Guido Beldi

Natural killer T cell dysfunction in CD39‐null mice protects against concanavalin A–induced hepatitis

Concanavalin A (Con A)–induced injury is an established natural killer T (NKT) cell–mediated model of inflammation that has been used in studies of immune liver disease. Extracellular nucleotides, such as adenosine triphosphate, are released by Con A–stimulated cells and bind to specific purinergic type 2 receptors to modulate immune activation responses. Levels of extracellular nucleotides are in turn closely regulated by ectonucleotidases, such as CD39/NTPDase1. Effects of extracellular nucleotides and CD39 on NKT cell activation and upon hepatic inflammation have been largely unexplored to date. Here, we show that NKT cells express both CD39 and CD73/ecto‐5′‐nucleotidase and can therefore generate adenosine from extracellular nucleotides, whereas natural killer cells do not express CD73. In vivo, mice null for CD39 are protected from Con A–induced liver injury and show substantively lower serum levels of interleukin‐4 and interferon‐γ when compared with matched wild‐type mice. Numbers of hepatic NKT cells are significantly decreased in CD39 null mice after Con A administration. Hepatic NKT cells express most P2X and P2Y receptors; exceptions include P2X3 and P2Y11. Heightened levels of apoptosis of CD39 null NKT cells in vivo and in vitro appear to be driven by unimpeded activation of the P2X7 receptor. Conclusion: CD39 and CD73 are novel phenotypic markers of NKT cells. Deletion of CD39 modulates nucleotide‐mediated cytokine production by, and limits apoptosis of, hepatic NKT cells providing protection against Con A–induced hepatitis. This study illustrates a further role for purinergic signaling in NKT‐mediated mechanisms that result in liver immune injury. (HEPATOLOGY 2008.)

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism.

Extracellular nucleotides (e.g. ATP, UTP, ADP) are released by activated endothelium, leukocytes and platelets within the injured vasculature and bind specific cell-surface type-2 purinergic (P2) receptors. This process drives vascular inflammation and thrombosis within grafted organs. Importantly, there are also vascular ectonucleotidases i.e. ectoenzymes that hydrolyze extracellular nucleotides in the blood to generate nucleosides (viz. adenosine). Endothelial cell NTPDase1/CD39 has been shown to critically modulate levels of circulating nucleotides. This process tends to limit the activation of platelet and leukocyte expressed P2 receptors and also generates adenosine to reverse inflammatory events. This vascular protective CD39 activity is rapidly inhibited by oxidative reactions, such as is observed with liver ischemia reperfusion injury. In this review, we chiefly address the impact of these signaling cascades following liver transplantation. Interestingly, the hepatic vasculature, hepatocytes and all non-parenchymal cell types express several components co-ordinating the purinergic signaling response. With hepatic and vascular dysfunction, we note heightened P2- expression and alterations in ectonucleotidase expression and function that may predispose to progression of disease. In addition to documented impacts upon the vasculature during engraftment, extracellular nucleotides also have direct influences upon liver function and bile flow (both under physiological and pathological states). We have recently shown that alterations in purinergic signaling mediated by altered CD39 expression have major impacts upon hepatic metabolism, repair mechanisms, regeneration and associated immune responses. Future clinical applications in transplantation might involve new therapeutic modalities using soluble recombinant forms of CD39, altering expression of this ectonucleotidase by drugs and/or using small molecules to inhibit deleterious P2-mediated signaling while augmenting beneficial adenosine-mediated effects within the transplanted liver.

Regulated catalysis of extracellular nucleotides by vascular CD39/ENTPD1 is required for liver regeneration.

BACKGROUND & AIMSnLittle is known about how endothelial cells respond to injury, regulate hepatocyte turnover and reconstitute the hepatic vasculature. We aimed to determine the effects of the vascular ectonucleotidase CD39 on sinusoidal endothelial cell responses following partial hepatectomy and to dissect purinergic and growth factor interactions in this model.nnnMETHODSnParameters of liver injury and regeneration, as well as the kinetics of hepatocellular and sinusoidal endothelial cell proliferation, were assessed following partial hepatectomy in mice that do not express CD39, that do not express ATP/UTP receptor P2Y2, and in controls. The effects of extracellular ATP on vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-6 responses were determined in vivo and in vitro. Phosphorylation of the endothelial VEGF receptor in response to extracellular nucleotides and growth factors was assessed in vitro.nnnRESULTSnAfter partial hepatectomy, expression of the vascular ectonucleotidase CD39 increased on sinusoidal endothelial cells. Targeted disruption of CD39 impaired hepatocellular regeneration, reduced angiogenesis, and increased hepatic injury, resulting in pronounced vascular endothelial apoptosis, and decreased survival. Decreased HGF release by sinusoidal endothelial cells, despite high levels of VEGF, reduced paracrine stimulation of hepatocytes. Failure of VEGF receptor-2/KDR transactivation by extracellular nucleotides on CD39-null endothelial cells was associated with P2Y2 receptor desensitization.nnnCONCLUSIONSnRegulated phosphohydrolysis of extracellular nucleotides by CD39 coordinates both hepatocyte and endothelial cell proliferation following partial hepatectomy. Lack of CD39 activity is associated with decreased hepatic regeneration and failure of vascular reconstitution.

Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto‐nucleoside triphosphate diphosphohydrolase 1 [E‐NTPD1]), a rate‐limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type‐2 purinergic (P2) receptors demonstrated on these cells. We note that CD39‐null mice are protected from acute vascular injury after single‐lobe warm IRI, and, relative to control wild‐type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end‐organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPγS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin‐12 and interleukin‐18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. Conclusion: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI. (HEPATOLOGY 2010.)

