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Little is known regarding lymph node (LN)-homing of immune cells via afferent lymphatics. Here, we show, using a photo-convertible Dendra-2 reporter, that recently activated CD4 T cells enter downstream LNs via afferent lymphatics at high frequencies. Intra-lymphatic immune cell transfer and live imaging data further show that activated T cells come to an instantaneous arrest mediated passively by the mechanical 3D-sieve barrier of the LN subcapsular sinus (SCS). Arrested T cells subsequently migrate randomly on the sinus floor independent of both chemokines and integrins. However, chemokine receptors are imperative for guiding cells out of the SCS, and for their subsequent directional translocation towards the T cell zone. By contrast, integrins are dispensable for LN homing, yet still contribute by increasing the dwell time within the SCS and by potentially enhancing T cell sensing of chemokine gradients. Together, these findings provide fundamental insights into mechanisms that control homing of lymph-derived immune cells. Immune cells mostly enter lymph nodes (LN) from blood circulation, but whether afferent lymphatics contributes to LN entry is unclear. Here, the authors show, using a photo-convertible reporter, that T cells in afferent lymphatics frequently enter LN and become arrested in the subcapsular sinus, with chemokines and integrins further guiding their migration in the LN.

Antigen-bearing dendritic cells transit through the lymphatics via chemokine receptor CCR7–dependent chemotactic cues. Rot and colleagues show that the atypical chemokine receptor CCRL1 establishes chemokine polarity in the subscapsular sinus that enables entry into the lymph node. Afferent lymph–borne dendritic cells essentially rely on the chemokine receptor CCR7 for their transition from the subcapsular lymph node sinus into the parenchyma, a migratory step driven by putative gradients of CCR7 ligands. We found that lymph node fringes indeed contained physiological gradients of the chemokine CCL21, which depended on the expression of CCRL1, the atypical receptor for the CCR7 ligands CCL19 and CCL21. Lymphatic endothelial cells lining the ceiling of the subcapsular sinus, but not those lining the floor, expressed CCRL1, which scavenged chemokines from the sinus lumen. This created chemokine gradients across the sinus floor and enabled the emigration of dendritic cells. In vitro live imaging revealed that spatially confined expression of CCRL1 was necessary and sufficient for the creation of functional chemokine gradients.

Key Points During embryonic development and postnatally, dendritic cell (DC) progenitors migrate into non-lymphoid organs and differentiate into immature DCs. Immature DCs form a dense network of sentinel cells at all outer and inner surfaces of the body, as well as in most organs. Immature DCs sample and process both self and foreign antigens. They subsequently undergo an activation process that is triggered by either an 'intrinsic programme' or in response to the recognition of molecular patterns associated with pathogens and the microbiota. As part of the activation programme, DCs upregulate CC-chemokine receptor 7 (CCR7) and increase their motility. The CCR7 ligand CC-chemokine ligand 21 (CCL21) is expressed on terminal lymphatics and CCR7–CCL21 interactions enable DCs to enter the lymphatic vasculature and eventually the draining lymph node, where they migrate into the T cell-rich paracortex. Within lymph nodes and other lymphoid organs, DCs present antigen to T cells, leading either to the induction of immunological tolerance or to the expansion of protective pro-inflammatory effector and memory T cell populations. In some cases, DC-mediated presentation of self or harmless foreign antigens leads to the formation of effector T cell populations; as such, DCs can contribute to the development of autoimmune or allergic diseases. Effector T cells that develop during protective immune responses home to the tissue site of infection and inflammation and frequently contribute to the recruitment of further DC progenitors. Following their differentiation, such progenitors can present antigen to T cells, either locally or — after mobilization — in draining lymph nodes, thus amplifying protective as well as detrimental immune responses. Dendritic cells (DCs) shape tolerogenic and pro-inflammatory immune responses by presenting antigens to T cells; their ability to localize in peripheral tissues and secondary lymphoid organs is crucial for these functions. This Review describes the different migratory pathways employed by subsets of DCs from distinct tissue sites, detailing their mechanistic basis and importance for maintaining health. Dendritic cells (DCs) are potent and versatile antigen-presenting cells, and their ability to migrate is key for the initiation of protective pro-inflammatory as well as tolerogenic immune responses. Recent comprehensive studies have highlighted the importance of DC migration in the maintenance of immune surveillance and tissue homeostasis, and also in the pathogenesis of a range of diseases. In this Review, we summarize the anatomical, cellular and molecular factors that regulate the migration of different DC subsets in health and disease. In particular, we focus on new insights concerning the role of migratory DCs in the pathogenesis of diseases of the skin, intestine, lung, and brain, as well as in autoimmunity and atherosclerosis.

