Impaired humoral immunity and tolerance in K14-VEGFR-3-Ig mice that lack dermal lymphatic drainage.
ABSTRACT: Lymphatic vessels transport interstitial fluid, soluble Ag, and immune cells from peripheral tissues to lymph nodes (LNs), yet the contribution of peripheral lymphatic drainage to adaptive immunity remains poorly understood. We examined immune responses to dermal vaccination and contact hypersensitivity (CHS) challenge in K14-VEGFR-3-Ig mice, which lack dermal lymphatic capillaries and experience markedly depressed transport of solutes and dendritic cells from the skin to draining LNs. In response to dermal immunization, K14-VEGFR-3-Ig mice produced lower Ab titers. In contrast, although delayed, T cell responses were robust after 21 d, including high levels of Ag-specific CD8+ T cells and production of IFN-?, IL-4, and IL-10 upon restimulation. T cell-mediated CHS responses were strong in K14-VEGFR-3-Ig mice, but importantly, their ability to induce CHS tolerance in the skin was impaired. In addition, 1-y-old mice displayed multiple signs of autoimmunity. These data suggest that lymphatic drainage plays more important roles in regulating humoral immunity and peripheral tolerance than in effector T cell immunity.
Project description:Lymphatic remodeling in tumor microenvironments correlates with progression and metastasis, and local lymphatic vessels play complex and poorly understood roles in tumor immunity. Tumor lymphangiogenesis is associated with increased immune suppression, yet lymphatic vessels are required for fluid drainage and immune cell trafficking to lymph nodes, where adaptive immune responses are mounted. Here, we examined the contribution of lymphatic drainage to tumor inflammation and immunity using a mouse model that lacks dermal lymphatic vessels (K14-VEGFR3-Ig mice). Melanomas implanted in these mice grew robustly, but exhibited drastically reduced cytokine expression and leukocyte infiltration compared with those implanted in control animals. In the absence of local immune suppression, transferred cytotoxic T cells more effectively controlled tumors in K14-VEGFR3-Ig mice than in control mice. Furthermore, gene expression analysis of human melanoma samples revealed that patient immune parameters are markedly stratified by levels of lymphatic markers. This work suggests that the establishment of tumor-associated inflammation and immunity critically depends on lymphatic vessel remodeling and drainage. Moreover, these results have implications for immunotherapies, the efficacies of which are regulated by the tumor immune microenvironment.
Project description: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.
Project description:This study was undertaken to investigate the effect of lymphatic inhibition on joint and draining lymph node (LN) pathology during the course of arthritis progression in mice.Tumor necrosis factor (TNF)-transgenic mice were used as a model of chronic inflammatory arthritis. Mice were subjected to contrast-enhanced magnetic resonance imaging to obtain ankle and knee joint synovial volumes and draining popliteal LN volumes before and after 8 weeks of treatment with vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody, VEGFR-2 neutralizing antibody, or isotype IgG. Animals were subjected to near-infrared lymphatic imaging to determine the effect of VEGFR-3 neutralization on lymph transport from paws to draining popliteal LNs. Histologic, immunohistochemical, and reverse transcriptase-polymerase chain reaction analyses were used to examine lymphatic vessel formation and the morphology of joints and popliteal LNs.Compared with IgG treatment, VEGFR-3 neutralizing antibody treatment significantly decreased the size of popliteal LNs, the number of lymphatic vessels in joints and popliteal LNs, lymphatic drainage from paws to popliteal LNs, and the number of VEGF-C-expressing CD11b+ myeloid cells in popliteal LNs. However, it increased the synovial volume and area of inflammation in ankle and knee joints. VEGFR-2 neutralizing antibody, in contrast, inhibited both lymphangiogenesis and joint inflammation.These findings indicate that lymphangiogenesis and lymphatic drainage are reciprocally related to the severity of joint lesions during the development of chronic arthritis. Lymphatic drainage plays a beneficial role in controlling the progression of chronic inflammation.
