Mst1 controls lymphocyte trafficking and interstitial motility within lymph nodes
2009; Springer Nature; Volume: 28; Issue: 9 Linguagem: Inglês
10.1038/emboj.2009.82
ISSN1460-2075
AutoresKoko Katagiri, Tomoya Katakai, Yukihiko Ebisuno, Yoshihiro Ueda, Takaharu Okada, Tatsuo Kinashi,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoArticle2 April 2009free access Mst1 controls lymphocyte trafficking and interstitial motility within lymph nodes Koko Katagiri Koko Katagiri Department of Molecular Genetics, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka, Japan Search for more papers by this author Tomoya Katakai Tomoya Katakai Department of Molecular Genetics, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka, Japan Search for more papers by this author Yukihiko Ebisuno Yukihiko Ebisuno Department of Molecular Genetics, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka, Japan Search for more papers by this author Yoshihiro Ueda Yoshihiro Ueda Department of Molecular Genetics, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka, Japan Search for more papers by this author Takaharu Okada Takaharu Okada Department of Synthetic Chemistry and Biological Chemistry, Innovative Techno-Hub for Integrated Medical Bio-imaging, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto, Japan Research Unit for Immunodynamics, RIKEN, Research Center for Allergy and Immunology, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, Japan Search for more papers by this author Tatsuo Kinashi Corresponding Author Tatsuo Kinashi Department of Molecular Genetics, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka, Japan Search for more papers by this author Koko Katagiri Koko Katagiri Department of Molecular Genetics, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka, Japan Search for more papers by this author Tomoya Katakai Tomoya Katakai Department of Molecular Genetics, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka, Japan Search for more papers by this author Yukihiko Ebisuno Yukihiko Ebisuno Department of Molecular Genetics, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka, Japan Search for more papers by this author Yoshihiro Ueda Yoshihiro Ueda Department of Molecular Genetics, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka, Japan Search for more papers by this author Takaharu Okada Takaharu Okada Department of Synthetic Chemistry and Biological Chemistry, Innovative Techno-Hub for Integrated Medical Bio-imaging, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto, Japan Research Unit for Immunodynamics, RIKEN, Research Center for Allergy and Immunology, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, Japan Search for more papers by this author Tatsuo Kinashi Corresponding Author Tatsuo Kinashi Department of Molecular Genetics, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka, Japan Search for more papers by this author Author Information Koko Katagiri1, Tomoya Katakai1, Yukihiko Ebisuno1, Yoshihiro Ueda1, Takaharu Okada2,3 and Tatsuo Kinashi 1 1Department of Molecular Genetics, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka, Japan 2Department of Synthetic Chemistry and Biological Chemistry, Innovative Techno-Hub for Integrated Medical Bio-imaging, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto, Japan 3Research Unit for Immunodynamics, RIKEN, Research Center for Allergy and Immunology, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, Japan *Corresponding author. Corresponding author. Department of Molecular Genetics, Kansai Medical University, Fumizono-cho 10-15, Moriguchi-City, Osaka, 570-8506, Japan. Tel.: +81 6 6993 9445; Fax: +81 6 6994 6099; E-mail: [email protected] The EMBO Journal (2009)28:1319-1331https://doi.org/10.1038/emboj.2009.82 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The regulation of lymphocyte adhesion and migration plays crucial roles in lymphocyte trafficking during immunosurveillance. However, our understanding of the intracellular signalling that regulates these processes is still limited. Here, we show that the Ste20-like kinase Mst1 plays crucial roles in lymphocyte trafficking in