Autoimmunity in CD73/Ecto-5'-nucleotidase deficient mice induces renal injury

Extracellular adenosine formed by 5'-ectonucleotidase (CD73) is involved in tubulo-glomerular feedback in the kidney but is also known to be an important immune modulator. Since CD73(-/-)mutant mice exhibit a vascular proinflammatory phenotype, we asked whether long term lack of CD73 causes inflammation related kidney pathologies. CD73(-/-)mice (13 weeks old) showed significantly increased low molecule proteinuria compared to C57BL6 wild type controls (4.8 ≥ 0.52 vs. 2.9 ± 0.54 mg/24 h, p<0.03). Total proteinuria increased to 5.97 ± 0.78 vs. 2.55 ± 0.35 mg/24 h at 30 weeks (p<0.01) whereas creatinine clearance decreased (0.161 ± 0.02 vs. 0.224 ± 0.02 ml/min). We observed autoimmune inflammation in CD73(-/-)mice with glomerulitis and peritubular capillaritis, showing glomerular deposition of IgG and C3 and enhanced presence of CD11b, CD8, CD25 as well as GR-1-positive cells in the interstitium. Vascular inflammation was associated with enhanced serum levels of the cytokines IL-18 and TNF-α as well as VEGF and the chemokine MIP-2 (CXCL-2) in CD73(-/-)mice, whereas chemokines and cytokines in the kidney tissue were unaltered or reduced. In CD73(-/-)mice glomeruli, we found a reduced number of podocytes and endothelial fenestrations, increased capillaries per glomeruli, endotheliosis and enhanced tubular fibrosis. Our results show that adult CD73(-/-)mice exhibit spontaneous proteinuria and renal functional deterioration even without exogenous stress factors. We have identified an autoimmune inflammatory phenotype comprising the glomerular endothelium, leading to glomeruli inflammation and injury and to a cellular infiltrate of the renal interstitium. Thus, long term lack of CD73 reduced renal function and is associated with autoimmune inflammation.     

Ecto-5'-nucleotidase (CD73)-mediated extracellular adenosine production plays a critical role in hepatic fibrosis

In previous studies, we have demonstrated that adenosine and its receptors play a role in hepatic fibrosis. Here, we review evidence that toxin-induced increases in hepatic adenosine concentrations are generated from adenine nucleotides by the action of ecto-5'nucleotidase and thus that adenosine-mediated, toxin-induced hepatic fibrosis depends on extracellular conversion of adenine nucleotides to adenosine.     

Ecto-5'-nucleotidase (CD73) decreases mortality and organ injury in sepsis

The extracellular concentrations of adenosine are increased during sepsis, and adenosine receptors regulate the host's response to sepsis. In this study, we investigated the role of the adenosine-generating ectoenzyme, ecto-5'-nucleotidase (CD73), in regulating immune and organ function during sepsis. Polymicrobial sepsis was induced by subjecting CD73 knockout (KO) and wild type (WT) mice to cecal ligation and puncture. CD73 KO mice showed increased mortality in comparison with WT mice, which was associated with increased bacterial counts and elevated inflammatory cytokine and chemokine concentrations in the blood and peritoneum. CD73 deficiency promoted lung injury, as indicated by increased myeloperoxidase activity and neutrophil infiltration, and elevated pulmonary cytokine levels. CD73 KO mice had increased apoptosis in the thymus, as evidenced by increased cleavage of caspase-3 and poly(ADP-ribose) polymerase and increased activation of NF-κB. Septic CD73 KO mice had higher blood urea nitrogen levels and increased cytokine levels in the kidney, indicating increased renal dysfunction. The increased kidney injury of CD73 KO mice was associated with augmented activation of p38 MAPK and decreased phosphorylation of Akt. Pharmacological inactivation of CD73 in WT mice using α, β-methylene ADP augmented cytokine levels in the blood and peritoneal lavage fluid. These findings suggest that CD73-derived adenosine may be beneficial in sepsis.     

