CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells.

Dendritic cell (DC) migration from peripheral tissues via afferent lymphatic vessels to draining lymph nodes (dLNs) is important for the organism's immune regulation and immune protection. Several lymphatic endothelial cell (LEC)-expressed adhesion molecules have thus far been found to support transmigration and movement within the lymphatic vasculature. In this study, we investigated the contribution of CD112, an adhesion molecule that we recently found to be highly expressed in murine LECs, to this process. Performing in vitro assays in the murine system, we found that transmigration of bone marrow-derived dendritic cells (BM-DCs) across or adhesion to murine LEC monolayers was reduced when CD112 was absent on LECs, DCs, or both cell types, suggesting the involvement of homophilic CD112-CD112 interactions. While CD112 was highly expressed in murine dermal LECs, CD112 levels were low in endogenous murine dermal DCs and BM-DCs. This might explain why we observed no defect in the in vivo lymphatic migration of adoptively transferred BM-DCs or endogenous DCs from the skin to dLNs. Compared to murine DCs, human monocyte-derived DCs expressed higher CD112 levels, and their migration across human CD112-expressing LECs was significantly reduced upon CD112 blockade. CD112 expression was also readily detected in endogenous human dermal DCs and LECs by flow cytometry and immunofluorescence. Upon incubating human skin punch biopsies in the presence of CD112-blocking antibodies, DC emigration from the tissue into the culture medium was significantly reduced, indicating impaired lymphatic migration. Overall, our data reveal a contribution of CD112 to human DC migration.

3-hydroxy-L-kynurenamine is an immunomodulatory biogenic amine.

Tryptophan catabolism is a major metabolic pathway utilized by several professional and non-professional antigen presenting cells to maintain immunological tolerance. Here we report that 3-hydroxy-L-kynurenamine (3-HKA) is a biogenic amine produced via an alternative pathway of tryptophan metabolism. In vitro, 3-HKA has an anti-inflammatory profile by inhibiting the IFN-γ mediated STAT1/NF-κΒ pathway in both mouse and human dendritic cells (DCs) with a consequent decrease in the release of pro-inflammatory chemokines and cytokines, most notably TNF, IL-6, and IL12p70. 3-HKA has protective effects in an experimental mouse model of psoriasis by decreasing skin thickness, erythema, scaling and fissuring, reducing TNF, IL-1ß, IFN-γ, and IL-17 production, and inhibiting generation of effector CD8+ T cells. Similarly, in a mouse model of nephrotoxic nephritis, besides reducing inflammatory cytokines, 3-HKA improves proteinuria and serum urea nitrogen, overall ameliorating immune-mediated glomerulonephritis and renal dysfunction. Overall, we propose that this biogenic amine is a crucial component of tryptophan-mediated immune tolerance.

CD112 Regulates Angiogenesis and T Cell Entry into the Spleen.

Junctional adhesion proteins play important roles in controlling angiogenesis, vascular permeability and leukocyte trafficking. CD112 (nectin-2) belongs to the immunoglobulin superfamily and was shown to engage in homophilic and heterophilic interactions with a variety of binding partners expressed on endothelial cells and on leukocytes. Recent in vitro studies suggested that CD112 regulates human endothelial cell migration and proliferation as well as transendothelial migration of leukocytes. However, so far, the role of CD112 in endothelial cell biology and in leukocyte trafficking has not been elucidated in vivo. We found CD112 to be expressed by lymphatic and blood endothelial cells in different murine tissues. In CD112-deficient mice, the blood vessel coverage in the retina and spleen was significantly enhanced. In functional in vitro studies, a blockade of CD112 modulated endothelial cell migration and significantly enhanced endothelial tube formation. An antibody-based blockade of CD112 also significantly reduced T cell transmigration across endothelial monolayers in vitro. Moreover, T cell homing to the spleen was significantly reduced in CD112-deficient mice. Overall, our results identify CD112 as a regulator of angiogenic processes in vivo and demonstrate a novel role for CD112 in T cell entry into the spleen.

In Vivo Imaging of Local Inflammation: Monitoring LPS-Induced CD80/CD86 Upregulation by PET.