Project description: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-7R? 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-7R?(-/-) 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-7R?(-/-) bone marrow chimeras demonstrated that the drainage-enhancing activity of IL-7 was exclusively dependent on IL-7R? expression in stromal but not in hematopoietic cells. Finally, near-infrared in vivo imaging performed in IL-7R?(-/-) 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.
Project description:In contrast to the established role of blood vessel remodeling in inflammation, the biologic function of the lymphatic vasculature in acute inflammation has remained less explored. We studied 2 established models of acute cutaneous inflammation, namely, oxazolone-induced delayed-type hypersensitivity reactions and ultraviolet B irradiation, in keratin 14-vascular endothelial growth factor (VEGF)-C and keratin 14-VEGF-D transgenic mice. These mice have an expanded network of cutaneous lymphatic vessels. Transgenic delivery of the lymphangiogenic factors VEGF-C and the VEGFR-3 specific ligand mouse VEGF-D significantly limited acute skin inflammation in both experimental models, with a strong reduction of dermal edema. Expression of VEGFR-3 by lymphatic endothelium was strongly down-regulated at the mRNA and protein level in acutely inflamed skin, and no VEGFR-3 expression was detectable on inflamed blood vessels and dermal macrophages. There was no major change of the inflammatory cell infiltrate or the composition of the inflammatory cytokine milieu in the inflamed skin of VEGF-C or VEGF-D transgenic mice. However, the increased network of lymphatic vessels in these mice significantly enhanced lymphatic drainage from the ear skin. These results provide evidence that specific lymphatic vessel activation limits acute skin inflammation via promotion of lymph flow from the skin and reduction of edema formation.
Project description:Lymphatic vessel plasticity and stability are of considerable importance when attempting to treat diseases associated with the lymphatic vasculature. Development of lymphatic vessels during embryogenesis is dependent on vascular endothelial growth factor (VEGF)-C but not VEGF-D. Using a recombinant adenovirus encoding a soluble form of their receptor VEGFR-3 (AdVEGFR-3-Ig), we studied lymphatic vessel dependency on VEGF-C and VEGF-D induced VEGFR-3 signaling in postnatal and adult mice. Transduction with AdVEGFR-3-Ig led to regression of lymphatic capillaries and medium-sized lymphatic vessels in mice under 2 weeks of age without affecting collecting lymphatic vessels or the blood vasculature. No effect was observed after this period. The lymphatic capillaries of neonatal mice also regressed partially in response to recombinant VEGFR-3-Ig or blocking antibodies against VEGFR-3, but not to adenovirus-encoded VEGFR-2-Ig. Despite sustained inhibitory VEGFR-3-Ig levels, lymphatic vessel regrowth was observed at 4 weeks of age. Interestingly, whereas transgenic expression of VEGF-C in the skin induced lymphatic hyperplasia even during embryogenesis, similar expression of VEGF-D resulted in lymphangiogenesis predominantly after birth. These results indicate considerable plasticity of lymphatic vessels during the early postnatal period but not thereafter, suggesting that anti-lymphangiogenic therapy can be safely applied in adults.
Project description:Langerhans cells (LCs) are antigen-presenting cells that reside in the epidermis of the skin and traffic to lymph nodes (LNs). The general role of these cells in skin immune responses is not clear because distinct models of LC depletion resulted in opposite conclusions about their role in contact hypersensitivity (CHS) responses. While comparing these models, we discovered a novel population of LCs that resides in the dermis and does not represent migrating epidermal LCs, as previously thought. Unlike epidermal LCs, dermal Langerin(+) dendritic cells (DCs) were radiosensitive and displayed a distinct cell surface phenotype. Dermal Langerin(+) DCs migrate from the skin to the LNs after inflammation and in the steady state, and represent the majority of Langerin(+) DCs in skin draining LNs. Both epidermal and dermal Langerin(+) DCs were depleted by treatment with diphtheria toxin in Lang-DTREGFP knock-in mice. In contrast, transgenic hLang-DTA mice lack epidermal LCs, but have normal numbers of dermal Langerin(+) DCs. CHS responses were abrogated upon depletion of both epidermal and dermal LCs, but were unaffected in the absence of only epidermal LCs. This suggests that dermal LCs can mediate CHS and provides an explanation for previous differences observed in the two-model systems.