vivo. Mst1−/− lymphocytes exhibited an impairment of firm adhesion to high endothelial venules, resulting in an inefficient homing capacity. In vitro lymphocyte adhesion cascade assays under physiological shear flow revealed that the stopping time of Mst1−/− lymphocytes on endothelium was markedly reduced, whereas their L-selectin-dependent rolling/tethering and transition to LFA-1-mediated arrest were not affected. Mst1−/− lymphocytes were also defective in the stabilization of adhesion through α4 integrins. Consequently, Mst1−/− mice had hypotrophic peripheral lymphoid tissues and reduced marginal zone B cells and dendritic cells in the spleen, and defective emigration of single positive thymocytes. Furthermore, Mst1−/− lymphocytes had impaired motility over lymph node-derived stromal cells and within lymph nodes. Thus, our data indicate that Mst1 is a key enzyme involved in lymphocyte entry and interstitial migration. Introduction Naive lymphocytes continuously circulate between secondary lymphoid tissues and vasculatures in search of foreign antigens (Butcher et al, 1999). Lymphocyte trafficking in the peripheral lymph nodes (LNs) is generally divided into four steps: entry through the high endothelial venules (HEV), interstitial migration, antigen scanning and exit through the efferent lymphatics (von Andrian and Mempel, 2003). Our understanding of the lymphocyte trafficking has been greatly advanced by the identification of adhesion molecules, chemokines, phospholipids and their receptors. During lymphocyte homing to the peripheral LN, naive lymphocytes are first captured by weak binding between L-selectin and a sulphated sialyl Lex-related carbohydrate, resulting in rolling on the HEV. When rolling lymphocytes are exposed to chemokines on the luminal side of the HEV, chemokine signalling coupled with Gi hetero-trimeric G proteins activates LFA-1, resulting in a complete stop. In gut-associated lymphoid tissues, α4β7 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) also support lymphocyte rolling and arrest. Within seconds to minutes, lymphocyte adhesion is stabilized and these cells transmigrate through the HEV into the tissues. Recent observations using multiphoton microscopy have revealed a robust random walk-like motility of naive lymphocytes within the LNs (Sumen et al, 2004). Lymphocytes appear to move in close proximity to intricate stromal networks composed of fibroblastic reticular cells in the paracortex and follicular dendritic cells (DCs) in the follicles (Bajenoff et al, 2006), suggesting that cell–cell interactions and/or tissue-derived factors enhance cell motility. Chemokines, in either gradients or nongradients, can activate integrins and induce lymphocyte polarized morphology, generating a leading edge and uropod and stimulating cell motility (Sanchez-Madrid and del Pozo, 1999; Stachowiak et al, 2006). Chemokine signalling is coupled with pertussis toxin-sensitive Gi/o hetero-trimeric G proteins, as illustrated by the ability of pertussis toxin treatment to inhibit chemokine-triggered integrin activation and attachment to the HEV (Butcher et al, 1999) as well as lymphocyte motility within the LN (Okada and Cyster, 2007). Gene targeting of Gαi2 impairs B-cell lymphocyte homing and interstitial motility (Han et al, 2005). Chemokines activate multiple signalling pathways, including the Ras/Rho family of small GTPases. For example, DOCK2 is a Rac guanine exchange factor that is critical for actin cytoskeletal rearrangements in lymphocytes (Fukui et al, 2001), integrin activation and trafficking in B cells (Nombela-Arrieta et al, 2004), and directional high-velocity lymphocyte movement within the LN (Nombela-Arrieta et al, 2007). Rho GTPase signalling also plays important roles in lymphocyte adhesion and migration (Laudanna et al, 2002). Indeed, the actin-nucleating and polymerization protein, mDia1, acts as a downstream Rho GTPase effector and is required for efficient chemokine-stimulated actin polymerization and T-cell trafficking in vivo (Sakata et al, 2007). Coronin1, an Arp2/3 inhibitory protein, is required for efficient T-cell trafficking in vivo, uropod formation