Ecto-5'-nucleotidase/CD73 inhibition by quercetin in the human U138MG glioma cell line

Gliomas are the most malignant of the primary brain tumors. Nucleotides represent an important class of extracellular molecules that are crucial for the normal function of the nervous system. ATP and adenosine can stimulate cell proliferation in different glioma cell lines; the events induced by extracellular adenine nucleotides are controlled by the action of ecto-nucleotidases, which hydrolyze ATP into adenosine in the extracellular space. Recent studies have shown that quercetin has an anti-proliferative effect on the U138MG glioma cell line. Since evidence suggests that purinergic signaling is involved in the growth and progression of glioma and, taking into consideration the anti-proliferative effect elicited by quercetin in this tumor type, the aim of the present study was to better investigate the extracellular metabolism of AMP and evaluate the effect of quercetin on this system in the human U138MG glioma cell line. The adenine products secreted by glioma cells were first characterized; extracellular AMP was efficiently metabolized by the glioma culture, demonstrating a very active ecto-5'-NT/CD73. Quercetin was able to inhibit the ecto-5'-NT/CD73 activity and modulate its expression. In addition, the cell treatment with APCP (alpha,beta-methyleneadenosine-5'-diphosphate), an ecto-5'-NT/CD73 inhibitor, led to a significant reduction in glioma cell proliferation. We suggest that the inhibition of ecto-5'-NT/CD73 may result in a decrease in extracellular adenosine production with a consequent reduction in tumor progression.     

Ecto-5'-nucleotidase (CD73)-mediated adenosine production is tissue protective in a model of bleomycin-induced lung injury

Adenosine signaling has diverse actions on inflammation and tissue injury. Levels of adenosine are rapidly elevated in response to tissue injury; however, the mechanisms responsible for adenosine production in response to injury are not well understood. In this study, we found that adenosine levels are elevated in the lungs of mice injured by the drug bleomycin. In addition, increased activity of ecto-5'-nucleotidase (CD73) was found in the lungs in conjunction with adenosine elevations. To determine the contribution of CD73 to the generation of adenosine in the lung, CD73(-/-) mice were subjected to bleomycin challenges. Results demonstrated that CD73(-/-) mice challenged with bleomycin no longer accumulated adenosine in their lungs, suggesting that the primary means of adenosine production following bleomycin injury resulted from the release and subsequent dephosphorylation of adenine nucleotides. CD73(-/-) mice challenged with bleomycin exhibited enhanced pulmonary inflammation and fibrosis as well as exaggerated expression of proinflammatory and profibrotic mediators in the lung. Intranasal instillations of exogenous nucleotidase restored the ability of lungs of CD73(-/-) mice to accumulate adenosine following bleomycin challenge. Furthermore, these treatments were associated with a decrease in pulmonary inflammation and fibrosis. CD73(+/+) animals challenged with bleomycin and supplemented with exogenous nucleotidase also exhibited reduced inflammation. Together, these findings suggest that CD73-dependent adenosine production contributes to anti-inflammatory pathways in bleomycin-induced lung injury.     

Coexpression of ecto-5'-nucleotidase/CD73 with specific NTPDases differentially regulates adenosine formation in the rat liver

Ectonucleotidases modulate purinergic signaling by hydrolyzing ATP to adenosine. Here we characterized the impact of the cellular distribution of hepatic ectonucleotidases, namely nucleoside triphosphate diphosphohydrolase (NTPDase)1/CD39, NTPDase2/CD39L1, NTPDase8, and ecto-5'-nucleotidase/CD73, and of their specific biochemical properties, on the levels of P1 and P2 receptor agonists, with an emphasis on adenosine-producing CD73. Immunostaining and enzyme histochemistry showed that the distribution of CD73 (protein and AMPase activity) overlaps partially with those of NTPDase1, -2, and -8 (protein levels and ATPase and ADPase activities) in normal rat liver. CD73 is expressed in fibroblastic cells located underneath vascular endothelial cells and smooth muscle cells, which both express NTPDase1, in portal spaces in a distinct fibroblast population next to NTPDase2-positive portal fibroblasts, and in bile canaliculi, together with NTPDase8. In fibrotic rat livers, CD73 protein expression and activity are redistributed but still overlap with the NTPDases mentioned. The ability of the observed combinations of ectonucleotidases to generate adenosine over time was evaluated by reverse-phase HPLC with the recombinant rat enzymes at high "inflammatory" (500 μM) and low "physiological" (1 μM) ATP concentrations. Overall, ATP was rapidly converted to adenosine by the NTPDase1+CD73 combination, but not by the NTPDase2+CD73 combination. In the presence of NTPDase8 and CD73, ATP was sequentially dephosphorylated to the CD73 inhibitor ADP, and then to AMP, thus resulting in a delayed formation of adenosine. In conclusion, the specific cellular cocompartmentalization of CD73 with hepatic NTPDases is not redundant and may lead to the differential activation of P1 and P2 receptors, under normal and fibrotic conditions.     

