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.

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.

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.

Robo4 contributes to the turnover of Peyer's patch B cells.

All leukocytes can get entrance into the draining lymph nodes via the afferent lymphatics but only lymphoid cells can leave the nodes. The molecular mechanisms behind this phenomenon have remained unknown. We employed genome wide microarray analyses of the subcapsular sinus and lymphatic sinus (LS) endothelial cells and found Robo4 to be selectively expressed on LS lymphatics. Further analyses showed high Robo4 expression in lymphatic vessels of Peyer's patches, which only have efferent lymphatic vessels. In functional assays, Robo4-deficient animals showed accumulation of naïve  B cells (CD19+/CD62Lhi/CD44lo) in Peyer's patches, whereas no difference was seen within other lymphocyte subtypes. Short-term lymphocyte homing via high endothelial venules to peripheral and mesenteric lymph nodes and Peyer's patches was also slightly impaired in Robo4 knockout animals. These results show for the first time, selective expression of Robo4 in the efferent arm of the lymphatics and its role in controlling the turnover of a subset of B lymphocytes from Peyer's patches.

Fenestral diaphragms and PLVAP associations in liver sinusoidal endothelial cells are developmentally regulated.

Endothelial cells contain several nanoscale domains such as caveolae, fenestrations and transendothelial channels, which regulate signaling and transendothelial permeability. These structures can be covered by filter-like diaphragms. A transmembrane PLVAP (plasmalemma vesicle associated protein) protein has been shown to be necessary for the formation of diaphragms. The expression, subcellular localization and fenestra-forming role of PLVAP in liver sinusoidal endothelial cells (LSEC) have remained controversial. Here we show that fenestrations in LSEC contain PLVAP-diaphragms during the fetal angiogenesis, but they lose the diaphragms at birth. Although it is thought that PLVAP only localizes to diaphragms, we found luminal localization of PLVAP in adult LSEC using several imaging techniques. Plvap-deficient mice revealed that the absence of PLVAP and diaphragms did not affect the morphology, the number of fenestrations or the overall vascular architecture in the liver sinusoids. Nevertheless, PLVAP in fetal LSEC (fenestrations with diaphragms) associated with LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1), neuropilin-1 and VEGFR2 (vascular endothelial growth factor receptor 2), whereas in the adult LSEC (fenestrations without diaphragms) these complexes disappeared. Collectively, our data show that PLVAP can be expressed on endothelial cells without diaphragms, contradict the prevailing concept that biogenesis of fenestrae would be PLVAP-dependent, and reveal previously unknown PLVAP-dependent molecular complexes in LSEC during angiogenesis.

Fetal-derived macrophages dominate in adult mammary glands.

Macrophages serve multiple functions including immune regulation, morphogenesis, tissue homeostasis and healing reactions. The current paradigm holds that mammary gland macrophages first arise postnatally during the prepubertal period from the bone marrow-derived monocytes. Here we delineate the origins of tissue-resident mammary gland macrophages using high-dimension phenotypic analyses, cell-fate mapping experiments, gene-deficient mice lacking selective macrophage subtypes, and antibody-based depletion strategies. We show that tissue-resident macrophages are found in mammary glands already before birth, and that the yolk sac-derived and fetal liver-derived macrophages outnumber the adult-derived macrophages in the mammary gland also in the adulthood. In addition, fetal-derived mammary gland macrophages have a characteristic phenotype, display preferential periductal and perivascular localization, and are highly active in scavenging. These findings identify fetal-derived macrophages as the predominant leukocyte type in the adult mammary gland stroma, and reveal previously unknown complexity of macrophage biology in the breast.

Enhanced Antibody Production in Clever-1/Stabilin-1-Deficient Mice.

Clever-1, encoded by the Stab1 gene, is a scavenger and leukocyte trafficking receptor expressed by subsets of vascular and lymphatic endothelial cells and immunosuppressive macrophages. Monocyte Clever-1 also modulates T cell activation. However, nothing is known about the possible links between B cell function and Clever-1. Here, we found that Stab1 knockout mice (Stab1-/-) lacking the Clever-1 protein from all cells present with abnormally high antibody levels under resting conditions and show enhanced humoral immune responses after immunization with protein and carbohydrate antigens. Removal of the spleen does not abolish the augmented basal and post-immunization antibody levels in Clever-1-deficient mice. The increased IgG production is also present in mice in which Clever-1 is selectively ablated from macrophages. When compared to wildtype macrophages, Clever-1-deficient macrophages show increased TNF-α synthesis. In co-culture experiments, monocytes/macrophages deficient of Clever-1 support higher IgM production by B cells, which is blocked by TNF-α depletion. Collectively, our data show that the excessive inflammatory activity of monocytes/macrophages in the absence of Clever-1 results in augmented humoral immune responses in vivo.