and cell survival (Foger et al, 2006). In addition to actin regulators, the Rap1 small GTPase activates integrins and stimulates lymphocyte polarization and motility (Bos et al, 2001; Kinashi, 2005). A deficiency in the Rap1-specific CalDAG-GEFI through gene targeting in mice (Crittenden et al, 2004; Bergmeier et al, 2007) or abnormal splicing in human LAD-III patients (Pasvolsky et al, 2007), severely impairs the functions of leukocyte and platelet integrins. We reported earlier that RAPL (also known as RASSF5b), a Rap1-GTP binding protein expressed predominantly in lymphoid tissues, was required for lymphocyte adhesion through LFA-1 and α4 integrins and cell polarization triggered by chemokines. RAPL−/− lymphocytes showed defective lymphocyte homing to peripheral LN (Katagiri et al, 2003, 2004). We identified mammalian Ste20-like kinase (Mst1, also known as Stk4) as a critical RAPL effector. RAPL associates with Mst1 and regulates the localization and kinase activity of Mst1. Knockdown of Mst1 showed that it is required for polarized morphology and integrin-dependent lymphocyte adhesion (Katagiri et al, 2006). Mst1 was originally identified as a protein kinase homologous to yeast sterile 20 that acts downstream of the pheromone-linked G protein in the mating pathway (Creasy and Chernoff, 1995). The mammalian Ste20 group of kinases regulates diverse biological functions including proliferation, differentiation, apoptosis, morphogenesis and cytoskeletal rearrangement. Mst1 was reported earlier to be involved in apoptosis through caspase-mediated proteolytic activation and histone H2B phosphorylation (Cheung et al, 2003), or in a proapoptotic pathway of Ki-Ras through Nore1 (Khokhlatchev et al, 2002). Hippo, the Drosophila ortholog of mammalian Mst1 and Mst2, has been shown to be involved in cell contact inhibition and the determination of organ size through negative regulation of cell proliferation and apoptosis (Zeng and Hong, 2008). To clarify the physiological roles of Mst1 in primary lymphocytes and trafficking in vivo, we generated Mst1-deficient mice. Mst1-deficient mice grew normally with no gross abnormalities. However, peripheral lymphoid organs were hypoplastic. Mst1−/− lymphocyte trafficking to the peripheral LN was defective due to impairment at a transition from transient arrest to stable attachment of lymphocytes on HEV. Furthermore, Mst1−/− lymphocytes exhibited defective motility on LN-derived stromal cells and interstitial migration within LNs. Our study also reveals that Mst1 is required for proper localization of marginal zone B (MZB) cells and splenic CD11c+ DC. Thus, these results show the crucial roles of Mst1 in regulating lymphocyte trafficking. Results Hypoplastic secondary lymphoid organs in Mst1−/− mice The Mst1 protein was expressed predominantly in lymphoid tissues, and both T and B cells expressed Mst1. Mst1 was also detected at lower levels in the lung and brain but was below detectable levels in the kidney, liver, heart and skeletal muscle (Figure 1A). To generate Mst1−/− mice, mice carrying floxed Mst1 alleles (Mst1f/f) were produced by gene targeting, in which exon 1 containing the initiation codon was flanked with loxP sites, and then mated with CAG-Cre transgenic mice to delete exon 1 ubiquitously (Supplementary Figure 1). Southern blot analysis confirmed that exon 1 was completely deleted in Cre+ Mst1f/f mice (Supplementary Figure 1). Although Mst1 was comparably expressed in wild-type and Mst1f/f mice, the Mst1 protein was not detected in the tissues of Cre+ Mst1f/f mice with no apparent generation of a truncated Mst1 protein (Figure 1A). We hereafter refer to Cre+ Mst1f/f mice as Mst1−/− mice. Mst2, which is homologous to Mst1 (78% amino acid identity), was expressed in all tissues examined, and there were no concomitant changes in expression with the Mst1 deficiency (Figure 1A). Mst1−/− mice were born with expected Mendelian frequencies and grew normally without gross abnormalities. Analysis of Mst1−/− tissues revealed hypoplastic lymphoid tissues, whereas there were no apparent abnormalities in other tissues including the lung and brain (data not