György Haskó

Adenosine receptors: therapeutic aspects for inflammatory and immune diseases

Adenosine is a key endogenous molecule that regulates tissue function by activating four G-protein-coupled adenosine receptors: A1, A2A, A2B and A3. Cells of the immune system express these receptors and are responsive to the modulatory effects of adenosine in an inflammatory environment. Animal models of asthma, ischaemia, arthritis, sepsis, inflammatory bowel disease and wound healing have helped to elucidate the regulatory roles of the various adenosine receptors in dictating the development and progression of disease. This recent heightened awareness of the role of adenosine in the control of immune and inflammatory systems has generated excitement regarding the potential use of adenosine-receptor-based therapies in the treatment of infection, autoimmunity, ischaemia and degenerative diseases.

NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential

Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impaired bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules, lack of response and the development of tolerance following prolonged administration. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages. Here we review the discovery, biochemistry, pharmacology and clinical potential of haem-dependent sGC stimulators (including YC-1, BAY 41-2272, BAY 41-8543, CFM-1571 and A-350619) and haem-independent sGC activators (including BAY 58-2667 and HMR-1766).

Adenosine inhibits IL-12 and TNF-α production via adenosine A2a receptor-dependent and independent mechanisms

Interleukin 12 (IL‐12) is a crucial cytokine in the regulation of T helper 1 vs. T helper 2 immune responses. In the present study, we investigated the effect of the endogenous purine nucleoside adenosine on the production of IL‐12. In mouse macrophages, adenosine suppressed IL‐12 production. Although the order of potency of adenosine receptor agonists suggested the involvement of A2a receptors, data obtained with A2a receptor‐deficient mice showed that the adenosine suppression of IL‐12 and even TNF‐α production is only partly mediated by A2a receptor ligation. Studies with adenosine receptor antagonists or the adenosine uptake blocker dipyridamole showed that adenosine released endogenously also decreases IL‐12. Although adenosine increases IL‐10 production, the inhibition of IL‐12 production is independent of the increased IL‐10. The mechanism of action of adenosine was not associated with alterations of the activation of the p38 and p42/p44 mitogen‐activated protein kinases or the phosphorylation of the c‐Jun terminal kinase. Adenosine failed to affect steady‐state levels of either IL‐12 p35 or p40 mRNA, but augmented IL‐10 mRNA levels. In summary, adenosine inhibits IL‐12 production via various adenosine receptors. These results support the notion that adenosinebased therapies might be useful in certain autoimmune and/or inflammatory diseases.—Haskó, G., Kuhel, D. G., Chen, J.‐F., Schwarzschild, M. A., Deitch, E. A., Mabley, J. G., Marton, A., Szabó, C. Adenosine inhibits IL‐12 and TNF‐a production via adenosine A2a receptor‐dependent and independent mechanisms. FASEB J. 14, 2065–2074 (2000)

CD39 and CD73 in immunity and inflammation

The enzymatic activities of CD39 and CD73 play strategic roles in calibrating the duration, magnitude, and chemical nature of purinergic signals delivered to immune cells through the conversion of ADP/ATP to AMP and AMP to adenosine, respectively. This drives a shift from an ATP-driven proinflammatory environment to an anti-inflammatory milieu induced by adenosine. The CD39/CD73 pathway changes dynamically with the pathophysiological context in which it is embedded. It is becoming increasingly appreciated that altering this catabolic machinery can change the course or dictate the outcome of several pathophysiological events, such as AIDS, autoimmune diseases, infections, atherosclerosis, ischemia-reperfusion injury, and cancer, suggesting these ectoenzymes are novel therapeutic targets for managing a variety of disorders.

Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy

Annexin V and Sytox Green are widely used markers to evaluate apoptosis in various cell types using flow cytometry and fluorescent microscopy. Recently, a novel fluoroprobe MitoSOX Red was introduced for selective detection of superoxide in the mitochondria of live cells and was validated for confocal microscopy and flow cytometry. This protocol describes simultaneous measurements of mitochondrial superoxide generation with apoptotic markers (Annexin V and Sytox Green) by both flow cytometry and confocal microscopy in endothelial cell lines. The advantages of the described flow cytometry method over other cell-based techniques are the tremendous speed (1–2 h), exquisite precision and the possibility of simultaneous quantitative measurements of mitochondrial superoxide generation and apoptotic (and other) markers, with maximal preservation of cellular functions. This method combined with fluorescent microscopy may be very useful to reveal important spatial–temporal changes in mitochondrial superoxide production and execution of programmed cell death in virtually any cell type.

Immunity, inflammation and cancer: A leading role for adenosine

Cancer is a complex disease that is dictated by both cancer cell-intrinsic and cell-extrinsic processes. Adenosine is an ancient extracellular signalling molecule that can regulate almost all aspects of tissue function. As such, several studies have recently highlighted a crucial role for adenosine signalling in regulating the various aspects of cell-intrinsic and cell-extrinsic processes of cancer development. This Review critically discusses the role of adenosine and its receptors in regulating the complex interplay among immune, inflammatory, endothelial and cancer cells during the course of neoplastic disease.

