Michael Sixt

Lifeact: a versatile marker to visualize F-actin.

Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.

Rapid leukocyte migration by integrin-independent flowing and squeezing

All metazoan cells carry transmembrane receptors of the integrin family, which couple the contractile force of the actomyosin cytoskeleton to the extracellular environment. In agreement with this principle, rapidly migrating leukocytes use integrin-mediated adhesion when moving over two-dimensional surfaces. As migration on two-dimensional substrates naturally overemphasizes the role of adhesion, the contribution of integrins during three-dimensional movement of leukocytes within tissues has remained controversial. We studied the interplay between adhesive, contractile and protrusive forces during interstitial leukocyte chemotaxis in vivo and in vitro. We ablated all integrin heterodimers from murine leukocytes, and show here that functional integrins do not contribute to migration in three-dimensional environments. Instead, these cells migrate by the sole force of actin-network expansion, which promotes protrusive flowing of the leading edge. Myosin II-dependent contraction is only required on passage through narrow gaps, where a squeezing contraction of the trailing edge propels the rigid nucleus.

Breaching multiple barriers: leukocyte motility through venular walls and the interstitium

The shuttling of leukocytes between the bloodstream and interstitial tissues involves different locomotion strategies that are governed by locally presented soluble and cell-bound signals. Recent studies have furthered our understanding of the rapidly advancing field of leukocyte migration, particularly regarding cellular and subcellular events at the level of the venular wall. Furthermore, emerging cellular models are now addressing the transition from an adherent mode to a non-adherent state, incorporating mechanisms that support an efficient migratory profile of leukocytes in the interstitial tissue beyond the venular wall.

Kindlin-3 is required for β2 integrin-mediated leukocyte adhesion to endothelial cells

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 β1 and β3 integrins on platelets. Impaired activation of β1, β2 and β3 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 β2 integrin cytoplasmic domain and is essential for neutrophil binding and spreading on β2 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 β1, β2 and β3 integrin classes, and loss of Kindlin-3 function is sufficient to cause a LAD-III–like phenotype in mice.

Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin

Neutrophil granulocytes form the bodys first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex-mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents.

Interstitial dendritic cell guidance by haptotactic chemokine gradients

A Well-Defined Path Although chemokines have long been thought to direct immune cell movements within tissues, a formal in vivo demonstration and detailed understanding are lacking. By tracking dendritic cell movements in the ears of mice, Weber et al. (p. 328) were able to provide both. Endogenous gradients of the chemokine CCL21 were observed in ear tissue and, at distances of up 90 µm, dendritic cells were able to use these gradients to migrate directionally toward lymphatic vessels. The CCL21 gradient was immobilized on heparan sulfates and disruption of the gradient inhibited dendritic cell migration. In mouse skin, immune cells migrate toward lymphatic vessels along an immobilized chemokine gradient. 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.

Immobilized Chemokine Fields and Soluble Chemokine Gradients Cooperatively Shape Migration Patterns of Dendritic Cells

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.

Preformed portals facilitate dendritic cell entry into afferent lymphatic vessels

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.

Lifeact mice for studying F-actin dynamics.

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The microanatomy of T‐cell responses

Summary: The priming of a T cell results from its physical interaction with a dendritic cell (DC) that presents the cognate antigenic peptide. The success rate of such interactions is extremely low, because the precursor frequency of a naive T cell recognizing a specific antigen is in the range of 1:105–106. To make this principle practicable, encounter frequencies between DCs and T cells are maximized within lymph nodes (LNs) that are compact immunological projections of the peripheral tissue they drain. But LNs are more than passive meeting places for DCs that immigrated from the tissue and lymphocytes that recirculated via the blood. The microanatomy of the LN stroma actively organizes the cellular encounters by providing preformed migration tracks that create dynamic but highly ordered movement patterns. LN architecture further acts as a sophisticated filtration system that sieves the incoming interstitial fluid at different levels and guarantees that immunologically relevant antigens are loaded on DCs or B cells while inert substances are channeled back into the blood circulation. This review focuses on the non‐hematopoietic infrastructure of the lymph node. We describe the association between fibroblastic reticular cell, conduit, DC, and T cell as the essential functional unit of the T‐cell cortex.