IFN-alpha induced adenosine production on the endothelium: a mechanism mediated by CD73 (ecto-5'-nucleotidase) up-regulation

CD73 (ecto-5'-nucleotidase; EC 3.1.3.5) participates in lymphocyte binding to endothelial cells and converts extracellular AMP into a potent anti-inflammatory substance adenosine. However, the regulation of expression and function of CD73 has remained largely unknown. In this study, we show that IFN-alpha produces a time- and dose-dependent long-term up-regulation of CD73 on endothelial cells, but not on lymphocytes both at protein and RNA levels. Moreover, CD73-mediated production of adenosine is increased after IFN-alpha treatment on endothelial cells, resulting in a decrease in the permeability of these cells. Subsequent to induction with PMA, FMLP, dibutyryl cAMP, thrombin, histamine, IL-1beta, TNF-alpha, and LPS, no marked changes in the level of CD73 expression on endothelial cells are observed. We also show that CD73 is up-regulated in vivo on the vasculature after intravesical treatment of urinary bladder cancers with IFN-alpha. In conclusion, distinct behavior of lymphocyte and endothelial CD73 subsequent to cytokine treatment further emphasizes the existence of cell type-specific mechanisms in the regulation of CD73 expression and function. Overall, these results suggest that IFN-alpha is a relevant in vivo regulator of CD73 in the endothelial-leukocyte microenvironment in infections/inflammations, and thus has a fundamental role in controlling the extent of inflammation via CD73-dependent adenosine production.     

Ecto-5'-nucleotidase (eN, CD73) is coexpressed with metastasis promoting antigens in human melanoma cells

Upregulated expression of eN has been found in the highly invasive human melanoma cell lines but neither in melanocytes nor in primary tumor cells. Membrane proteins associated with cell adhesion and metastasis: alpha5-, beta1-, beta3-integrins, and CD44 were elevated gradually in accordance with increasing metastatic potential. alphav-integrin was seen mostly in aggressive melanomas. The expression of eN correlated with a number of metastasis-related markers and thus may have a function in the process. eN activity went parallel with its amount in all cells. Concanavalin A strongly inhibited the enzyme in a noncompetitive way. Clustering of eN protein in overexpressing cells by ConA-treatment increased the enzyme association with the heavy cytoskeletal complexes. A similar shift towards cytoskeletal fractions took also place with other membrane proteins coexpressed with eN. This ConA-induced association may reflect a putative interaction of eN with physiological ligand, that upon interaction, aggregates protein components of lipid rafts and triggers signaling pathway that may be intrinsically involved in cell-stroma adhesion.     

Ecto-5′-nucleotidase/CD73 inhibition by quercetin in the human U138MG glioma cell line

Gliomas are the most malignant of the primary brain tumors. Nucleotides represent an important class of extracellular molecules that are crucial for the normal function of the nervous system. ATP and adenosine can stimulate cell proliferation in different glioma cell lines; the events induced by extracellular adenine nucleotides are controlled by the action of ecto-nucleotidases, which hydrolyze ATP into adenosine in the extracellular space. Recent studies have shown that quercetin has an anti-proliferative effect on the U138MG glioma cell line. Since evidence suggests that purinergic signaling is involved in the growth and progression of glioma and, taking into consideration the anti-proliferative effect elicited by quercetin in this tumor type, the aim of the present study was to better investigate the extracellular metabolism of AMP and evaluate the effect of quercetin on this system in the human U138MG glioma cell line. The adenine products secreted by glioma cells were first characterized; extracellular AMP was efficiently metabolized by the glioma culture, demonstrating a very active ecto-5′-NT/CD73. Quercetin was able to inhibit the ecto-5′-NT/CD73 activity and modulate its expression. In addition, the cell treatment with APCP (α,β-methyleneadenosine-5′-diphosphate), an ecto-5′-NT/CD73 inhibitor, led to a significant reduction in glioma cell proliferation. We suggest that the inhibition of ecto-5′-NT/CD73 may result in a decrease in extracellular adenosine production with a consequent reduction in tumor progression.     

