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ABSTRACT: Directional guidance of cells via gradients of chemokines is considered crucial for embryonic development, cancer dissemination, and immune responses. Nevertheless, the concept still lacks direct experimental confirmation in vivo. Here, we identify endogenous gradients of the chemokine CCL21 within mouse skin and show that they guide dendritic cells toward lymphatic vessels. Quantitative imaging reveals depots of CCL21 within lymphatic endothelial cells and steeply decaying gradients within the perilymphatic interstitium. These gradients match the migratory patterns of the dendritic cells, which directionally approach vessels from a distance of up to 90-micrometers. Interstitial CCL21 is immobilized to heparan sulfates, and its experimental delocalization or swamping the endogenous gradients abolishes directed migration. These findings functionally establish the concept of haptotaxis, directed migration along immobilized gradients, in tissues.
Science 01/2013; 339(6117):328-332. · 31.20 Impact Factor
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ABSTRACT: Leukocyte migration through the interstitial space is crucial for the maintenance of tolerance and immunity. The main cues for leukocyte trafficking are chemokines thought to directionally guide these cells towards their targets. However, model systems that facilitate quantification of chemokine-guided leukocyte migration in vivo are uncommon. Here we describe an ex vivo crawl-in assay using explanted mouse ears that allows the visualization of chemokine-dependent dendritic cell (DC) motility in the dermal interstitium in real time. We present methods for the preparation of mouse ear sheets and their use in multidimensional confocal imaging experiments to monitor and analyze the directional migration of fluorescently labelled DCs through the dermis and into afferent lymphatic vessels. The assay provides a more physiological approach to study leukocyte migration than in vitro three-dimensional (3D) or 2-dimensional (2D) migration assays such as collagen gels and transwell assays.
Methods in molecular biology (Clifton, N.J.) 01/2013; 1013:215-26.
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ABSTRACT: Recently, the mammalian actin-binding protein 1 (mAbp1; Hip-55, SH3P7, debrin-like protein) was identified as a novel component of the β(2) integrin-mediated signaling cascade during complement-mediated phagocytosis and firm adhesion of polymorphonuclear neutrophils (PMN) under physiological shear stress conditions. In this study, we found that the genetic ablation of mAbp1 severely compromised not only the induction of adhesion, but also subsequent spreading of leukocytes to the endothelium as assessed by intravital microscopy of inflamed vessels of the cremaster muscle of mice. In vitro studies using murine PMN confirmed that mAbp1 was required for β(2) integrin-mediated spreading under shear stress conditions, whereas mAbp1 was dispensable for spreading under static conditions. Upon β(2) integrin-mediated adhesion and chemotactic migration of human neutrophil-like differentiated HL-60 cells, mAbp1 was enriched at the leading edge of the polarized cell. Total internal reflection fluorescence microscopy revealed that mAbp1 formed propagating waves toward the front of the lamellipodium, which are characteristic for dynamic reorganization of the cytoskeleton. Accordingly, binding of mAbp1 to actin was increased upon β(2) integrin-mediated adhesion, as shown by coimmunoprecipitation experiments. However, chemotactic migration under static conditions was unaffected in the absence of mAbp1. In contrast, the downregulation of mAbp1 by RNA interference technique in neutrophil-like differentiated HL-60 cells or the genetic ablation of mAbp1 in leukocytes led to defective migration under flow conditions in vitro and in inflamed cremaster muscle venules in the situation in vivo. In conclusion, mAbp1 is of fundamental importance for spreading and migration under shear stress conditions, which are critical prerequisites for efficient PMN extravasation during inflammation.
The Journal of Immunology 03/2012; 188(9):4590-601. · 5.79 Impact Factor
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Meike Wendland,
Stefanie Willenzon,
Jessica Kocks,
Ana Clara Davalos-Misslitz,
Swantje I Hammerschmidt,
Kathrin Schumann,
Elisabeth Kremmer, Michael Sixt,
Angelika Hoffmeyer,
Oliver Pabst,
Reinhold Förster
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ABSTRACT: Little is known about mechanisms determining the homeostasis of lymphocytes within lymphoid organs. Applying different mouse models, including conditionally proficient Ccr7 gene-targeted mice, we now show that semimature steady state dendritic cells (sDCs) constitutively trafficking into lymph nodes (LNs) were essential contributors to T cell homeostasis in these organs. sDCs provided vascular endothelial growth factor known to support high endothelial venule formation, thus facilitating enhanced homing of T cells to LNs. The presence of sDCs led to increased CCL21 production in T-zone fibroblastic reticular cells. CCL21 is a ligand for CCR7 known to regulate homing as well as retention of T cells in LNs. In addition, we provide evidence that CCL21 binds to the surface of DCs via its heparin-binding domain, further explaining why T cells leave LNs more rapidly in the absence of sDCs. Together, these data reveal multiple roles for sDCs in regulating T cell homeostasis in LNs.
