprint). A tug-of-war mechanism, with an interplay between coverslips were first treated with aminopropyltriethoxysilane strong adhesion and high traction, is therefore irrelevant for (APTES), then sequentially incubated with solutions of protein A, amoeboid cells, and the existence of adhesive haptotaxis is all but bovine serum albumin (BSA) and chimeric Fc–ICAM-1 or Fc– evident for leukocytes. VCAM-1 (Fig. 1B). UV illumination was then used to degrade the Adhesive haptotaxis might, however, rely on mechanisms more functionality of ICAM-1 or VCAM-1 in presence of the sophisticated than a tug-of-war, especially when integrins are involved photoactivator. Degradation was UV-dose dependent, and gray- (King et al., 2016). Integrins are indeed more than adhesion molecules, leveled illumination allowed us to obtain substrates with gradual as they can transduce signal when they engage to their ligands and are adhesion properties. Fig. 1C shows an immunofluorescence submitted to a force – a phenomenon called mechanotransduction (Ross microscopy image of an ICAM-1 printed substrate, illuminated et al., 2013; Zaidel-Bar et al., 2005). Adhesive haptotaxis mediated by with stripes of different UV doses. The maximum dose was of 800 integrins can therefore involve signal transduction by integrins relayed mJ/mm2, and the pattern of illumination corresponded to nine by internal signaling pathways to transform external cues into stripes of doses ranging between 10, 20, 30, 40, 50, 60, 70, 80, 90 cytoskeletal reorganization, as in chemotaxis (Artemenko et al., 2016; and 100% of the maximum dose intercalated with stripes without Aznavoorian et al., 1990; Klominek et
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