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.

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.

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.

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.

Primary Amine Oxidase of Escherichia coli Is a Metabolic Enzyme that Can Use a Human Leukocyte Molecule as a Substrate.

Escherichia coli amine oxidase (ECAO), encoded by the tynA gene, catalyzes the oxidative deamination of aromatic amines into aldehydes through a well-established mechanism, but its exact biological role is unknown. We investigated the role of ECAO by screening environmental and human isolates for tynA and characterizing a tynA-deletion strain using microarray analysis and biochemical studies. The presence of tynA did not correlate with pathogenicity. In tynA+ Escherichia coli strains, ECAO enabled bacterial growth in phenylethylamine, and the resultant H2O2 was released into the growth medium. Some aminoglycoside antibiotics inhibited the enzymatic activity of ECAO, which could affect the growth of tynA+ bacteria. Our results suggest that tynA is a reserve gene used under stringent environmental conditions in which ECAO may, due to its production of H2O2, provide a growth advantage over other bacteria that are unable to manage high levels of this oxidant. In addition, ECAO, which resembles the human homolog hAOC3, is able to process an unknown substrate on human leukocytes.

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.

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.

Clever-1/stabilin-1 controls cancer growth and metastasis.

PURPOSE: Immunosuppressive leukocytes and vasculature are important host cell components regulating tumor progression. Clever-1/Stabilin-1, a multifunctional scavenger and adhesion receptor, is constitutively present on a subset of type II macrophages and lymphatic endothelium, but its functional role in cancer is unknown. EXPERIMENTAL DESIGN: Here, we generated full Clever-1 knockout mice and cell-specific ones lacking Clever-1 either on macrophages or endothelium. We also used anti-Clever-1 antibody therapy to treat B16 melanoma and EL-4 lymphoma. RESULTS: Clever-1-deficient mice had smaller primary and metastatic tumors than wild-type (WT) controls. Growth of primary tumors, but not of metastases, was attenuated also in mice lacking Clever-1 selectively in macrophages or in vascular endothelium. Anti-Clever-1 antibody treatment inhibited tumor progression in WT mice. Both genetically and therapeutically induced absence of functional Clever-1 led to diminished numbers of immunosuppressive leukocyte types in tumors. Functionally Clever-1 mediated binding of immunosuppressive leukocytes to the intratumoral blood vessels aberrantly expressing Clever-1, and tumor cell traffic via the lymphatics. The antibody therapy did not aggravate autoimmunity. CONCLUSION: This work identifies Clever-1 in type II macrophages and in tumor vasculature as a new immunosuppressive molecule in cancer. Our finding that Clever-1 supports binding of tumor-infiltrating lymphocytes to tumor vasculature increases our understanding of leukocyte immigration to tumors. The ability of anti-Clever-1 antibody treatment to attenuate tumor progression in WT mice in vivo is therapeutically relevant. Thus, Clever-1 may be an emerging new target for modulating immune evasion and lymphatic spread in cancer.

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.

Adenosine inhibits tumor cell invasion via receptor-independent mechanisms.

UNLABELLED: Extracellular adenosine mediates diverse anti-inflammatory, angiogenic, and other signaling effects via binding to adenosine receptors, and it also regulates cell proliferation and death via activation of the intrinsic signaling pathways. Given the emerging role of adenosine and other purines in tumor growth and metastasis, this study evaluated the effects of adenosine on the invasion of metastatic prostate and breast cancer cells. Treatment with low micromolar concentrations of adenosine, but not other nucleosides or adenosine receptor agonists, inhibited subsequent cell invasion and migration through Matrigel- and laminin-coated inserts. These inhibitory effects occurred via intrinsic receptor-independent mechanisms, despite the abundant expression of A2B adenosine receptors (ADORA2B). Extracellular nucleotides and adenosine were shown to be rapidly metabolized on tumor cell surfaces via sequential ecto-5'-nucleotidase (CD73/NT5E) and adenosine deaminase reactions with subsequent cellular uptake of nucleoside metabolites and their intracellular interconversion into ADP/ATP. This was accompanied by concurrent inhibition of AMP-activated protein kinase and other signaling pathways. No differences in the proliferation rates, cytoskeleton assembly, expression of major adhesion molecules [integrin-1ß (ITGB1), CD44, focal adhesion kinase], and secretion of matrix metalloproteinases were detected between the control and treated cells, thus excluding the contribution of these components of invasion cascade to the inhibitory effects of adenosine. These data provide a novel insight into the ability of adenosine to dampen immune responses and prevent tumor invasion via two different, adenosine receptor-dependent and -independent mechanisms. IMPLICATIONS: This study suggests that the combined targeting of adenosine receptors and modulation of intracellular purine levels can affect tumor growth and metastasis phenotypes.