The endothelial protein PLVAP in lymphatics controls the entry of lymphocytes and antigens into lymph nodes.

In the lymphatic sinuses of draining lymph nodes, soluble lymph-borne antigens enter the reticular conduits in a size-selective manner and lymphocytes transmigrate to the parenchyma. The molecular mechanisms that control these processes are unknown. Here we unexpectedly found that PLVAP, a prototypic endothelial protein of blood vessels, was synthesized in the sinus-lining lymphatic endothelial cells covering the distal conduits. In PLVAP-deficient mice, both small antigens and large antigens entered the conduit system, and the transmigration of lymphocytes through the sinus floor was augmented. Mechanistically, the filtering function of the lymphatic sinus endothelium was dependent on diaphragms formed by PLVAP fibrils in transendothelial channels. Thus, in the lymphatic sinus, PLVAP forms a physical sieve that regulates the parenchymal entry of lymphocytes and soluble antigens.

Polymorphism in interferon alpha/beta receptor contributes to glucocorticoid response and outcome of ARDS and COVID-19.

BACKGROUND: The use of glucocorticoids has given contradictory results for treating acute respiratory distress syndrome (ARDS). The use of intravenous Interferon beta (IFN ß) for the treatment of ARDS was recently tested in a phase III ARDS trial (INTEREST), in which more than half of the patients simultaneously received glucocorticoids. Trial results showed deleterious effects of glucocorticoids when administered together with IFN ß, and therefore, we aimed at finding the reason behind this. METHODS: We first sequenced the genes encoding the IFN α/ß receptor of the patients, who participated in the INTEREST study (ClinicalTrials.gov Identifier:  NCT02622724 , November 24, 2015) in which the patients were randomized to receive an intravenous injection of IFN ß-1a (144 patients) or placebo (152 patients). Genetic background was analyzed against clinical outcome, concomitant medication, and pro-inflammatory cytokine levels. Thereafter, we tested the influence of the genetic background on IFN α/ß receptor expression in lung organ cultures and whether, it has any effect on transcription factors STAT1 and STAT2 involved in IFN signaling. RESULTS: We found a novel disease association of a SNP rs9984273, which is situated in the interferon α/ß receptor subunit 2 (IFNAR2) gene in an area corresponding to a binding motif of the glucocorticoid receptor (GR). The minor allele of SNP rs9984273 associates with higher IFNAR expression, more rapid decrease of IFN γ and interleukin-6 (IL-6) levels and better outcome in IFN ß treated patients with ARDS, while the major allele associates with a poor outcome especially under concomitant IFN ß and glucocorticoid treatment. Moreover, the minor allele of rs9984273 associates with a less severe form of coronavirus diseases (COVID-19) according to the COVID-19 Host Genetics Initiative database. CONCLUSIONS: The distribution of this SNP within clinical study arms may explain the contradictory results of multiple ARDS studies and outcomes in COVID-19 concerning type I IFN signaling and glucocorticoids.

Fetal liver endothelium regulates the seeding of tissue-resident macrophages.

Macrophages are required for normal embryogenesis, tissue homeostasis and immunity against microorganisms and tumours. Adult tissue-resident macrophages largely originate from long-lived, self-renewing embryonic precursors and not from haematopoietic stem-cell activity in the bone marrow. Although fate-mapping studies have uncovered a great amount of detail on the origin and kinetics of fetal macrophage development in the yolk sac and liver, the molecules that govern the tissue-specific migration of these cells remain completely unknown. Here we show that an endothelium-specific molecule, plasmalemma vesicle-associated protein (PLVAP), regulates the seeding of fetal monocyte-derived macrophages to tissues in mice. We found that PLVAP-deficient mice have completely normal levels of both yolk-sac- and bone-marrow-derived macrophages, but that fetal liver monocyte-derived macrophage populations were practically missing from tissues. Adult PLVAP-deficient mice show major alterations in macrophage-dependent iron recycling and mammary branching morphogenesis. PLVAP forms diaphragms in the fenestrae of liver sinusoidal endothelium during embryogenesis, interacts with chemoattractants and adhesion molecules and regulates the egress of fetal liver monocytes to the systemic vasculature. Thus, PLVAP selectively controls the exit of macrophage precursors from the fetal liver and, to our knowledge, is the first molecule identified in any organ as regulating the migratory events during embryonic macrophage ontogeny.