Limited contribution of claudin-5-dependent tight junction strands to endothelial barrier function

Members of the claudin family are involved in formation of barriers that control access to the paracellular space of epithelia. Likewise, endothelium-specific claudin-5 is involved in the function of the blood-brain barrier (BBB). Here, we assessed the role of claudin-5 in non-BBB endothelial barriers using lentiviral-driven overexpression and silencing of claudin-5 in its native environment of primary vascular endothelial cells. Effects were monitored using macromolecular tracers between 342Da and 40kDa. Measurements were made both in absence and presence of transmigrating leukocytes. Freeze-fracture preparations were analyzed for effects at the ultrastructural level. We show that overexpression of claudin-5 leads to formation of elaborate networks of junction strands, which are absent in untransduced endothelial cells. Concomitantly, a modest, non-size-selective enhancement of the barrier function was observed. In contrast, silencing of endogenous claudin-5 does not influence barrier function. The efficient sealing of the endothelium during diapedesis of monocytes or granulocytes is also claudin-5 independent. Collectively, these data provide evidence for a limited contribution of claudin-5 to the barrier function of human umbilical vein endothelial cells (HUVEC), implying that, unlike selective barriers in epithelia, the barrier of non-BBB endothelium seems largely independent of claudin-directed tight junction structures.     

CD73 engagement promotes lymphocyte binding to endothelial cells via a lymphocyte function-associated antigen-1-dependent mechanism

CD73 is a GPI-anchored lymphocyte adhesion molecule possessing an ecto-5'-nucleotidase enzyme activity. In this work, we show that engagement of lymphocyte CD73 increases lymphocyte binding to cultured endothelial cells (EC) in an LFA-1-dependent fashion. Engagement of CD73 by an anti-CD73 mAb 4G4 increases the adhesion of lymphocytes to cultured EC by about 80% compared with that of lymphocytes treated with a negative control Ab, and the increased adhesion can be blocked by an anti-CD18 mAb. The CD73-regulated increase in lymphocyte adhesion is not due to a conformational change leading to high-affinity LFA-1 receptors as assayed using mAb 24 against an activation-induced epitope of the molecule. Instead, CD73 engagement induces clustering of LFA-1 that is inhibitable by calpeptin, indicating involvement of Ca(2+)-dependent activation of a calpain-like enzyme in this process. In conclusion, the results shown here demonstrate that CD73 regulates the avidity of LFA-1 by clustering. This indicates a previously undescribed role for CD73 in controlling the poorly characterized activation step in the multistep cascade of lymphocyte extravasation. Moreover, these results suggest that in physiological conditions the activation step may result in clustering of LFA-1 rather than in an affinity change of the molecule.     

VEGF-C alters barrier function of cultured lymphatic endothelial cells through a VEGFR-3-dependent mechanism

BACKGROUND: The lymphatic endothelium is an important semi-permeable barrier separating lymph from the interstitial space. However, there is currently a limited understanding of the lymphatic endothelial barrier and the mechanisms of lymph formation. The objectives of this study were to investigate the potential active role of lymphatic endothelial cells in barrier regulation, and to test whether the endothelial cell agonists VEGF-A and VEGF-C can alter lymphatic endothelial barrier function. METHODS AND RESULTS: Cultured adult human dermal microlymphatic endothelial cells (HMLEC-d) and human umbilical vein endothelial cells (HUVEC) were respectively used as models of lymphatic and vascular endothelium. Transendothelial electrical resistance (TER) of endothelial monolayers served as an index of barrier function. Cells were treated with VEGF-A, VEGF-C, or the VEGFR-3 selective mutant VEGF-C156S. MAZ51 was used to inhibit VEGFR-3 signaling. The results show that while VEGF-A causes a time-dependent decrease in TER in HUVEC, there is no response in HMLEC-d. In contrast, VEGF-C and VEGF-C156S cause a similar decrease in TER in HMLEC-d that is not observed in HUVEC. These results corresponded to the protein expression of VEGFR-2 and VEGFR-3 in these cell types, determined by Western blotting. In addition, the VEGF-C- and VEGF-C156S-induced TER changes were inhibited by MAZ51. CONCLUSIONS: The results indicate differential responses of the lymphatic and vascular endothelial barriers to VEGF-A and VEGF-C. Furthermore, our data suggest that VEGF-C alters lymphatic endothelial function through a mechanism involving VEGFR-3.     