Immunity 12/2011; 35(6):945-57. · 21.64 Impact Factor
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ABSTRACT: 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, nonactivated 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 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 contraction during lymphocyte recirculation.
The Journal of Immunology 09/2011; 187(5):2356-64. · 5.79 Impact Factor
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Daniel Schraivogel,
Lasse Weinmann,
Dagmar Beier,
Ghazaleh Tabatabai,
Alexander Eichner,
Jia Yun Zhu,
Martina Anton, Michael Sixt,
Michael Weller,
Christoph P Beier,
Gunter Meister
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ABSTRACT: Cancer stem cells or cancer initiating cells are believed to contribute to cancer recurrence after therapy. MicroRNAs (miRNAs) are short RNA molecules with fundamental roles in gene regulation. The role of miRNAs in cancer stem cells is only poorly understood. Here, we report miRNA expression profiles of glioblastoma stem cell-containing CD133(+) cell populations. We find that miR-9, miR-9(*) (referred to as miR-9/9(*)), miR-17 and miR-106b are highly abundant in CD133(+) cells. Furthermore, inhibition of miR-9/9(*) or miR-17 leads to reduced neurosphere formation and stimulates cell differentiation. Calmodulin-binding transcription activator 1 (CAMTA1) is a putative transcription factor, which induces the expression of the anti-proliferative cardiac hormone natriuretic peptide A (NPPA). We identify CAMTA1 as an miR-9/9(*) and miR-17 target. CAMTA1 expression leads to reduced neurosphere formation and tumour growth in nude mice, suggesting that CAMTA1 can function as tumour suppressor. Consistently, CAMTA1 and NPPA expression correlate with patient survival. Our findings could provide a basis for novel strategies of glioblastoma therapy.
The EMBO Journal 08/2011; 30(20):4309-22. · 9.20 Impact Factor
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ABSTRACT: Ectopic or tertiary lymphoid tissues (TLTs) are often induced at sites of chronic inflammation. They typically contain various hematopoietic cell types, high endothelial venules, and follicular dendritic cells; and are organized in lymph node-like structures. Although fibroblastic stromal cells may play a role in TLT induction and persistence, they have remained poorly defined. Herein, we report that TLTs arising during inflammation in mice and humans in a variety of tissues (eg, pancreas, kidney, liver, and salivary gland) contain stromal cell networks consisting of podoplanin(+) T-zone fibroblastic reticular cells (TRCs), distinct from follicular dendritic cells. Similar to lymph nodes, TRCs were present throughout T-cell-rich areas and had dendritic cells associated with them. They expressed lymphotoxin (LT) β receptor (LTβR), produced CCL21, and formed a functional conduit system. In rat insulin promoter-CXCL13-transgenic pancreas, the maintenance of TRC networks and conduits was partially dependent on LTβR and on lymphoid tissue inducer cells expressing LTβR ligands. In conclusion, TRCs and conduits are hallmarks of secondary lymphoid organs and of well-developed TLTs, in both mice and humans, and are likely to act as important scaffold and organizer cells of the T-cell-rich zone.
American Journal Of Pathology 04/2011; 178(4):1662-75. · 4.89 Impact Factor
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ABSTRACT: Cell migration on two-dimensional (2D) substrates follows entirely different rules than cell migration in three-dimensional (3D) environments. This is especially relevant for leukocytes that are able to migrate in the absence of adhesion receptors within the confined geometry of artificial 3D extracellular matrix scaffolds and within the interstitial space in vivo. Here, we describe in detail a simple and economical protocol to visualize dendritic cell migration in 3D collagen scaffolds along chemotactic gradients. This method can be adapted to other cell types and may serve as a physiologically relevant paradigm for the directed locomotion of most amoeboid cells.