Fetal-derived macrophages persist and sequentially maturate in ovaries after birth in mice.

Macrophages, which are highly diverse in different tissues, play a complex and vital role in tissue development, homeostasis, and inflammation. The origin and heterogeneity of tissue-resident monocytes and macrophages in ovaries remains unknown. Here we identify three tissue-resident monocyte populations and five macrophage populations in the adult ovaries using high-dimensional single cell mass cytometry. Ontogenic analyses using cell fate mapping models and cell depletion experiments revealed the infiltration of ovaries by both yolk sac and fetal liver-derived macrophages already during the embryonic development. Moreover, we found that both embryonic and bone marrow-derived macrophages contribute to the distinct ovarian macrophage subpopulations in the adults. These assays also showed that fetal-derived MHC II-negative macrophages differentiate postnatally in the maturing ovary to MHC II-positive cells. Our analyses further unraveled that the developmentally distinct macrophage types share overlapping distribution and scavenging function in the ovaries under homeostatic conditions. In conclusion, we report here the first comprehensive analyses of ovarian monocytes and macrophages. In addition, we show that the mechanisms controlling monocyte immigration, the phenotype of different pools of interstitial macrophages, and the interconversion capacity of fetal-derived macrophages in ovaries are remarkably different from those seen in other tissue niches.

Effect of Intravenous Interferon ß-1a on Death and Days Free From Mechanical Ventilation Among Patients With Moderate to Severe Acute Respiratory Distress Syndrome: A Randomized Clinical Trial.

Importance: Acute respiratory distress syndrome (ARDS) is associated with high mortality. Interferon (IFN) ß-1a may prevent the underlying event of vascular leakage. Objective: To determine the efficacy and adverse events of IFN-ß-1a in patients with moderate to severe ARDS. Design, Setting, and Participants: Multicenter, randomized, double-blind, parallel-group trial conducted at 74 intensive care units in 8 European countries (December 2015-December 2017) that included 301 adults with moderate to severe ARDS according to the Berlin definition. The radiological and partial pressure of oxygen, arterial (Pao2)/fraction of inspired oxygen (Fio2) criteria for ARDS had to be met within a 24-hour period, and the administration of the first dose of the study drug had to occur within 48 hours of the diagnosis of ARDS. The last patient visit was on March 6, 2018. Interventions: Patients were randomized to receive an intravenous injection of 10 µg of IFN-ß-1a (144 patients) or placebo (152 patients) once daily for 6 days. Main Outcomes and Measures: The primary outcome was a score combining death and number of ventilator-free days at day 28 (score ranged from -1 for death to 27 if the patient was off ventilator on the first day). There were 16 secondary outcomes, including 28-day mortality, which were tested hierarchically to control type I error. Results: Among 301 patients who were randomized (mean age, 58 years; 103 women [34.2%]), 296 (98.3%) completed the trial and were included in the primary analysis. At 28 days, the median composite score of death and number of ventilator-free days at day 28 was 10 days (interquartile range, -1 to 20) in the IFN-ß-1a group and 8.5 days (interquartile range, 0 to 20) in the placebo group (P = .82). There was no significant difference in 28-day mortality between the IFN-ß-1a vs placebo groups (26.4% vs 23.0%; difference, 3.4% [95% CI, -8.1% to 14.8%]; P = .53). Seventy-four patients (25.0%) experienced adverse events considered to be related to treatment during the study (41 patients [28.5%] in the IFN-ß-1a group and 33 [21.7%] in the placebo group). Conclusions and Relevance: Among adults with moderate or severe ARDS, intravenous IFN-ß-1a administered for 6 days, compared with placebo, resulted in no significant difference in a composite score that included death and number of ventilator-free days over 28 days. These results do not support the use of IFN-ß-1a in the management of ARDS. Trial Registration: ClinicalTrials.gov Identifier: NCT02622724.

Gene-expression profiling of different arms of lymphatic vasculature identifies candidates for manipulation of cell traffic.