Regulation of ecto-5'-nucleotidase (CD73) in cultured cortical astrocytes by different inflammatory factors

Ecto-5'-nucleotidase (e-5NT) is a cell-surface located, rate-limiting enzyme in the extracellular metabolism of ATP, catalyzing the final step of the conversion of AMP to adenosine. Since this enzyme shifts the balance from pro-inflammatory ATP to anti-inflammatory adenosine, it is considered to be an important regulator of inflammation. Although up-regulation of e-5NT was repeatedly reported in several in vivo models of brain injury, the regulation of its expression and function remains largely unknown. We have studied effects of several pro-inflammatory factors, namely, bacterial endotoxin lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), glutamate (Glu) and hydrogen peroxide (H(2)O(2)) on e-5NT (i) activity, (ii) mRNA expression and (iii) membrane protein abundance in primary cultured cortical astrocytes. We are clearly able to demonstrate a stimulus-specific regulation of the e-5NT pathway. IFN-γ, LPS, Glu and H(2)O(2) decrease, while TNF-α increases e-5NT activity. The analysis of e-5NT gene expression and e-5NT membrane protein levels revealed that tested factors regulate e-5NT at different levels and by employing different mechanisms. In summary, we provide evidence that e-5NT activity is tightly regulated in a stimulus-specific manner.     

Podosome formation impairs endothelial barrier function by sequestering zonula occludens proteins

Podosomes and tight junctions (TJs) are subcellular compartments that both exist in endothelial cells and localize at cell surfaces. In contrast to the well-characterized role of TJs in maintaining cerebrovascular integrity, the specific function of endothelial podosomes remains unknown. Intriguingly, we discovered cross-talk between podosomes and TJs in human brain endothelial cells. Tight junction scaffold proteins ZO-1 and ZO-2 localize at podosomes in response to phorbol-12-myristate-13-acetate treatment. We found that both ZO proteins are essential for podosome formation and function. Rather than being derived from new protein synthesis, podosomal ZO-1 and ZO-2 are relocated from a pre-existing pool found at the peripheral plasma membrane with enhanced physical interaction with cortactin, a known protein marker for podosomes. Sequestration of ZO proteins in podosomes weakens tight junction complex formation, leading to increased endothelial cell permeability. This effect can be further attenuated by podosome inhibitor PP2. Altogether, our data revealed a novel cellular function of podosomes, specifically, their ability to negatively regulate tight junction and endothelial barrier integrity, which have been linked to a variety of cerebrovascular diseases.     

Vesicle formation and trafficking in endothelial cells and regulation of endothelial barrier function

Endothelial barrier function is regulated in part by the transcellular transport of albumin and other macromolecules via endothelial caveolae (i.e., this process is defined as transcytosis). Using pulmonary microvascular endothelial cells, we have identified the specific interactions between a cell surface albumin-docking protein gp60 and caveolin-1 as well as components of the signaling machinery, heterotrimeric G protein (G(i))- and Src-family tyrosine kinase. Ligation of gp60 on the apical membrane induces the release of caveolae from the apical membrane and activation of endocytosis. The formed vesicles contain the gp60-bound albumin and also albumin and other solutes present in the fluid phase. Vesicles are transported in a polarized manner to the basolateral membrane, releasing their contents by exocytosis into the subendothelial space. The signaling functions of G(i) and Src are important in the release of caveolae from the plasma membrane. The Src-induced phosphorylation of caveolin-1 is crucial in regulating interactions of caveolin-1 with other components of the signaling machinery such as G(i), and key signaling entry of caveolae into the cytoplasm and endocytosis of albumin and other solutes. This review addresses the basis of transcytosis in endothelial cells, its central role as a determinant of endothelial barrier function, and signaling mechanisms involved in regulating fission of caveolae and trafficking of the formed vesicles from the luminal to abluminal side of the endothelial barrier.     