PURPOSE: The co-stimulatory molecules CD80 and CD86 are upregulated on activated antigen-presenting cells (APC). We investigated whether local APC activation, induced by subcutaneous (s.c.) inoculation of lipopolysaccharides (LPS), can be imaged by positron emission tomography (PET) with CD80/CD86-targeting 64Cu-labelled abatacept. PROCEDURES: Mice were inoculated s.c. with extracellular-matrix gel containing either LPS or vehicle (PBS). Immune cell populations were analysed by flow cytometry and marker expression by RT-qPCR. 64Cu-NODAGA-abatacept distribution was analysed using PET/CT and ex vivo biodistribution. RESULTS: The number of CD80+ and CD86+ immune cells at the LPS inoculation site significantly increased a few days after inoculation. CD68 and CD86 expression were higher at the LPS than the PBS inoculation site, and CD80 was only increased at the LPS inoculation site. CTLA-4 was highest 10 days after LPS inoculation, when CD80/CD86 decreased again. A few days after inoculation, 64Cu-NODAGA-abatacept distribution to the inoculation site was significantly higher for LPS than PBS (4.2-fold). Co-administration of unlabelled abatacept or human immunoglobulin reduced tracer uptake. The latter reduced the number of CD86+ immune cells at the LPS inoculation site. CONCLUSIONS: CD80 and CD86 are upregulated in an LPS-induced local inflammation, indicating invasion of activated APCs. 64Cu-NODAGA-abatacept PET allowed following APC activation over time.

Dendritic Cells and T Cells Interact Within Murine Afferent Lymphatic Capillaries.

Afferent lymphatic vessels contribute to immunity by transporting antigen and leukocytes to draining lymph nodes (LNs) and are emerging as new players in the regulation of peripheral tolerance. Performing intravital microscopy in inflamed murine ear skin we found that migrating dendritic cells (DCs) and antigen-experienced effector T cells spend considerable time arresting or clustering within afferent lymphatic capillaries. We also observed that intralymphatic T cells frequently interacted with DCs. When imaging polyclonal T cells during an ongoing contact-hypersensitivity response, most intralymphatic DC-T cell interactions were short-lived. Conversely, during a delayed-type-hypersensitivity response, cognate antigen-bearing DCs engaged in long-lived MHCII-(I-A/I-E)-dependent interactions with antigen-specific T cells. Long-lived intralymphatic DC-T cell interactions reduced the speed of DC crawling but did not delay overall DC migration to draining LNs. While further consequences of these intralymphatic interactions still need to be explored, our findings suggest that lymphatic capillaries represent a unique compartment in which adaptive immune interaction and modulation occur.

Cellular traffic through afferent lymphatic vessels.

The lymphatic system has long been known to serve as a highway for migrating leukocytes from peripheral tissue to draining lymph nodes (dLNs) and back to circulation, thereby contributing to the induction of adaptive immunity and immunesurveillance. Lymphatic vessels (LVs) present in peripheral tissues upstream of a first dLN are generally referred to as afferent LVs. In contrast to migration through blood vessels (BVs), the detailed molecular and cellular requirements of cellular traffic through afferent LVs have only recently started to be unraveled. Progress in our ability to track the migration of lymph-borne cell populations, in combination with cutting-edge imaging technologies, nowadays allows the investigation and visualization of lymphatic migration of endogenous leukocytes, both at the population and at the single-cell level. These studies have revealed that leukocyte trafficking through afferent LVs generally follows a step-wise migration pattern, relying on the active interplay of numerous molecules. In this review, we will summarize and discuss current knowledge of cellular traffic through afferent LVs. We will first outline how the structure of the afferent LV network supports leukocyte migration and highlight important molecules involved in the migration of dendritic cells (DCs), T cells and neutrophils, i.e. the most prominent cell types trafficking through afferent LVs. Additionally, we will describe how tumor cells hijack the lymphatic system for their dissemination to draining LNs. Finally, we will summarize and discuss our current understanding of the functional significance as well as the therapeutic implications of cell traffic through afferent LVs.

AutoTube: a novel software for the automated morphometric analysis of vascular networks in tissues.

Due to their involvement in many physiologic and pathologic processes, there is a great interest in identifying new molecular pathways that mediate the formation and function of blood and lymphatic vessels. Vascular research increasingly involves the image-based analysis and quantification of vessel networks in tissue whole-mounts or of tube-like structures formed by cultured endothelial cells in vitro. While both types of experiments deliver important mechanistic insights into (lymph)angiogenic processes, the manual analysis and quantification of such experiments are typically labour-intensive and affected by inter-experimenter variability. To bypass these problems, we developed AutoTube, a new software that quantifies parameters like the area covered by vessels, vessel width, skeleton length and branching or crossing points of vascular networks in tissues and in in vitro assays. AutoTube is freely downloadable, comprises an intuitive graphical user interface and helps to perform otherwise highly time-consuming image analyses in a rapid, automated and reproducible manner. By analysing lymphatic and blood vascular networks in whole-mounts prepared from different tissues or from gene-targeted mice with known vascular abnormalities, we demonstrate the ability of AutoTube to determine vascular parameters in close agreement to the manual analyses and to identify statistically significant differences in vascular morphology in tissues and in vascular networks formed in in vitro assays.