Afferent lymphatic vessels bring antigens and diverse populations of leukocytes to draining lymph nodes, whereas efferent lymphatics allow only lymphocytes and antigens to leave the nodes. Despite the fundamental importance of afferent vs. efferent lymphatics in immune response and cancer spread, the molecular characteristics of these different arms of the lymphatic vasculature are largely unknown. The objective of this work was to explore molecular differences behind the distinct functions of afferent and efferent lymphatic vessels, and find possible molecules mediating lymphocyte traffic. We used laser-capture microdissection and cell sorting to isolate lymphatic endothelial cells (LECs) from the subcapsular sinus (SS, afferent) and lymphatic sinus (LS, efferent) for transcriptional analyses. The results reveal marked differences between afferent and efferent LECs and identify molecules on lymphatic vessels. Further characterizations of Siglec-1 (CD169) and macrophage scavenger receptor 1 (MSR1/CD204), show that they are discriminatively expressed on lymphatic endothelium of the SS but not on lymphatic vasculature of the LS. In contrast, endomucin (EMCN) is present on the LS endothelium and not on lymphatic endothelium of the SS. Moreover, both murine and human MSR1 on lymphatic endothelium of the SS bind lymphocytes and in in vivo studies MSR1 regulates entrance of lymphocytes from the SS to the lymph node parenchyma. In conclusion, this paper reports surprisingly distinct molecular profiles for afferent and efferent lymphatics and a function for MSR1. These results may open avenues to explore some of the now-identified molecules as targets to manipulate the function of lymphatic vessels.

Stabilin-1 expression defines a subset of macrophages that mediate tissue homeostasis and prevent fibrosis in chronic liver injury.

Macrophages are key regulators of fibrosis development and resolution. Elucidating the mechanisms by which they mediate this process is crucial for establishing their therapeutic potential. Here, we use experimental models of liver fibrosis to show that deficiency of the scavenger receptor, stabilin-1, exacerbates fibrosis and delays resolution during the recovery phase. We detected a subset of stabilin-1(+) macrophages that were induced at sites of cellular injury close to the hepatic scar in mouse models of liver fibrosis and in human liver disease. Stabilin-1 deficiency abrogated malondialdehyde-LDL (MDA-LDL) uptake by hepatic macrophages and was associated with excess collagen III deposition. Mechanistically, the lack of stabilin-1 led to elevated intrahepatic levels of the profibrogenic chemokine CCL3 and an increase in GFAP(+) fibrogenic cells. Stabilin-1(-/-) macrophages demonstrated a proinflammatory phenotype during liver injury and the normal induction of Ly6C(lo) monocytes during resolution was absent in stabilin-1 knockouts leading to persistence of fibrosis. Human stabilin-1(+) monocytes efficiently internalized MDA-LDL and this suppressed their ability to secrete CCL3, suggesting that loss of stabilin-1 removes a brake to CCL3 secretion. Experiments with cell-lineage-specific knockouts revealed that stabilin-1 expression in myeloid cells is required for the induction of this subset of macrophages and that increased fibrosis occurs in their absence. This study demonstrates a previously unidentified regulatory pathway in fibrogenesis in which a macrophage scavenger receptor protects against organ fibrosis by removing fibrogenic products of lipid peroxidation. Thus, stabilin-1(+) macrophages shape the tissue microenvironment during liver injury and healing.

Amine oxidase activity regulates the development of pulmonary fibrosis.

In pulmonary fibrosis, an inflammatory reaction and differentiation of myofibroblasts culminate in pathologic deposition of collagen. Amine oxidase copper containing-3 (AOC3) is a cell-surface-expressed oxidase that regulates leukocyte extravasation. Here we analyzed the potential role of AOC3 using gene-modified and inhibitor-treated mice in a bleomycin-induced pulmonary fibrosis model. Inflammation and fibrosis of lungs were assessed by histologic, flow cytometric, and quantitative PCR analysis. AOC3-deficient mice showed a 30-50% reduction in fibrosis, collagen synthesis, numbers of myofibroblasts, and accumulation of CD4+ lymphocytes, NK T cells, macrophages, and type 2 innate lymphoid cells compared with wild-type control mice. AOC3-knock-in mice, which express a catalytically inactive form of AOC3, were also protected from lung fibrosis. In wild-type mice, a small-molecule AOC3 inhibitor treatment reduced leukocyte infiltration, myofibroblast differentiation, and fibrotic injury both in prophylactic and early therapeutic settings by about 50% but was unable to reverse the established fibrosis. AOC3 was also induced in myofibroblasts in human idiopathic pulmonary fibrosis. Thus, the oxidase activity of AOC3 contributes to the development of lung fibrosis mainly by regulating the accumulation of pathogenic leukocyte subtypes, which drive the fibrotic response.-Marttila-Ichihara, F., Elima, K., Auvinen, K., Veres, T. Z., Rantakari, P., Weston, C., Miyasaka, M., Adams, D., Jalkanen, S., Salmi, M. Amine oxidase activity regulates the development of pulmonary fibrosis.