Heightened NTPDase-1/CD39 expression and angiogenesis in radiation proctitis

Radiation proctitis is an inflammatory process associated with persistent and refractory lower gastrointestinal bleeding. Purinergic signaling regulates hemostasis, inflammation, and angiogenesis. For example, CD39, the vascular ectonucleotidase, blocks platelet activation and is required for angiogenesis. Whether CD39 expression is affected by radiation injury is unknown. The aim of this work was to study CD39 expression patterns after clinical radiation injury to the rectum. We prospectively enrolled eight patients with radiation proctitis and five gender-matched controls. Biopsies were taken from normal-appearing rectal mucosa of controls and from the normal sigmoid and abnormal rectum of patients. Expression patterns of CD39, P2Y2 receptor, CD31, CD61 integrin, and vascular endothelial growth factor receptor 2 were examined by immunostaining; levels of CD39 were further evaluated by Western blots. Chronic inflammatory lesions of radiation proctitis were associated with heightened levels of angiogenesis. Immunohistochemical stains showed increased vascular expression of CD39, as confirmed by Western blots. CD39 was co-localized with vascular endothelial markers CD31 and CD61 integrin, as well as expressed by stromal tissues. Development of neovasculature and associated CD39 expression in radiation proctitis may be associated with the chronic, refractory bleeding observed in this condition.

Externalized percutaneous stent vs. internal double J stent: Short- and long-term complications after kidney transplantation

BACKGROUNDnIn patients undergoing kidney transplantation, ureteral stents are an established technique to reduce major urologic complications such as leakage and stenosis of the ureter. However, the best technique for ureteral stenting remains unclear. The aim of this study was to compare the outcome of percutaneous ureteral stents (PS) with internal double J stents (JJS) after kidney transplantation.nnnMETHODSnAll patients undergoing kidney transplantation between 2005 and 2014 were retrospectively analyzed. After excluding patientsxa0<18 years old, patients without stenting, and patients who underwent multiorgan transplantation, a total of 308 patients were included in the study. Two consecutive cohorts of patients were compared. In the cohort transplanted between 2005 and 2010, stenting was routinely performed using PS (216 patients), and in the second cohort, those transplanted after 2011, stenting was routinely performed using JJS (92 patients). For ureteric anastomosis, the Lich-Grégoir technique was used in all patients.nnnRESULTSnThere was no statistical difference in postoperative urinary tract infections (Pxa0=xa0.239) between the 2 cohorts. In patients with PS, the incidence of major urologic complications (11.6% vs 3.3%; Pxa0= .018), vesicoureteral reflux (14.3% vs 2.2%; Pxa0< .001), and urologic reinterventions (14.4% vs 5.4%; Pxa0= .031) was significantly higher when compared with JJS patients. Multivariable logistic regression revealed increased incidence of major urologic complications (odds ratio [OR] 3.66, 95% confidence interval [CI] 1.07-12.55, Pxa0= .039) and vesicoureteral reflux (OR 5.29, 95% CI 1.21-23.10, Pxa0= .027) in patients with PS compared with JJS.nnnCONCLUSIONnStenting of ureterovesical anastomosis using JJS is associated with reduced complications compared with PS after kidney transplantation.

Lebertransplantation – wer, wann, wie?

Die orthotope Lebertransplantation ist mittlerweile ein standardisiertes Verfahren mit geringer Morbiditat und Mortalitat und fuhrt bei korrekter Indikationsstellung zu einer ausgezeichneten Uberlebensrate und verbesserter Lebensqualitat.

89 DELETION OF TRAIL ON NK CELLS IS ASSOCIATED WITH EXCESSIVE HEPATIC ISCHEMIA-REPERFUSION INJURY IN MICE

Background and Aims: Ischemia-reperfusion injury (IRI) is a key factor that contributes to early and late dysfunction of liver grafts. Recent studies reveal that natural killer (NK) cells play an important role in liver injury post IRI. We hypothesized that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand with high expression on NK cells significantly impacts IRI. Methods: C57/BL6 wild-type and TRAIL knock-out mice (TRAIL−/−) were subjected to hepatic IRI for one hour. Adoptive transfer of wild-type and TRAIL−/− NK cells was performed into RAG2/common gamma null mice that lack T, B and NK cells. Liver injury was assessed by hepatic neutrophil infiltration, alanine aminotransferase (ALT), aspartate transaminase (AST), hepatic neutrophil activation by myeloperoxidase (MPO) activity. NK cell subsets of the ischemic liver lobe were analysed by flow cytometry. NK cell cytotoxicity and interferon gamma secretion were performed in vitro studies. Results: TRAIL−/− mice exhibit significantly more hepatic damage assessed by AST and ALT levels 24 hours post IRI compared to wildtype mice. Adoptive transfer of TRAIL−/− NK cells to Rag2/common gamma-null mice was associated with significantly increased IRI compared to transfer with wild-type NK cells. Hepatic neutrophil activation was significantly increased in TRAIL−/− compared to wild-type mice. Staining for CD107a, a marker of degranulation and cytotoxicity on NK cells was significantly elevated in ischemic liver lobes of TRAIL−/− compared to wild-type mice. In vitro NK cell cytotoxicity to a Yac-1 cancer cell line was significantly increased in sorted TRAIL−/− NK cells compared to wild-type NK cells. Systemic cytokine levels (TNF alpha, IL-1beta, IL-6) and interferon gamma secretion of NK cells after stimulation with IL-12/IL-18 in vitro were not significantly different between the groups. Conclusions: These results show that expression of TRAIL on NK cells is protective in a murine model of hepatic IRI via the modulation of NK cell mediated cytotoxicity.