Evidence for coordinated induction and repression of ecto-5'-nucleotidase (CD73) and the A2a adenosine receptor in a human B cell line

In the human B cell line P493-6 two mitogenic signals, the Epstein-Barr virus nuclear antigen 2 (EBNA2) and myc, can be independently regulated by means of an estrogen receptor fusion construct or an inducible expression vector, respectively. Shut off of EBNA2, either in the presence or absence of myc, leads to a significant increase in enzymatic activity and surface expression of ecto-5'-nucleotidase (CD73) as well as an increased adenosine receptor response in cyclic AMP formation. Shut off of myc expression has a small additional positive effect on CD73 activity. Among the four different subtypes of adenosine receptors, the A2a receptor exclusively is subject to regulation in this system, which is substantiated by pharmacologic data (specific agonists and inhibitors), as well as on the mRNA level. With up-regulated CD73 and A2a, cells also respond to 5'-AMP with increased cyclic AMP formation. Turn on of EBNA2 has the reverse effect of repression of CD73 and A2a expression. The time course of both induction and repression of CD73 and A2a is rather slow.     

Human T cell activation. Synergy between CD73 (ecto-5'-nucleotidase) and signals delivered through CD3 and CD2 molecules

Interaction of the glycosyl phosphatidylinositol-linked differentiation Ag CD73 (ecto-5'-nucleotidase) with the CD73-specific mAb 1E9 generates agonistic signals that strongly synergize with T cell activation induced by CD3 and CD2 mAb. This synergy is observed only when 1E9 is immobilized on plastic and occurs in the absence of accessory cells or exogenous lymphokines. 1E9 induces a rapid (though transient) increase in [Ca2+]i in a minor proportion (20 to 30%) of unfractionated T lymphocytes (presumably CD73+ cells). However, this [Ca2+]i mobilization is not sufficient to fully activate CD73+ T cells, as shown by the requirement of additional signals such as CD3 or CD2 stimulation to initiate T cell proliferation. These signals cannot be substituted by the exogenous lymphokines, rIL-1, rIL-2, or rIL-4, or PMA (when T cells are rigorously depleted of monocytes). These data indicate that CD73 may behave as an accessory molecule regulating interactions between T cells and antigens or APC. A comparison was carried out with mAb 9.3 to the differentiation Ag CD28, another agonistic molecule with activating properties similar to CD73. Despite their lower percentage, the ability of CD73+ T cells to amplify the proliferation induced by CD3 or CD2 mAb was equivalent or even greater than that of CD28+ T cells. Once activated, CD73+ cells may recruit the remaining (CD73-) cells primed by CD3 or CD2 stimulation. Based on these data, we suggest that CD73+ T lymphocytes may be a specialized subset to amplify immune responses originated by the CD3 and CD2 activation pathways. Finally, the functional association between CD73 and integral membrane molecules like CD3 and CD2 suggests that GPI-anchored molecules may play a role in transmembrane signaling mediated by conventional second messenger systems.     

Neurotransmitter acetylcholine negatively regulates neuromuscular synapse formation by a Cdk5-dependent mechanism

Synapse formation requires interactions between pre- and postsynaptic cells to establish the connection of a presynaptic nerve terminal with the neurotransmitter receptor-rich postsynaptic apparatus. At developing vertebrate neuromuscular junctions, acetylcholine receptor (AChR) clusters of nascent postsynaptic apparatus are not apposed by presynaptic nerve terminals. Two opposing activities subsequently promote the formation of synapses: positive signals stabilize the innervated AChR clusters, whereas negative signals disperse those that are not innervated. Although the nerve-derived protein agrin has been suggested to be a positive signal, the negative signals remain elusive. Here, we show that cyclin-dependent kinase 5 (Cdk5) is activated by ACh agonists and is required for the ACh agonist-induced dispersion of the AChR clusters that have not been stabilized by agrin. Genetic elimination of Cdk5 or blocking ACh production prevents the dispersion of AChR clusters in agrin mutants. Therefore, we propose that ACh negatively regulates neuromuscular synapse formation through a Cdk5-dependent mechanism.