Monocyte Stabilin-1 Suppresses the Activation of Th1 Lymphocytes.

In this study, we analyzed the putative functions of stabilin-1 in blood monocytes. Microarray analysis revealed downregulation of several proinflammatory genes in the stabilin-1(high) monocytes when compared with stabilin-1(low) monocytes. When cocultured with stabilin-1(high) monocytes, IFN-γ synthesis by T cells was diminished in Ag-recall assays. Knockdown of stabilin-1 in monocytes increased the synthesis of several proinflammatory molecules, including TNF-α, and supported high IFN-γ and low IL-4 and IL-5 production by T cells in Ag-specific stimulation assays. Anti-stabilin-1 Ab treatment also led to increased IFN-γ synthesis in the recall assays. In clinical settings, the expression of stabilin-1 was diminished on blood monocytes and tissue macrophages under proinflammatory conditions. These data define stabilin-1 as a new immunosuppressive molecule and suggest that stabilin-1(high) monocytes may dampen proinflammatory reactions in vivo.

Ecto-5'-nucleotidase/CD73 enhances endothelial barrier function and sprouting in blood but not lymphatic vasculature.

CD73/ecto-5'-nucleotidase is a key enzyme in the regulation of purinergic signaling and inflammatory reactions. It hydrolyzes extracellular AMP into adenosine, which dampens immune cell activation, and reduces leukocyte trafficking. By comparing CD73 expression and function in mononuclear and endothelial cells (ECs) of blood and lymph, we show that extracellular purines and CD73 activity have differential effects in these two vascular systems. We found that CD8-positive T lymphocytes and CD19-positive B lymphocytes in human lymph expressed high levels of CD73 and other purinergic enzymes and adenosine receptors. Soluble CD73 was less abundant in human lymph than in serum, whereas CD73 activity was higher in afferent lymphatic ECs than in blood ECs. Adenosine signaling improved barrier function and induced sprouting of human blood, but not lymphatic, ECs in vitro. Similarly, using CD73-deficient mice we found that CD73 controls only blood vascular permeability at selected lymphoid organs under physiological conditions. Thus, both vascular and lymphatic arms of the immune system synthesize the components of purinergic signaling system, but surprisingly they use CD73 differentially to control endothelial permeability and sprouting.

CD44 binds to macrophage mannose receptor on lymphatic endothelium and supports lymphocyte migration via afferent lymphatics.

RATIONALE: Macrophage mannose receptor (MRC) is one of the few molecules known to be involved in lymphocyte trafficking via the lymphatic vessels. In endothelial cells of efferent lymphatics, it binds L-selectin on lymphocytes. In afferent lymphatics, MRC mediates trafficking of both normal and malignant L-selectin-negative cells to the draining lymph nodes. OBJECTIVE: This work was designed to search for additional lymphocyte ligands of MRC to elucidate how lymphocytes migrate into the draining lymph nodes. METHODS AND RESULTS: Using immunoprecipitation and binding studies with natural and recombinant proteins, we show that MRC and CD44 can interact with each other. Fine mapping revealed that the cysteine-rich domain of MRC binds to the chondroitin sulfate side chains of CD44. In vivo homing experiments with MRC- and CD44-deficient mice verified that MRC and CD44 function as a receptor-ligand pair in supporting lymphocyte migration via the afferent lymphatics into the draining lymph nodes. CONCLUSIONS: These data identify a new counter-receptor for MRC and reveal CD44 as a new molecule involved in the poorly understood process of lymphocyte transit via the lymphatic vasculature.

Early prediction of persistent organ failure by soluble CD73 in patients with acute pancreatitis*.

