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.

Stromal Expression of Activated Leukocyte Cell Adhesion Molecule Promotes Lung Tumor Growth and Metastasis.

Activated leukocyte cell adhesion molecule (ALCAM) is expressed on various cell types, including leukocytes, endothelial cells, and certain tumor cells. Although ALCAM expression on tumor cells has been linked to tumor invasion and metastatic spread, the contribution of ALCAM expressed in cells forming the tumor stroma to cancer progression has not been investigated. In this study, ALCAM-deficient (ALCAM-/-) mice were used to evaluate the role of ALCAM in lung tumor growth and metastasis. ALCAM-/- mice displayed an altered blood vascular network in the lung and the diaphragm, indicative of an angiogenetic defect. The absence of ALCAM expression in cells forming the stromal tumor microenvironment profoundly affected lung tumor growth in three different i.v. metastasis models. In the case of Lewis lung carcinoma (LLC), an additional defect in tumor cell homing to the lungs and a resulting reduction in the number of lung tumor nodules were observed. Similarly, when LLC cells were implanted subcutaneously for the study of spontaneous tumor cell metastasis, the rate of LLC metastasis to the lungs was profoundly reduced in ALCAM-/- mice. Taken together, our work demonstrates for the first time the in vivo contribution of ALCAM to angiogenesis and reveals a novel role of stromally expressed ALCAM in supporting tumor growth and metastatic spread.

Interleukin-7 is produced by afferent lymphatic vessels and supports lymphatic drainage.

The cytokine interleukin (IL)-7 exerts essential roles in lymph node (LN) organogenesis and lymphocyte development and homeostasis. Recent studies have identified lymphatic endothelial cells (LECs) as a major source of IL-7 in LNs. Here, we report that LECs not only produce IL-7, but also express the IL-7 receptor chains IL-7Rα and CD132. Stimulation with recombinant IL-7 enhanced LEC in vitro activity and induced lymphangiogenesis in the cornea of wild-type (WT) mice. Whereas in IL-7Rα(-/-) mice, dermal lymphatic vessels (LVs) were abnormally organized and lymphatic drainage was compromised, transgenic overexpression of IL-7 in mice resulted in an expanded dermal LV network with increased drainage function. Moreover, systemic treatment with recombinant IL-7 enhanced lymphatic drainage in the skin of WT mice and of mice devoid of lymphocytes. Experiments in IL-7Rα(-/-) bone marrow chimeras demonstrated that the drainage-enhancing activity of IL-7 was exclusively dependent on IL-7Rα expression in stromal but not in hematopoietic cells. Finally, near-infrared in vivo imaging performed in IL-7Rα(-/-) mice revealed that the pumping activity of collecting vessels was normal but fluid uptake into lymphatic capillaries was defective. Overall, our data point toward an unexpected new role for IL-7 as a potential autocrine mediator of lymphatic drainage.

Novel role for ALCAM in lymphatic network formation and function.

Adhesion molecules play an important role in vascular biology because they mediate vascular stability, permeability, and leukocyte trafficking to and from tissues. Performing microarray analyses, we have recently identified activated leukocyte cell adhesion molecule (ALCAM) as an inflammation-induced gene in lymphatic endothelial cells (LECs). ALCAM belongs to the immunoglobulin superfamily and engages in homophilic as well as heterophilic interactions. In this study, we found ALCAM to be expressed at the protein level in human and murine lymphatic and blood vascular endothelial cells in vitro and in the vasculature of human and murine tissues in vivo. Functional in vitro experiments revealed that ALCAM mediates adhesive interactions, migration, and tube formation in LECs, suggesting a role for ALCAM in lymphatic vessel (LV) stability and in lymphangiogenesis. Furthermore, ALCAM supported dendritic cell (DC) adhesion to lymphatic endothelium. In agreement with these findings, experiments performed in ALCAM mice detected reduced LEC numbers in various tissues and defects in the formation of an organized LV network. Moreover, DC migration from lung to draining lymph nodes was compromised in ALCAM mice. Collectively, our data reveal a novel role for ALCAM in stabilizing LEC-LEC interactions and in the organization and function of the LV network.