shown). The number of both T and B cells was decreased in the peripheral LN, Peyer's patches and spleen (Figure 1B, D and F). Immunohistology showed decreases in the sizes and cellular densities of B-cell follicles and T-cell areas of these secondary lymphoid tissues (Figure 1C, E and G). The architecture of lymphoid tissues, including the segregation of T and B cells, distribution of the HEV, and stromal networks appeared normal (Figure 1; Supplementary Figure 2). The proliferative response of Mst1−/− B cells stimulated with BCR ligation was normal, whereas T-cell growth responses were rather augmented compared with control cells when stimulated with TCR ligation (Supplementary Figure 3). There were no significant differences in spontaneous apoptosis of mature T and B cells, although double-positive thymocytes from Mst1−/− mice displayed slightly enhanced cell survival (see below). Therefore, proliferation and apoptosis could not account for the reduced lymphocyte numbers in lymphoid tissues. Figure 1.Hypoplastic lymphoid organs in Mst1-deficient mice. (A) Expression of Mst1 and Mst2 in organs of wild-type (+/+), Mst1flox/flox (f/f) and Cre+ Mst1flox/flox (−/−) mice. Tubulin served as a loading control. (B) Total and CD3+ and B220+ subset cell numbers in the inguinal lymph nodes. Total and subset numbers of axillary, popliteal, cervical and mesenteric lymph nodes in Mst1-deficient (−/−) mice were decreased to similar extents. n=5 for each, *P<0.001, **P<0.005, compared with the corresponding Mst1flox/flox (f/f) fractions. (C) Immunofluorescence staining of frozen tissue sections of axillary lymph nodes for B cells (B220; green), T cells (CD3; red) and laminin (blue). (D) Total and CD3+ and B220+ subset cell numbers in Peyer's patches. n=5 for each, *P<0.001, compared with the corresponding Mst1flox/flox (f/f) fractions. (E) Immunofluorescence staining of frozen tissue sections of Peyer's patches for B cells (B220; green), T cells (CD3; red) and laminin (blue). (F) Total and CD3+ and B220+ subset cell numbers in spleens. n=5 for each, *P<0.01, **P<0.005, compared with the corresponding Mst1flox/flox (f/f) fractions. (G) Immunofluorescence staining of frozen tissue sections of the spleen for B cells (B220; green), T cells (CD3; red) and laminin (blue). Download figure Download PowerPoint Defective lymphocyte trafficking to the peripheral LN As the cellularity in peripheral lymphoid tissues was reduced, we examined whether an Mst1 deficiency could impair lymphocyte homing to secondary lymphoid organs. T and B lymphocytes were isolated from the LNs and spleens of Mst1f/f and Mst1−/− mice, both of which exhibited naive phenotypes for T cells (CD62LhiCD44loCD69−) and B cells (CD62 L+IgM+IgDhi). Control Mst1f/f and Mst1−/− lymphocytes were differentially labelled and adoptively transferred into normal mice. Trafficking of Mst1−/− T cells to the peripheral LNs and spleen was reduced to one fourth and one third, respectively, of control T cells (Figure 2A). Mst1−/− B-cell trafficking to the LN was also reduced to one fourth compared with that of control B cells (Figure 2A). These data suggest that hypoplastic lymphoid tissues are due to impaired homing capacity of Mst1-deficient lymphocytes. Figure 2.Defective homing of Mst1-deficient lymphocytes. (A) Adoptive transfer of T cells. T cells from Mst1flox/flox (f/f) and Mst1-deficient (−/−) mice were labelled with CFSE and CMTMR, respectively. They were mixed in equal numbers and injected into the tail veins of wild-type (Wt) mice. After 1 h, lymphocytes from the peripheral lymph nodes, spleen and blood were analysed by flow cytometry. Representative flow cytometry profiles of blood, lymph nodes, and spleen are shown. Numbers beside the boxed areas indicate the ratio of Mst1-deficient cells to Mst1flox/flox (f/f) cells (upper panel). Adoptive transfer of B cells. B cells from Mst1flox/flox (f/f) and Mst1-deficient (−/−) mice were similarly analysed as the T cells (lower panel). (B) Appearance of lymphocyte attachment to the HEV of the mesenteric lymph node. Intravital images of lymphocyte attachment to the HEV were taken 20 min after intravenous transfer of lymphocytes from Mst1flox/flox (f/f) (green) and Mst1-deficient (−/−) (red) mice (top). Representative images of three independent experiments are shown. The number of attached Mst1flox/flox (f/f) or Mst1−/− (−/−) T and B cells to the HEV. The number of attached cells were counted using images of more than five microscopic fields taken 30 min after cell transfer (bottom). Representative data of three independent experiments are shown. *P<0.01, **P<0.005, compared with the corresponding Mst1flox/flox (f/f) fractions. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint We examined attachment to the HEV for control and Mst1−/− lymphocytes simultaneously by intravital microscopy. Although accumulation of attached control T cells (green) on the HEV in the mesenteric LN was obvious 20 min after transfer, Mst1−/− T cells (red) poorly attached to the HEV (Figure 2B). We quantified the number of attached cells using images of several microscopic fields taken 30 min after cell transfer. The number of attached Mst1−/− T cells was decreased by approximately 65% compared with control cells (Figure 2B). Although B cells tended to be less efficient than T cells in attaching to the HEV, attachment of Mst1−/− B cells to the HEV was reduced by approximately 75% compared with control B cells (Figure 2B). Impaired integrin-dependent firm adhesion of Mst1−/− T and B cells Naive lymphocyte interaction to the HEV in peripheral LN is regulated by adhesive cascades initiated by L-selectin-mediated tethering and rolling, followed by chemokine-triggered integrin activation and integrin-dependent arrest. Expression of L-selectin, LFA-1, α4 integrin and chemokine receptors CCR7 and CXCR4 was not affected in Mst1−/− lymphocytes (Supplementary Figure 4). To clarify specifically which step in the interaction with the HEV is impaired in Mst1−/− lymphocytes, we established an in vitro assay that reconstitute the lymphocyte adhesion cascade using endothelial cells (Kimura et al, 1999; Shamri et al, 2005) that express peripheral node addressin (PNAd) and ICAM-1, as intravital microscopic experiment was not found to be suitable for dissection of each step of adhesion cascades quantitatively. T cells were perfused into a parallel plate flow chamber coated with the endothelial monolayer with immobilized CCL21. The interactive processes were video-recorded and digitized with 30-ms intervals for a frame-by-frame cell tracking analysis. Representative profiles of the interactive processes were shown in Figure 3A. A fraction of T cell transiently attached, rolled and stopped under physiological shear stress (2–6 dyne/cm2), whereas low shear stress ( 500 μm/s) (Figure 3A, B and D; Supplementary video 1). L-selectin-dependent interactions in the presence of anti-LFA-1 antibody resulted in a brief stop, which was 1 s. A few cells detached within 10 s, but 94% of the attached cells stopped for >10 s, mostly over 2-min observation time (Figure 3C, D; Supplementary video 3). Therefore, we categorized the LFA-1-dependent adhesion into the transient (0.5–10 s) and stable arrest (>10 s), depending on dwell time on endothelial cells. As expected, PTX treatment inhibited the arrest, both transient and stable, without affecting tether/rolling (Figure 3D), indicating that LFA-1 is activated by the intracellular signalling mediated through the Gi family. Mst1−/− T cells tethered and rolled normally, indicating that L-selectin-dependent adhesive interaction is not affected. However, the LFA-1-dependent adhesion was found to be unstable with >80% of Mst1−/− T cells were detached within 5 s (Figure 3C, D; Supplementary video 4), indicating that Mst1 plays a critical role in stabilization of the transient arrest. Figure 3.Defective integrin-dependent stable adhesion of Mst1-deficient lymphocytes. (A) Time-displacement profiles of individual T-cell movement over LS12 endothelial monolayers under shear flow. Primary T cells from control mice perfused at 2 dyne/cm2 on LS12 monolayers immobilized with CCL21. Representative profiles of the cellular displacements over time were shown in four categories (no interaction, rolling, tether, transient and