Methods in molecular biology (Clifton, N.J.) 01/2011; 769:149-65.
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ABSTRACT: In their search for antigens, lymphocytes continuously shuttle among blood vessels, lymph vessels, and lymphatic tissues. Chemokines mediate entry of lymphocytes into lymphatic tissues, and sphingosine 1-phosphate (S1P) promotes localization of lymphocytes to the vasculature. Both signals are sensed through G protein-coupled receptors (GPCRs). Most GPCRs undergo ligand-dependent homologous receptor desensitization, a process that decreases their signaling output after previous exposure to high ligand concentration. Such desensitization can explain why lymphocytes do not take an intermediate position between two signals but rather oscillate between them. The desensitization of S1P receptor 1 (S1PR1) is mediated by GPCR kinase 2 (GRK2). Deletion of GRK2 in lymphocytes compromises desensitization by high vascular S1P concentrations, thereby reducing responsiveness to the chemokine signal and trapping the cells in the vascular compartment. The desensitization kinetics of S1PR1 allows lymphocytes to dynamically shuttle between vasculature and lymphatic tissue, although the positional information in both compartments is static.
Science Signaling 01/2011; 4(198):pe43. · 7.50 Impact Factor
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ABSTRACT: For innate and adaptive immune responses it is essential that inflammatory cells use quick and flexible locomotion strategies. Accordingly, most leukocytes can efficiently infiltrate and traverse almost every physiological or artificial environment. Here, we review how leukocytes might achieve this task mechanistically, and summarize recent findings on the principles of cytoskeletal force generation and transduction at the leading edge of leukocytes. We propose a model in which the cells switch between adhesion-receptor-mediated force transmission and locomotion modes that are based on cellular deformations, but independent of adhesion receptors. This plasticity in migration strategies allows leukocytes to adapt to the geometry and molecular composition of their environment.
EMBO Reports 10/2010; 11(10):744-50. · 7.36 Impact Factor
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The EMBO Journal 09/2010; 29(17):2861-3. · 9.20 Impact Factor
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Kathrin Schumann,
Tim Lämmermann,
Markus Bruckner,
Daniel F Legler,
Julien Polleux,
Joachim P Spatz,
Gerold Schuler,
Reinhold Förster,
Manfred B Lutz,
Lydia Sorokin, Michael Sixt
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ABSTRACT: Chemokines orchestrate immune cell trafficking by eliciting either directed or random migration and by activating integrins in order to induce cell adhesion. Analyzing dendritic cell (DC) migration, we showed that these distinct cellular responses depended on the mode of chemokine presentation within tissues. The surface-immobilized form of the chemokine CCL21, the heparan sulfate-anchoring ligand of the CC-chemokine receptor 7 (CCR7), caused random movement of DCs that was confined to the chemokine-presenting surface because it triggered integrin-mediated adhesion. Upon direct contact with CCL21, DCs truncated the anchoring residues of CCL21, thereby releasing it from the solid phase. Soluble CCL21 functionally resembles the second CCR7 ligand, CCL19, which lacks anchoring residues and forms soluble gradients. Both soluble CCR7 ligands triggered chemotactic movement, but not surface adhesion. Adhesive random migration and directional steering cooperate to produce dynamic but spatially restricted locomotion patterns closely resembling the cellular dynamics observed in secondary lymphoid organs.
Immunity 05/2010; 32(5):703-13. · 21.64 Impact Factor
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Julia Riedl,
Kevin C Flynn,
Aurelia Raducanu,
Florian Gärtner,
Gisela Beck,
Michael Bösl,
Frank Bradke,
Steffen Massberg,
Attila Aszodi, Michael Sixt,
Roland Wedlich-Söldner
Nature Methods 03/2010; 7(3):168-9. · 19.28 Impact Factor
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ABSTRACT: Although both processes occur at similar rates, leukocyte extravasation from the blood circulation is well investigated, whereas intravasation into lymphatic vessels has hardly been studied. In contrast to a common assumption-that intra- and extravasation follow similar molecular principles-we previously showed that lymphatic entry of dendritic cells (DCs) does not require integrin-mediated adhesive interactions. In this study, we demonstrate that DC-entry is also independent of pericellular proteolysis, raising the question of whether lymphatic vessels offer preexisting entry routes. We find that the perilymphatic basement membrane of initial lymphatic vessels is discontinuous and therefore leaves gaps for entering cells. Using a newly developed in situ live cell imaging approach that allows us to dynamically visualize the cells and their extracellular environment, we demonstrate that DCs enter through these discontinuities, which are transiently mechanically dilated by the passaging cells. We further show that penetration of the underlying lymphatic endothelial layer occurs through flap valves lacking continuous intercellular junctions. Together, we demonstrate free cellular communication between interstitium and lymphatic lumen.