Opposing roles of endothelial and leukocyte-expressed IL-7Rα in the regulation of psoriasis-like skin inflammation.

The interleukin 7 receptor alpha chain (IL-7Rα) is predominately expressed by lymphocytes, and activation by its ligand IL-7 supports the development and maintenance of T cells and boosts T-cell mediated immunity. We recently reported that lymphatic endothelial cells (LECs) in dermal lymphatics also express IL-7 and its receptor chains (IL-7Rα and CD132) and that IL-7 supports lymphatic drainage. This suggested that activation of IL-7Rα signaling in lymphatics could exert inflammation-resolving activity, by promoting the clearance of excess tissue fluid. Here we investigated how the potentially opposing effects of IL-7Rα signaling in immune cells and in the lymphatic vasculature would affect the development and progression of psoriasis-like skin inflammation. We found that during acute and chronic skin inflammation mice with an endothelial-specific deletion of IL-7Rα (IL-7RαΔEC mice) developed more edema compared to control mice, as a consequence of impaired lymphatic drainage. However, systemic treatment of wild-type mice with IL-7 exacerbated edema and immune cell infiltration in spite of increasing lymphatic drainage, whereas treatment with IL-7Rα blocking antibody ameliorated inflammatory symptoms. These data identify IL-7Rα signaling as a new pathway in psoriasis-like skin inflammation and show that its pro-inflammatory effects on the immune compartment override its anti-inflammatory, drainage-enhancing effects on the endothelium.

ALCAM Mediates DC Migration Through Afferent Lymphatics and Promotes Allospecific Immune Reactions.

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is a cell adhesion molecule of the immunoglobulin superfamily and has been implicated in diverse pathophysiological processes including T cell activation, leukocyte trafficking, and (lymph)angiogenesis. However, exploring the therapeutic potential of ALCAM blockade in immune-mediated inflammatory disorders has been difficult due to the lack of antibodies with blocking activity toward murine ALCAM. In this study, we identified and characterized a monoclonal antibody with high affinity and specificity for murine ALCAM. This antibody reduced in vitro T cell activation induced by antigen-presenting dendritic cells (DCs) as well as (trans)migration of murine DCs across lymphatic endothelial monolayers. Moreover, it reduced emigration of DCs from in vitro-cultured human skin biopsies. Similarly, antibody-based blockade of ALCAM reduced (lymph)angiogenic processes in vitro and decreased developmental lymphangiogenesis in vivo to levels observed in ALCAM-deficient mice. Since corneal allograft rejection is an important medical condition that also involves (lymph)angiogenesis, DC migration and T cell activation, we investigated the therapeutic potential of ALCAM blockade in murine corneal disease. Blocking ALCAM lead to DC retention in corneas and effectively prevented corneal allograft rejection. Considering that we also detected ALCAM expression in human corneal DCs and lymphatics, our findings identify ALCAM as a potential novel therapeutic target in human corneal allograft rejection.

Multiple roles of lymphatic vessels in peripheral lymph node development.

The mammalian lymphatic system consists of strategically located lymph nodes (LNs) embedded into a lymphatic vascular network. Mechanisms underlying development of this highly organized system are not fully understood. Using high-resolution imaging, we show that lymphoid tissue inducer (LTi) cells initially transmigrate from veins at LN development sites using gaps in venous mural coverage. This process is independent of lymphatic vasculature, but lymphatic vessels are indispensable for the transport of LTi cells that egress from blood capillaries elsewhere and serve as an essential LN expansion reservoir. At later stages, lymphatic collecting vessels ensure efficient LTi cell transport and formation of the LN capsule and subcapsular sinus. Perinodal lymphatics also promote local interstitial flow, which cooperates with lymphotoxin-ß signaling to amplify stromal CXCL13 production and thereby promote LTi cell retention. Our data unify previous models of LN development by showing that lymphatics intervene at multiple points to assist LN expansion and identify a new role for mechanical forces in LN development.

Intralymphatic CCL21 Promotes Tissue Egress of Dendritic Cells through Afferent Lymphatic Vessels.