An angiogenic switch in macrophages involving synergy between toll-like receptors 2, 4, 7, and 9 and adenosine A2A receptors

Adenosine A(2A) receptor (A(2A)R) agonists synergize with Escherichia coli (E. coli) LPS [toll-like receptor (TLR)4 agonist] to up-regulate vascular endothelial growth factor (VEGF) expression in murine macrophages. Here, we demonstrate that TLR2, TLR7, and TLR9, but not TLR3 and TLR5 agonists, also synergize with A(2A)R agonists and adenosine to up-regulate VEGF, while simultaneously strongly down-regulating TNFalpha expression. In the absence of adenosine or A(2A)R agonists, Porphyromonas gingivalis (P. gingivalis) LPS and PAM(3)CAG (TLR2 agonists), resiquimod (R848) (TLR7 agonist), and non-methylated CpG DNA (TLR9 agonist) strongly up-regulate TNFalpha expression, with no effect on VEGF. In the presence of adenosine or A(2A)R agonists, but not A(1)R agonists, TLR2, 4, 7, and 9 agonists strongly up-regulate VEGF expression, while simultaneously down-regulating TNFalpha. C57BL/10ScN (TLR4 deletion mutant) macrophages produce TNFalpha in response to TLR2, 3, 7, and 9 agonists, but not the TLR4 agonist E. coli LPS. With adenosine or A(2A)R agonists, TLR2, 7, and 9, but not TLR4 agonists, also synergistically up-regulate VEGF, while down-regulating TNFalpha expression. Polyinosinic-polycytidilic acid (poly(I:C)) (TLR3 agonist) stimulates TNFalpha expression in macrophages from both C57BL/10ScSn and C57BL/10ScN mice, but has little effect on VEGF expression in the presence of adenosine or A(2A)R agonists. R-flagellins from Serratia marcescens (S. marcescens) and Salmonella muenchen (S. muenchen) do not stimulate TNFalpha expression in either C57BL/10ScSn or C57BL10/ScN mice, and have no effect on VEGF production in the presence of adenosine or A(2A)R agonists. While adenosine and A(2A)R agonists strongly down-regulate TNFalpha protein expression induced by TLR2, 3, 4, 7, and 9 agonists, TNFalpha mRNA and NF-kappaB activation are not reduced. We propose a novel signaling pathway in murine macrophages involving synergy between TLRs 2, 4, 7, and 9 and A(2A)Rs, that up-regulates VEGF and down-regulates TNFalpha expression, thus acting as an angiogenic switch. This angiogenic switch may play an important role in ischemia when TLR agonists are present, providing an interface between innate immunity and wound healing.

Inosine Inhibits Inflammatory Cytokine Production by a Posttranscriptional Mechanism and Protects Against Endotoxin-Induced Shock

Extracellular purines, including adenosine and ATP, are potent endogenous immunomodulatory molecules. Inosine, a degradation product of these purines, can reach high concentrations in the extracellular space under conditions associated with cellular metabolic stress such as inflammation or ischemia. In the present study, we investigated whether extracellular inosine can affect inflammatory/immune processes. In immunostimulated macrophages and spleen cells, inosine potently inhibited the production of the proinflammatory cytokines TNF-α, IL-1, IL-12, macrophage-inflammatory protein-1α, and IFN-γ, but failed to alter the production of the anti-inflammatory cytokine IL-10. The effect of inosine did not require cellular uptake by nucleoside transporters and was partially reversed by blockade of adenosine A1 and A2 receptors. Inosine inhibited cytokine production by a posttranscriptional mechanism. The activity of inosine was independent of activation of the p38 and p42/p44 mitogen-activated protein kinases, the phosphorylation of the c-Jun terminal kinase, the degradation of inhibitory factor κB, and elevation of intracellular cAMP. Inosine suppressed proinflammatory cytokine production and mortality in a mouse endotoxemic model. Taken together, inosine has multiple anti-inflammatory effects. These findings, coupled with the fact that inosine has very low toxicity, suggest that this agent may be useful in the treatment of inflammatory/ischemic diseases.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

Cannabinoid-2 receptor mediates protection against hepatic ischemia/reperfusion injury

Hepatic ischemia‐reperfusion (I/R) injury continues to be a fatal complication that can follow liver surgery or transplantation. We have investigated the involvement of the endocannabinoid system in hepatic I/R injury using an in vivo mouse model. Here we report that I/R triggers several‐fold increases in the hepatic levels of the endocannabinoids anandamide and 2‐arachidonoylglycerol, which originate from hepa‐tocytes, Kupffer, and endothelial cells. The I/R‐in‐duced increased tissue endocannabinoid levels positively correlate with the degree of hepatic damage and serum TNF‐α, MIP‐1α, and MIP‐2 levels. Furthermore, a brief exposure of hepatocytes to various oxidants (H2O2 and peroxynitrite) or inflammatory stimuli (endotoxin and TNF‐α) also increases endocannabinoid levels. Activation of CB2 cannabinoid receptors by JWH133 protects against I/R damage by decreasing inflammatory cell infiltration, tissue and serum TNF‐α, MIP‐1a and MIP‐2 levels, tissue lipid peroxidation, and expression of adhesion molecule ICAM‐1 in vivo. JWH133 also attenuates the TNF‐a‐induced ICAM‐1 and VCAM‐1 expression in human liver sinusoidal endothelial cells (HLSECs) and the adhesion of human neutrophils to HLSECs in vitro. Consistent with the protective role of CB2 receptor activation, CB2_/_ mice develop increased I/R‐induced tissue damage and proinflammatory phenotype. These findings suggest that oxidative/nitrosative stress and inflammatory stimuli may trigger endocannabinoid production, and indicate that targeting CB2 cannabinoid receptors may represent a novel protective strategy against I/R injury. We also demonstrate that CB2−/− mice have a normal hemodynamic profile.—Bátkai, S., Osei‐Hyiaman, D., Pan, H., El‐Assal, O., Rajesh, M., Mukhopadhyay, P., Hong, F., Harvey‐White, J., Jafri, A., Hasko, G., Huffman, J. W., Gao, B., Kunos, G., Pacher, P. Cannabi‐noid‐2 receptor mediates protection against hepatic ischemia/reperfusion injury. FASEB J. 21, 1788–1800 (2007)