Dendritic cell traffic across lymphatic endothelium under inflammatory conditions

Zusammenfassung

The thesis project is sum up in three different objectives OBJ 1: Migration of DC into lymphatic vessels ferries antigenic cargo and pro-inflammatory stimuli into the draining LN. Given that tissues under the influence of viral infections produce type I IFN, it is conceivable that these cytokines enhance DC migration in order to facilitate an antiviral immune response. Cultured LEC monolayers pretreated with TNF-a were used to model this phenomenon under inflammatory conditions. DC differentiated in the presence of either IFN-a2b or IFN-a5 showed enhanced adhesion to LEC. These pro-adhesive effects were mediated by DC, not the LE, and correlated with increased DC transmigration across LEC monolayers. Transmigration was guided by chemokines acting on DC, and blocking experiments with mAb indicated a role for LFA-1. Furthermore, incubation of DC with IFN-a led to the appearance of active conformation epitopes on the CD11a integrin chains expressed by DC. Differentiation of mouse DC in the presence of IFN-α also increased DC migration from inflamed footpads toward popliteal LN. Collectively, these results indicate a role for type I IFN in directing DC toward LN under inflammatory conditions. OBJ2: CD137 was originally described as a surface molecule present on activated T cells. However, its expression is also detected on hypoxic endothelial cells and inflamed blood vessels. Here, we demonstrate LECs up-regulate CD137 expression from undetectable levels on stimulation with TNF-a. CD137 cross-linking with an agonistic mAb results in NF-κB nuclear translocation, followed by up-regulation of VCAM and an increase in the production of the chemokine CCL21. Accordingly, there is an increase in CCR7-dependent migration toward conditioned medium from activated LECs on cross-linking with the agonistic mAb or the natural ligand (CD137L). Such an enhancement is also observed with monocyte-derived dendritic cells transmigrating across CD137-activated LEC monolayers. Using explanted human dermal tissue, we found that inflamed skin contains abundant CD137(+) lymphatic vessels and that ex vivo incubation of explanted human dermis with TNF-α induces CD137 expression in lymphatic capillaries. More interestingly, treatment with CD137 agonistic antibody induces CCL21 expression and DC accumulation close to lymphatic vessels. Collectively, our results demonstrate that the inflammatory function of lymphatic vessels can be regulated by CD137. OBJ 3: Dendritic cell (DC) transmigration across LEC is critical for the initiation and sustenance of immune responses. Under basal conditions DC transit across LEC has been shown to be integrin-independent. This is mainly due to the intermittent distribution of adherent junctions on LEC surface. In contrast, increasing evidences accumulate on the participation of integrins and their ligands in DC transit across lymphatics under inflammation. In this sense, it has been reported that under sustained inflammation, lung initial lymphatic vessels modify the distribution of the adherent junctions towards a less permissive-continuous zipper-like phenotype. Using confocal imaging, we show how skin lymphatic vessels presented altered VE-cadherin distribution after acute inflammation in mouse ear samples. Consequently, we described for the first time, the formation of ICAM-1 -enriched 3D structures around DC adhered and in transit across mouse and human ex-vivo inflamed skin lymphatic vessels. Ex vivo and in vitro DC adhesion to LEC under inflammation was dependent on the presence of LFA-1 integrin on DC surface. Besides, in vitro experiments demonstrated the formation of microvilli-like structures on LEC close to CCL21 depots on inflamed LEC. Therefore, under inflammatory conditions, integrin ligands structure in 3D membrane projections on the surface LEC. These structures, offer docking sites for DC transit from the tissue towards the lymphatic lumen and can be modulated by chemokine exposure.