Helicobacter hepaticus can lead to chronic hepatitis and hepatocellular carcinoma in certain strains of mice. Until now the pathogenic role of Helicobacter species on human liver tissue is still not clarified though Helicobacter species identification in human liver cancer was successful in case controlled studies. Therefore we established an in vitro model to investigate the interaction of primary human hepatocytes (PHH) with Helicobacter hepaticus. Successful co-culturing of PHH with Helicobacter hepaticus was confirmed by visualization of motile bacteria by two-photon-microscopy. Isolated human monocytes were stimulated with PHH conditioned media. Changes in mRNA expression of acute phase cytokines and proteins in PHH and stimulated monocytes were determined by Real-time PCR. Furthermore, cytokines and proteins were analyzed in PHH culture supernatants by ELISA. Co-cultivation with Helicobacter hepaticus induced mRNA expression of Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha, Interleukin-8 (IL-8) and Monocyte chemotactic protein-1 (MCP-1) in PHH (p<0.05) resulting in a corresponding increase of IL-8 and MCP-1 concentrations in PHH supernatants (p<0.05). IL-8 and IL-1β mRNA expression was induced in monocytes stimulated with Helicobacter hepaticus infected PHH conditioned media (p<0.05). An increase of Cyclooxygenase-2 mRNA expression was observed, with a concomitant increase of prostaglandin E2 concentration in PHH supernatants at 24 and 48 h (p<0.05). In contrast, at day 7 of co-culture, no persistent elevation of cytokine mRNA could be detected. High expression of intercellular adhesion molecule-1 on PHH cell membranes after co-culture was shown by two-photon-microscopy and confirmed by flow-cytometry. Finally, expression of Cytochrome P450 3A4 and albumin mRNA were downregulated, indicating an impairment of hepatocyte synthesis function by Helicobacter hepaticus presence. This is the first in vitro model demonstrating a pathogenic effect of a Helicobacter spp. on human liver cells, resulting in an inflammatory response with increased synthesis of inflammatory mediators and consecutive monocyte activation.

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. An extra dimension Leukocytes are remarkable in their ability to infiltrate any type of tissue almost anywhere in the body. On flat surfaces they migrate using transmembrane receptors of the integrin family — present in all metazoan cells — that couple the contractile force of the actomyosin cytoskeleton to the extracellular environment. But studies of cell migration on two-dimensional surfaces overemphasize the role of adhesion. The rapid movement and extreme flexibility of leukocytes in three dimensions is now shown — by genetic knockout — not to involve integrins. Instead, the cells migrate using an amoeba-like flowing and squeezing action, powered by actin network expansion alone. Leukocyte migration over two-dimensional surfaces is dependent on the integrin family of adhesion receptors, which couple the contractile force of the actomyosin cytoskeleton to the extracellular environment. In this study, all integrin heterodimers from mouse leukocytes were ablated and it is shown that integrins are not required for migration in 3D environments, in vitro and in vivo . Such non-adhesive migration renders leukocytes autonomous from the tissue environment.

Lymphatic vessels provide conduits that channel leukocytes to draining lymph nodes. Förster and colleagues show that lymph-derived dendritic cells and T cells take different paths to enter draining lymph nodes. Little is known about the molecular mechanisms that determine the entry into the lymph node and intranodal positioning of lymph-derived cells. By injecting cells directly into afferent lymph vessels of popliteal lymph nodes, we demonstrate that lymph-derived T cells entered lymph-node parenchyma mainly from peripheral medullary sinuses, whereas dendritic cells (DCs) transmigrated through the floor of the subcapsular sinus on the afferent side. Transmigrating DCs induced local changes that allowed the concomitant entry of T cells at these sites. Signals mediated by the chemokine receptor CCR7 were absolutely required for the directional migration of both DCs and T cells into the T cell zone but were dispensable for the parenchymal entry of lymph-derived T cells and dendrite probing of DCs. Our findings provide insight into the molecular and structural requirements for the entry into lymph nodes and intranodal migration of lymph-derived cells of the immune system.

By establishing micromanipulator-guided injections into afferent lymph vessels of the popliteal lymph node (LN) in living mice, we demonstrate, using two-photon microscopy, that lymph-derived T cells entered LN parenchyma primarily from peripheral medullary sinuses while dendritic cells (DCs) transmigrated through the floor of the afferent side subcapsular sinus. Transmigrating DCs induced local changes of the subcapsular sinus floor thereby allowing the concomitant entry of T cells at these sites. Signals mediated by the chemokine receptor CCR7 were absolutely required for the directional migration of both DC and T cells into the T cell zone but were dispensable for parenchymal entry of lymph-derived T cells and dendrite probing of DC.

I hearby nominate President Reagan for the \"Golden Skate Award\" of the decade for his outstanding performance on TV (\"'Better Off Than We Were,' Reagan Says or Recession.\" Times, Sept. 29).