Journal of Experimental Medicine 12/2009; 206(13):2925-35. · 13.85 Impact Factor
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ABSTRACT: The leading front of a cell can either protrude as an actin-free membrane bleb that is inflated by actomyosin-driven contractile forces, or as an actin-rich pseudopodium, a site where polymerizing actin filaments push out the membrane. Pushing filaments can only cause the membrane to protrude if the expanding actin network experiences a retrograde counter-force, which is usually provided by transmembrane receptors of the integrin family. Here we show that chemotactic dendritic cells mechanically adapt to the adhesive properties of their substrate by switching between integrin-mediated and integrin-independent locomotion. We found that on engaging the integrin-actin clutch, actin polymerization was entirely turned into protrusion, whereas on disengagement actin underwent slippage and retrograde flow. Remarkably, accelerated retrograde flow was balanced by an increased actin polymerization rate; therefore, cell shape and protrusion velocity remained constant on alternating substrates. Due to this adaptive response in polymerization dynamics, tracks of adhesive substrate did not dictate the path of the cells. Instead, directional guidance was exclusively provided by a soluble gradient of chemoattractant, which endowed these 'amoeboid' cells with extraordinary flexibility, enabling them to traverse almost every type of tissue.
Nature Cell Biology 11/2009; 11(12):1438-43. · 19.49 Impact Factor
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Jürgen Schymeinsky,
Ronald Gerstl,
Ingrid Mannigel,
Katy Niedung,
David Frommhold,
Klaus Panthel,
Jürgen Heesemann, Michael Sixt,
Thomas Quast,
Waldemar Kolanus,
Attila Mocsai,
Jürgen Wienands,
Markus Sperandio,
Barbara Walzog
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ABSTRACT: The mammalian actin-binding protein 1 (mAbp1, Hip-55, SH3P7) is phosphorylated by the nonreceptor tyrosine kinase Syk that has a fundamental effect for several beta(2) integrin (CD11/CD18)-mediated neutrophil functions. Live cell imaging showed a dynamic enrichment of enhanced green fluorescence protein-tagged mAbp1 at the phagocytic cup of neutrophil-like differentiated HL-60 cells during beta(2) integrin-mediated phagocytosis of serum-opsonized Escherichia coli. The genetic absence of Syk or its pharmacologic inhibition using piceatannol abrogated the proper localization of mAbp1 at the phagocytic cup. The genetic absence or down-regulation of mAbp1 using the RNA interference technique significantly compromised beta(2) integrin-mediated phagocytosis of serum-opsonized E coli or Salmonella typhimurium in vitro as well as clearance of S typhimurium infection in vivo. Moreover, the genetic absence of mAbp1 almost completely abrogated firm neutrophil adhesion under physiologic shear stress conditions in vitro as well as leukocyte adhesion and extravasation in inflamed cremaster muscle venules of mice treated with tumor-necrosis factor alpha. Functional analysis showed that the down-regulation of mAbp1 diminished the number of beta(2) integrin clusters in the high-affinity conformation under flow conditions. These unanticipated results define mAbp1 as a novel molecular player in integrin biology that is critical for phagocytosis and firm neutrophil adhesion under flow conditions.
Blood 08/2009; 114(19):4209-20. · 9.90 Impact Factor
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ABSTRACT: The morphological term 'amoeboid' migration subsumes a number of rather distinct biophysical modes of cellular locomotion that range from blebbing motility to entirely actin-polymerization-based gliding. Here, we discuss the diverse principles of force generation and force transduction that lead to the distinct amoeboid phenotypes. We argue that shifting the balance between actin protrusion, actomyosin contraction, and adhesion to the extracellular substrate can explain the different modes of amoeboid movement and that blebbing and gliding are barely extreme variants of one common migration strategy. Depending on the cell type, physiological conditions or experimental manipulation, amoeboid cells can adopt the distinct mechanical modes of amoeboid migration.