OBJECTIVE: New biomarkers are needed to better predict the severity of acute pancreatitis. CD73/ecto-5'-nucleotidase is an enzyme that generates adenosine, which dampens inflammation and improves vascular barrier function in several disease models. CD73 also circulates in a soluble form in the blood. We studied whether levels of soluble form of CD73 predict the development of organ failure in acute pancreatitis. DESIGN: A prospective cohort study of patients with acute pancreatitis from 2003 to 2007. SETTING: Admissions to the biggest tertiary care hospital in Finland. PATIENTS: One hundred sixty-one patients with acute pancreatitis, of which 107 were subclassified according to the revised Atlanta criteria into mild, 29 into moderately severe and 25 into severe. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Serum and blood cell samples were collected at admission. Protein levels of soluble form of CD73 in serum were determined using a novel enzyme-linked immunosorbent assay, activity of soluble form of CD73 using radioactive enzyme assays, and CD73 messenger RNA levels from leukocytes using quantitative polymerase chain reaction. Activity and protein concentration of soluble form of CD73, and messenger RNA level of CD73 all decreased along with the disease severity (p ≤ 0.01 for all). The activity of soluble form of CD73 at admission predicted the development of the severe pancreatitis in different groups of the patients. The area under the receiver-operating characteristic curve value for activity of soluble form of CD73 was 0.65 (95% CI, 0.51-0.80) among a subgroup of patients comprising moderately severe and severe disease, 0.79 (95% CI, 0.69-0.88) among all patients including mild pancreatitis, and 0.75 (95% CI, 0.60-0.89) among patients who had no signs of organ failure (modified Marshall score < 2) at admission. Especially, in the last-mentioned group, activity of soluble form of CD73 was better than C-reactive protein or creatinine in predicting the severe pancreat CONCLUSIONS: : Activity of soluble form of CD73 at admission to hospital has prognostic value in predicting the development of the severe form of acute pancreatitis.

PV-1 is recognized by the PAL-E antibody and forms complexes with NRP-1.

Pathologische anatomie leiden endothelium (PAL-E) antibody has been used for more than 20 years as a prototype marker for vascular endothelium. The elusive target of this antibody has been claimed to be plasmalemma vesicle-associated protein-1 (PV-1) and neuropilin-1 (NRP-1). Using immunofluorescence, we show that PAL-E, anti-PV-1, anti-NRP-1, and anti-CD31 antibodies show largely identical staining patterns in the vasculature of different tissues. However, PV-1-transfected cells only bind PAL-E and anti-PV-1 antibodies, whereas NRP-1 transfectants stain with anti-NRP-1 antibodies in flow cytometry. Using lysates from tissues and transfected cells, we further confirm that the molecule recognized by PAL-E and anti-PV-1 antibodies is not NRP-1 but PV-1. Nevertheless, coimmunoprecipitation studies unambiguously demonstrate that NRP-1 can form complexes with PV-1. This connects, for the first time, 2 molecules involved in leukocyte trafficking and angiogenesis, thereby opening interesting possibilities for future research in this field.

Consequences of the lack of CD73 and prostatic acid phosphatase in the lymphoid organs.

CD73, ecto-5'-nucleotidase, is the key enzyme catalyzing the conversion of extracellular AMP to adenosine that controls vascular permeability and immunosuppression. Also prostatic acid phosphatase (PAP) possesses ecto-5'-nucleotidase/AMPase activity and is present in leukocytes. However, its role related to immune system is unknown. Therefore, we analyzed enzymatic activities and leukocyte subtypes of CD73 and PAP knockouts and generated CD73/PAP double knockout mice to elucidate the contribution of CD73 and PAP to immunological parameters. Enzymatic assays confirmed the ability of recombinant human PAP to hydrolyze [(3)H]AMP, although at much lower rate than human CD73. Nevertheless, 5'-nucleotidase/AMPase activity in splenocytes and lymphocytes from PAP(-/-) mice tended to be lower than in wild-type controls, suggesting potential contribution of PAP, along with CD73, into lymphoid AMP metabolism ex vivo. Single knockouts had decreased number of CD4(+)/CD25(+)/FoxP3 (+) regulatory T cells in thymus and CD73/PAP double knockouts exhibited reduced percentages of CD4(+) cells in spleen, regulatory T cells in lymph nodes and thymus, and CD4(+) and CD8(+) cells in blood. These findings suggest that PAP has a synergistic role together with CD73 in the immune system by contributing to the balance of leukocyte subpopulations and especially to the number of regulatory T cells in lymph nodes and thymus.

Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 and can be used in PET imaging of inflammation and cancer.

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1-dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

Altered purinergic signaling in CD73-deficient mice inhibits tumor progression.

CD73/ecto-5'-nucleotidase dephosphorylates extracellular AMP into adenosine, and it is a key enzyme in the regulation of adenosinergic signaling. The contribution of host CD73 to tumor growth and anti-tumor immunity has not been studied. Here, we show that under physiological conditions CD73-deficient mice had significantly elevated ATPase and ADPase activities in LN T cells. In a melanoma model, the growth of primary tumors and formation of metastasis were significantly attenuated in mice lacking CD73. Among tumor-infiltrating leukocytes there were fewer Tregs and mannose receptor-positive macrophages, and increased IFN-γ and NOS2 mRNA production in CD73-deficient mice. Treatment of tumor-bearing animals with soluble apyrase, an enzyme hydrolyzing ATP and ADP, significantly inhibited tumor growth and accumulation of intratumoral Tregs and mannose receptor-positive macrophages in the WT C57BL/6 mice but not in the CD73-deficient mice. Pharmacological inhibition of CD73 with α,ß-methylene-adenosine-5'-diphosphate in WT mice retarded tumor progression similarly to the genetic deletion of CD73. Together these data show that increased pericellular ATP degradation in the absence of CD73 activity in the host cells is a novel mechanism controlling anti-tumor immunity and tumor progression, and that the purinergic balance can be manipulated therapeutically to inhibit tumor growth.

Human Siglec-10 can bind to vascular adhesion protein-1 and serves as its substrate.

Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular adhesion protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counterreceptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays, and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production, indicating that Siglec-10 serves as a substrate for VAP-1. Thus, the Siglec-10-VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products.

Lymphatic Endothelial Cell Activation and Dendritic Cell Transmigration Is Modified by Genetic Deletion of Clever-1.

Clever-1 also known as Stabilin-1 and FEEL-1 is a scavenger molecule expressed on a subpopulation of anti-inflammatory macrophages and lymphatic endothelial cells (LECs). However, its role in regulating dendritic cell (DC) trafficking and subsequent effects on immunity have remained unexplored. In this study, we demonstrate that DC trafficking from the skin into the draining lymph nodes is compromised in the absence of Clever-1. By adoptive transfer approaches we further show that the poor trafficking is due to the impaired entrance of DCs into afferent lymphatics. Despite this, injections of ovalbumin-loaded DCs into the footpads induced a stronger proliferative response of OT II T cells in the draining lymph nodes. This could be explained by the increased MHC II expression on DCs and a less tolerogenic phenotype of LECs in lymph nodes of Clever-1 knockout mice. Thus, although fewer DCs reach the nodes, they are more active in creating antigen-specific immune responses. This suggests that the DCs migrating to the draining lymph node within Clever-1 positive lymphatics experience immunosuppressive interactions with LECs. In conclusion, besides being a trafficking molecule on lymphatic vasculature Clever-1 is immunosuppressive towards migrating DCs and thus, regulates the magnitude of immune responses created by incoming DCs in the draining lymph nodes.

Clever-1/Stabilin-1 regulates lymphocyte migration within lymphatics and leukocyte entrance to sites of inflammation.

Clever-1/Stabilin-1 is a scavenger receptor present on lymphatic and sinusoidal endothelium as well as on a subset of type II macrophages. It is also induced on vasculature at sites of inflammation. However, its in vivo function has remained practically unknown and this work addresses those unknown aspects. We demonstrate using in vivo models that Clever-1/Stabilin-1 mediates migration of T and B lymphocytes to the draining lymph nodes in vivo and identify the adhesive epitope of the Clever-1/Stabilin-1 molecule responsible for the interaction between lymphocytes and lymphatic endothelium. Moreover, we demonstrate that Ab blocking of Clever-1/Stabilin-1 efficiently inhibits peritonitis in mice by decreasing the entrance of granulocytes by 50%, while migration of monocytes and lymphocytes into the inflamed peritoneum is prevented almost completely. Despite efficient anti-inflammatory activity the Ab therapy does not dramatically dampen immune responses against the bacterial and foreign protein Ag tested and bacterial clearance. These results indicate that anti-Clever-1/Stabilin-1 treatment can target two different arms of the vasculature--traffic via lymphatics and inflamed blood vessels.