stable arrest), as described in the text. (B) The noninteracting and rolling velocities of control T cells movements on LS12 in the presence of anti-L-selectin and anti-LFA-1 antibody. (C) Stopping time of Mst1flox/flox (f/f) or Mst1-deficient (−/−) T cells arrested on LS12 endothelial cells were shown. More than 100 cells were measured in three independent experiments, and representative distribution of stopping time is shown. (D) Effects of anti-L-selectin, anti-LFA-1, PTX and Mst1-deficiency on the interactions of T cells with LS12 endothelial cells. Control Mst1flox/flox (f/f) T cells were pretreated with anti-L-selectin, LFA-1 and pertussis toxin (PTX), as described in Materials and methods. Mst1flox/flox (f/f) T cells and Mst1-deficient (−/−) T cells perfused at 2 dyne/cm2 on LS12 monolayers, which was immobilized with CCL21. The adhesive events of >100 cells were measured and categorized as described in (A). Data represent the means and s.e.m. of three independent experiments. *P<0.001, compared with Mst1flox/flox (f/f) lymphocytes. Download figure Download PowerPoint We also examined under flow adhesion to the α4β7 ligand MAdCAM-1, the major ligand for homing to Peyer's patches. Control T and B cells displayed tethering/rolling, which efficiently resulted in stable arrest in the presence of chemokines (Supplementary Figure 5). Although the frequencies of the transient arrest were rather increased, both Mst1−/− T and B cells had defective stable arrest that was reduced to approximately one third of control cells (Supplementary Figure 5). Taken together, these data indicate that the reduced homing capacity of Mst1−/− lymphocytes is due to the impairment in stabilization of integrin-dependent arrest on HEV. Mst1−/− lymphocytes are defective in LFA-1 clustering and talin accumulation at the contact sites Under flow conditions, lymphocytes have to develop integrin-dependent stable adhesion within seconds. We also examined roles of Mst1 in the stabilization of lymphocyte adhesion under static conditions, in which cells might develop stable adhesion by other mechanisms. The lymphocytes were allowed to adhere to immobilized integrin ligands for several minutes before subjected to shear flow. Compared with control lymphocytes, there were few Mst1−/− T and B cells that exhibited shear resistant, firm attachment to ICAM-1 after a 10-min incubation in the presence of CCL21 for T cells and CXCR4 ligand CXCL12 for B cells (Figure 4A). The stable adhesion of both Mst1−/− T and B cells to the VLA-4 ligand VCAM-1 were also severely decreased (Figure 4B), compared with those of control T and B cells. Thus, Mst1 plays a nonredundant role in adhesion stabilization under static as well as flow conditions. Figure 4.Defective stable adhesion and LFA-1 clustering in Mst1-deficient cells. (A) CCL21-stimulated T-cell adhesion (left) or CXCL12-stimulated B-cell adhesion (right) to ICAM-1. After incubation with 100 nM CCL21 or CXCL12 for 10 min, shear stress-resistant adhesion was measured as described in Materials and methods. Data represent the means and s.e.m. of triplicate experiments. None, no stimulation. *P<0.001, compared with Mst1flox/flox (f/f) T cells stimulated with CCL21; **P<0.001, compared with Mst1flox/flox (f/f) B cells stimulated with CXCL12. (B) CCL21-stimulated T-cell adhesion (left) or CXCL12-stimulated B-cell adhesion (right) to VCAM-1. Shear stress-resistant adhesion was measured as described above. Data represent the mean and s.e.m. of triplicate experiments. None, no stimulation. *P< 0.002, compared with Mst1flox/flox (f/f) T cells stimulated with CCL21; **P<0.002, compared with Mst1flox/flox (f/f) B cells stimulated with CXCL12. (C) Redistribution of LFA-1 (red) and CD44 (green). Mst1flox/flox (f/f) and Mst1-deficient (−/−) T and B cells were stimulated with CCL21 or CXCL12 for 5 min, then fixed and analysed by confocal microscopy quantitatively for cells showing a polarized distribution of LFA-1 and CD44 (top). Representative cell morphology and distribution of LFA-1 and CD44 (bottom). Data represent the means and s.e.m. of triplicate experiments. *P<0.001, compared with Mst1flox/flox (f/f) lymphocytes. (D) Confocal microscopic analysis of LFA-1 and talin distribution of T cells from Mst1flox/flox (f/f) (left panel, upper) and Mst1-deficient (−/−) (left panel, bottom) mice. T cells were incubated on cover glass coated with ICAM-1 in the presence of CCL21 for 5 min, and then fixed and stained for LFA-1 and talin. DAPI was used for nuclear staining. A series of Z-stack images at 1-μm intervals from the glass surface are shown above (left panels). Right panels showed the LFA-1 and talin distribution on contact sites of Mst1flox/flox (f/f) and Mst1-deficient (−/−) T cells on ICAM-1. Download figure Download PowerPoint We reported earlier that Mst1 is required for cell polarization and LFA-1 clustering triggered by chemokines but not involved in the regulation of LFA-1 affinity changes measured by binding to soluble ICAM-1 (Katagiri et al, 2006). We examined whether an Mst1 deficiency in primary lymphocytes affected LFA-1 clustering and lymphocyte polarization in response to chemokines. T and B cells were treated with CCL21 and CXCL12 for 5 min in suspension and were then fixed and stained for LFA-1 and CD44. Approximately 20–25% of chemokine-stimulated T and B cells from control mice showed polarized morphologies with a leading edge and uropod, to which LFA-1 and CD44 were clustered, respectively (Figure 4C). Although talin tended to be accumulated at the leading edge, it was not precisely colocalized with clustered LFA-1 (data not shown). The majority of Mst1−/− T and B cells remained unpolarized, and the redistribution of LFA-1 was not clearly observed (Figure 4C). The defects in cell polarization and LFA-1 clustering were also observed when incubated on ICAM-1 in the presence of chemokines (Figure 4D). In control cells, LFA-1 clustering was observed at the contact sites on ICAM-1, where talin was colocalized (Figure 4D), in agreement with the important role of talin in the final common step of integrin activation (Tadokoro et al, 2003). In contrast, colocalization of LFA-1 with talin was not observed clearly on the contact site of Mst1−/− cells upon attachment to ICAM-1 (Figure 4D). These data suggest that the chemokine-triggered lymphocytes attach to ICAM-1 through LFA-1 clustering, and the impaired LFA-1 clustering in Mst1−/− cells result in defective talin recruitment to the contact sites, leading to unstable adhesion. Reduced B-cell subsets and DC in the spleen Segregation of T cells and follicular B cells in the peripheral LN requires chemokine signalling (von Andrian and Mempel, 2003), but the contribution of integrins to this process is unclear. In contrast, MZB cells were reported to localize in the marginal sinus of the spleen in a manner dependent on ICAM-1 and VCAM-1 (Lu and Cyster, 2002). MZB cells are characterized by high IgM and CD21 expression and low IgD and CD23 expression (Martin and Kearney, 2002). FACS analysis revealed that the B220+ CD21hi CD23lo population corresponding to MZB cells was scarcely present in Mst1−/− mice (Figure 5A). In control mice, IgMhi IgDlo MZB cells were clearly detected in the marginal sinus at the border between the white and red pulp of the spleen (Figure 5B). However, there were few cells at the corresponding sites in the spleens of Mst1−/− mice (Figure 5B). There were no irregular structures of the marginal sinus, which normally express ICAM-1, MAdCAM-1 and VCAM-1 (Supplementary Figure 6). These results support the notion that defective adhesion to ICAM-1 and VCAM-1 results in a MZB cell deficiency in Mst1−/− mice. Figure 5.Deficient numbers of MZB cells and dendritic cells in the spleen of Mst1−/− mice. (A) Flow cytometry profiles of B220+ splenic B cells from Mst1flox/flox (f/f) and Mst1-deficient (−/−) mice stained with anti-CD21 and anti-CD23. The numbers beside the boxed areas indicate the percentages of CD21hiCD23low MZB cells, CD21hiCD23hi mature B cells and CD21−CD23− immature B cells of the total number of B220+ cells. (B) Spleen sections stained with IgM (green), IgD (red) and laminin (blue). IgMhi and IgD− marginal zone B cells were not observed in Mst1-defi
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