The role of chemotactic gradients in dendritic cell migration Dissertation Thesis

Author(s): Schumann, Kathrin
Advisor(s): Sixt, Michael
Committee Member(s): Bollenbach, Tobias; Luther, Sanjiv
Title: The role of chemotactic gradients in dendritic cell migration
Affiliation IST Austria
Abstract: Chemokines organize immune cell trafficking by inducing either directed (tactic) or random (kinetic) migration and by activating integrins in order to support surface adhesion (haptic). Beyond that the same chemokines can establish clearly defined functional areas in secondary lymphoid organs. Until now it is unclear how chemokines can fulfill such diverse functions. One decisive prerequisite to explain these capacities is to know how chemokines are presented in tissue. In theory chemokines could occur either soluble or immobilized, and could be distributed either homogenously or as a concentration gradient. To dissect if and how the presenting mode of chemokines influences immune cells, I tested the response of dendritic cells (DCs) to differentially displayed chemokines. DCs are antigen presenting cells that reside in the periphery and migrate into draining lymph nodes (LNs) once exposed to inflammatory stimuli to activate naïve T cells. DCs are guided to and within the LN by the chemokine receptor CCR7, which has two ligands, the chemokines CCL19 and CCL21. Both CCR7 ligands are expressed by fibroblastic reticular cells in the LN, but differ in their ability to bind to heparan sulfate residues. CCL21 has a highly charged C-terminal extension, which mediates binding to anionic surfaces, whereas CCL19 is lacking such residues and likely distributes as a soluble molecule. This study shows that surface-bound CCL21 causes random, haptokinetic DC motility, which is confined to the chemokine coated area by insideout activation of β2 integrins that mediate cell binding to the surface. CCL19 on the other hand forms concentration gradients which trigger directional, chemotactic movement, but no surface adhesion. In addition DCs can actively manipulate this system by recruiting and activating serine proteases on their surfaces, which create - by proteolytically removing the adhesive C-terminus - a solubilized variant of CCL21 that functionally resembles CCL19. By generating a CCL21 concentration gradient DCs establish a positive feedback loop to recruit further DCs from the periphery to the CCL21 coated region. In addition DCs can sense chemotactic gradients as well as immobilized haptokinetic fields at the same time and integrate these signals. The result is chemotactically biased haptokinesis - directional migration confined to a chemokine coated track or area - which could explain the dynamic but spatially tightly controlled swarming leukocyte locomotion patterns that have been observed in lymphatic organs by intravital microscopists. The finding that DCs can approach soluble cues in a non-adhesive manner while they attach to surfaces coated with immobilized cues raises the question how these cells transmit intracellular forces to the environment, especially in the non-adherent migration mode. In order to migrate, cells have to generate and transmit force to the extracellular substrate. Force transmission is the prerequisite to procure an expansion of the leading edge and a forward motion of the whole cell body. In the current conceptions actin polymerization at the leading edge is coupled to extracellular ligands via the integrin family of transmembrane receptors, which allows the transmission of intracellular force. Against the paradigm of force transmission during migration, leukocytes, like DCs, are able to migrate in threedimensional environments without using integrin transmembrane receptors (Lämmermann et al., 2008). This reflects the biological function of leukocytes, as they can invade almost all tissues, whereby their migration has to be independent from the extracellular environment. How the cells can achieve this is unclear. For this study I examined DC migration in a defined threedimensional environment and highlighted actin-dynamics with the probe Lifeact-GFP. The result was that chemotactic DCs can switch between integrin-dependent and integrin- independent locomotion and can thereby adapt to the adhesive properties of their environment. If the cells are able to couple their actin cytoskeleton to the substrate, actin polymerization is entirely converted into protrusion. Without coupling the actin cortex undergoes slippage and retrograde actin flow can be observed. But retrograde actin flow can be completely compensated by higher actin polymerization rate keeping the migration velocity and the shape of the cells unaltered. Mesenchymal cells like fibroblast cannot balance the loss of adhesive interaction, cannot protrude into open space and, therefore, strictly depend on integrinmediated force coupling. This leukocyte specific phenomenon of “adaptive force transmission” endows these cells with the unique ability to transit and invade almost every type of tissue.
Publication Title: IST Dissertation
Degree Granting Institution: IST Austria  
Degree: PhD
Degree Date: 2011-03-01
Start Page: 1
Total Pages: 141
Notes: I would like to express my sincere gratitude to the following people who made with their continuous support and encouragement this thesis possible: First, I want to thank Prof. Dr. Michael Sixt for his excellent supervision and mentoring, especially for the nice, relaxed working atmosphere, a lot of brilliant ideas and the freedom to work in my own way. Prof. Dr. Reinhard Fässler for his constant support of the Sixt lab and for providing excellent working conditions. Prof. Dr. Sanjiv Luther and Prof. Dr. Tobias Bollenbach for agreeing to be member of my thesis committee and to evaluate my work. Dr. Walther Göhring, Carmen Schmitz, the Recombinant Protein Production core facility and the animal care takers for providing the “infrastructure” for this thesis. Prof. Dr. Daniel Legler, Markus Bruckner and Dr. Julien Polleux for very fruitful collaborations and discussions. My labmates for their help, a lot of discussions and to make the Sixt lab to a convenient place to work : Karin Hirsch, Tim Lämmeramnn, Holger Pflicke, Jörg Renkawitz, Michele Weber and Alexander Eichner All members of the Department of Molecular Medicine for their help. Especially I want to thank Sarah Schmidt, Karin Hirsch and Raphael Ruppert for their friendship, nice chats and their uncensored point of view.
Open access: no