To induce adaptive immunity, dendritic cells (DCs) migrate through afferent lymphatic vessels (LVs) to draining lymph nodes (dLNs). This process occurs in several consecutive steps. Upon entry into lymphatic capillaries, DCs first actively crawl into downstream collecting vessels. From there, they are next passively and rapidly transported to the dLN by lymph flow. Here, we describe a role for the chemokine CCL21 in intralymphatic DC crawling. Performing time-lapse imaging in murine skin, we found that blockade of CCL21-but not the absence of lymph flow-completely abolished DC migration from capillaries toward collecting vessels and reduced the ability of intralymphatic DCs to emigrate from skin. Moreover, we found that in vitro low laminar flow established a CCL21 gradient along lymphatic endothelial monolayers, thereby inducing downstream-directed DC migration. These findings reveal a role for intralymphatic CCL21 in promoting DC trafficking to dLNs, through the formation of a flow-induced gradient.

Oxazolone-induced contact hypersensitivity reduces lymphatic drainage but enhances the induction of adaptive immunity.

Contact hypersensitivity (CHS) induced by topical application of haptens is a commonly used model to study dermal inflammatory responses in mice. Several recent studies have indicated that CHS-induced skin inflammation triggers lymphangiogenesis but may negatively impact the immune-function of lymphatic vessels, namely fluid drainage and dendritic cell (DC) migration to draining lymph nodes (dLNs). On the other hand, haptens have been shown to exert immune-stimulatory activity by inducing DC maturation. In this study we investigated how the presence of pre-established CHS-induced skin inflammation affects the induction of adaptive immunity in dLNs. Using a mouse model of oxazolone-induced skin inflammation we observed that lymphatic drainage was reduced and DC migration from skin to dLNs was partially compromised. At the same time, a significantly stronger adaptive immune response towards ovalbumin (OVA) was induced when immunization had occurred in CHS-inflamed skin as compared to uninflamed control skin. In fact, immunization with sterile OVA in CHS-inflamed skin evoked a delayed-type hypersensitivity (DTH) response comparable to the one induced by conventional immunization with OVA and adjuvant in uninflamed skin. Striking phenotypic and functional differences were observed when comparing DCs from LNs draining uninflamed or CHS-inflamed skin. DCs from LNs draining CHS-inflamed skin expressed higher levels of co-stimulatory molecules and MHC molecules, produced higher levels of the interleukin-12/23 p40 subunit (IL-12/23-p40) and more potently induced T cell activation in vitro. Immunization experiments revealed that blockade of IL-12/23-p40 during the priming phase partially reverted the CHS-induced enhancement of the adaptive immune response. Collectively, our findings indicate that CHS-induced skin inflammation generates an overall immune-stimulatory milieu, which outweighs the potentially suppressive effect of reduced lymphatic vessel function.

Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice.

The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/"metabolizing acquired dioxin-induced skin hamartomas" (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis.

Nrf2 links epidermal barrier function with antioxidant defense.

The skin provides an efficient permeability barrier and protects from microbial invasion and oxidative stress. Here, we show that these essential functions are linked through the Nrf2 transcription factor. To test the hypothesis that activation of Nrf2 provides skin protection under stress conditions, we determined the consequences of pharmacological or genetic activation of Nrf2 in keratinocytes. Surprisingly, mice with enhanced Nrf2 activity in keratinocytes developed epidermal thickening, hyperkeratosis and inflammation resembling lamellar ichthyosis. This resulted from upregulation of the cornified envelope proteins small proline-rich proteins (Sprr) 2d and 2h and of secretory leukocyte peptidase inhibitor (Slpi), which we identified as novel Nrf2 targets in keratinocytes. Since Sprrs are potent scavengers of reactive oxygen species and since Slpi has antimicrobial activities, their upregulation contributes to Nrf2's protective function. However, it also caused corneocyte fragility and impaired desquamation, followed by alterations in the epidermal lipid barrier, inflammation and overexpression of mitogens that induced keratinocyte hyperproliferation. These results identify an unexpected role of Nrf2 in epidermal barrier function, which needs to be considered for pharmacological use of Nrf2 activators.