Current opinion in cell biology 07/2009; 21(5):636-44. · 14.15 Impact Factor
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Thomas Quast,
Barbara Tappertzhofen,
Cora Schild,
Jessica Grell,
Niklas Czeloth,
Reinhold Förster,
Ronen Alon,
Line Fraemohs,
Katrin Dreck,
Christian Weber,
Tim Lämmermann, Michael Sixt,
Waldemar Kolanus
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ABSTRACT: Adhesion and motility of mammalian leukocytes are essential requirements for innate and adaptive immune defense mechanisms. We show here that the guanine nucleotide exchange factor cytohesin-1, which had previously been demonstrated to be an important component of beta-2 integrin activation in lymphocytes, regulates the activation of the small GTPase RhoA in primary dendritic cells (DCs). Cytohesin-1 and RhoA are both required for the induction of chemokine-dependent conformational changes of the integrin beta-2 subunit of DCs during adhesion under physiological flow conditions. Furthermore, use of RNAi in murine bone marrow DCs (BM-DCs) revealed that interference with cytohesin-1 signaling impairs migration of wild-type dendritic cells in complex 3D environments and in vivo. This phenotype was not observed in the complete absence of integrins. We thus demonstrate an essential role of cytohesin-1/RhoA during ameboid migration in the presence of integrins and further suggest that DCs without integrins switch to a different migration mode.
Blood 05/2009; 113(23):5801-10. · 9.90 Impact Factor
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ABSTRACT: Mature dendritic cells (DCs) moving from the skin to the lymph node are a prototypic example of rapidly migrating amoeboid leukocytes. Interstitial DC migration is directionally guided by chemokines, but independent of specific adhesive interactions with the tissue as well as pericellular proteolysis. Instead, the protrusive flow of the actin cytoskeleton directly drives a basal mode of locomotion that is occasionally supported by actomyosin contractions at the trailing edge to propel the cell's rigid nucleus. We here delete the small GTPase Cdc42 in DCs and find that actin flow and actomyosin contraction are still initiated in response to chemotactic cues. Accordingly, the cells are able to polarize and form protrusions. However, in the absence of Cdc42 the protrusions are temporally and spatially dysregulated, which leads to impaired leading edge coordination. Although this defect still allows the cells to move on 2-dimensional surfaces, their in vivo motility is completely abrogated. We show that this difference is entirely caused by the geometric complexity of the environment, as multiple competing protrusions lead to instantaneous entanglement within 3-dimensional extracellular matrix scaffolds. This demonstrates that the decisive factor for migrating DCs is not specific interaction with the extracellular environment, but adequate coordination of cytoskeletal flow.
Blood 03/2009; 113(23):5703-10. · 9.90 Impact Factor
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ABSTRACT: Integrin activation is essential for the function of all blood cells, including platelets and leukocytes. The blood cell-specific FERM domain protein Kindlin-3 is required for the activation of the beta1 and beta3 integrins on platelets. Impaired activation of beta1, beta2 and beta3 integrins on platelets and leukocytes is the hallmark of a rare autosomal recessive leukocyte adhesion deficiency syndrome in humans called LAD-III, characterized by severe bleeding and impaired adhesion of leukocytes to inflamed endothelia. Here we show that Kindlin-3 also binds the beta2 integrin cytoplasmic domain and is essential for neutrophil binding and spreading on beta2 integrin-dependent ligands such as intercellular adhesion molecule-1 and the complement C3 activation product iC3b. Moreover, loss of Kindlin-3 expression abolished firm adhesion and arrest of neutrophils on activated endothelial cells in vitro and in vivo, whereas selectin-mediated rolling was unaffected. Thus, Kindlin-3 is essential to activate the beta1, beta2 and beta3 integrin classes, and loss of Kindlin-3 function is sufficient to cause a LAD-III-like phenotype in mice.
Nature medicine 03/2009; 15(3):300-5. · 27.14 Impact Factor