Project description:Lymphatic vessels play an important role in the regulation of tissue fluid balance, immune responses, and fat adsorption and are involved in diseases including lymphedema and tumor metastasis. Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3) is necessary for development of the blood vasculature during early embryogenesis, but later, VEGFR-3 expression becomes restricted to the lymphatic vasculature. We analyzed mice deficient in both of the known VEGFR-3 ligands, VEGF-C and VEGF-D. Unlike the Vegfr3(-/-) embryos, the Vegfc(-/-); Vegfd(-/-) embryos displayed normal blood vasculature after embryonic day 9.5. Deletion of Vegfr3 in the epiblast, using keratin 19 (K19) Cre, resulted in a phenotype identical to that of the Vegfr3(-/-) embryos, suggesting that this phenotype is due to defects in the embryo proper and not in placental development. Interestingly, the Vegfr3(neo) hypomorphic mutant mice carrying the neomycin cassette between exons 1 and 2 showed defective lymphatic development. Overexpression of human or mouse VEGF-D in the skin, under the K14 promoter, rescued the lymphatic hypoplasia of the Vegfc(+/-) mice in the K14-VEGF-D; Vegfc(+/-) compound mice, suggesting that VEGF-D is functionally redundant with VEGF-C in the stimulation of developmental lymphangiogenesis. Our results suggest VEGF-C- and VEGF-D-independent functions for VEGFR-3 in the early embryo.
Project description:OBJECTIVE:B cell depletion therapy ameliorates rheumatoid arthritis by mechanisms that are incompletely understood. Arthritis flare in tumor necrosis factor (TNF)-transgenic mice is associated with efferent lymph node (LN) "collapse," triggered by B cell translocation into lymphatic spaces and decreased lymphatic drainage. The aim of this study was to examine whether the efficacy of B cell depletion therapy is associated with restoration of lymphatic drainage due to removal of obstructing nodal B cells. METHODS:We used contrast-enhanced magnetic resonance imaging, indocyanine green near-infrared imaging, and intravital immunofluorescence imaging to longitudinally assess synovitis, lymphatic flow, and cell migration in lymphatic vessels in TNF-transgenic mice. We conducted tests to determine whether the efficacy of B cell depletion therapy is associated with restoration of lymphatic draining and cell egress from arthritic joints. RESULTS:Unlike active lymphatics to normal and prearthritic knees, afferent lymphatic vessels to collapsed LNs in inflamed knees do not pulse. Intravital immunofluorescence imaging demonstrated that CD11b+ monocyte/macrophages in lymphatic vessels afferent to expanding LNs travel at high velocity (mean±SD 186±37 ?m/second), while these cells are stationary in lymphatic vessels afferent to collapsed popliteal LNs. B cell depletion therapy for arthritis flares in TNF-transgenic mice significantly decreased knee synovium volume (by 50% from the baseline level) and significantly increased lymphatic clearance compared with placebo (P<0.05). This increased lymphatic drainage restored macrophage egress from inflamed joints without recovery of the lymphatic pulse. CONCLUSION:These results support a novel mechanism in which B cell depletion therapy for joint arthritis flares lessens inflammation by increasing lymphatic drainage and subsequent migration of cells and cytokines from the synovial space.
Project description:Lymph nodes are secondary lymphoid tissues that play a critical role in filtering the lymph and supporting adaptive immune responses. Surgical resection of LNs, radiation therapy or infections may damage lymphatic vasculature and compromised immune functions. Here, we describe the generation of functional synthetic lympho-organoids (LOs) using LN stromal progenitors and decellularized extra cellular matrix-based scaffolds. We show that upon transplantation at the site of resected LNs, LOs become integrated into the endogenous lymphatic vasculature and efficiently restore lymphatic drainage and perfusion. Upon immunization, LOs support the activation of antigen-specific immune responses, thus acquiring properties of native lymphoid tissues. These findings provide the first proof-of-concept for the development of synthetic lympho-organoids suitable to restore lymphatic and immune cell functions.