Zsolt Selmeczy

Adenosine A2A receptor activation inhibits T helper 1 and T helper 2 cell development and effector function

Adenosine is an immunosuppressive nucleoside, and adenosine A2A receptors inhibit T‐cell activation. We investigated the role of A2A receptors in regulating T helper (Th)1‐ and Th2‐cell development and effector function. A2A‐receptor stimulation suppressed the development of T‐cell receptor (TCR) ‐stimulated naive T cells into both Th1 and Th2 cells, as indicated by decreased IFN‐γ production by cells developed under Th1‐skewing conditions and decreased interleukin (IL) −4, IL5, and IL10 production by cells developed under Th2‐skewing conditions. Using A2A receptor‐deficient mice, we demonstrate that A2A receptor activation inhibits Th1‐ and Th2‐cell development by decreasing the proliferation and IL2 production of naive T cells, irrespective of whether the cells are expanded under Th1‐ or Th2‐skewing environment. Using in vivo established Th1 and Th2 cells, we further demonstrate the nonselective nature of A2A receptor‐mediated immunosuppressive effects, because A2A receptor activation decreased IFN‐γ and IL4 secretion and mRNA level of TCR‐stimulated effector Th1 and Th2 cells, respectively. A2A receptor mRNA expression in both Th1 and Th2 effector cells increased following TCR stimulation. In summary, these data demonstrate that A2A receptor activation has strong inhibitory actions during early developmental, as well as late effector, stages of Th1‐ and Th2‐cell responses.—Csóka, B., Himer, L., Selmeczy, Z., Vizi, E. S., Pacher, P., Ledent, C., Deitch, E. A., Spolarics, Z., Németh, Z. H., Haskó, G. Adenosine A2A receptor activation inhibits T helper 1 and T helper 2 cell development and effector function. FASEB J. 22, 3491–3499 (2008)

A2B Adenosine Receptors Protect against Sepsis-Induced Mortality by Dampening Excessive Inflammation

Despite intensive research, efforts to reduce the mortality of septic patients have failed. Adenosine is a potent extracellular signaling molecule, and its levels are elevated in sepsis. Adenosine signals through G-protein–coupled receptors and can regulate the host’s response to sepsis. In this study, we studied the role of A2B adenosine receptors in regulating the mortality and inflammatory response of mice following polymicrobial sepsis. Genetic deficiency of A2B receptors increased the mortality of mice suffering from cecal ligation and puncture-induced sepsis. The increased mortality of A2B knockout mice was associated with increased levels of inflammatory cytokines and chemokines and augmented NF-κB and p38 activation in the spleen, heart, and plasma in comparison with wild-type animals. In addition, A2B receptor knockout mice showed increased splenic apoptosis and phosphatase and tensin homolog activation and decreased Akt activation. Experiments using bone-marrow chimeras revealed that it is the lack of A2B receptors on nonhematopoietic cells that is primarily responsible for the increased inflammation of septic A2B receptor-deficient mice. These results indicate that A2B receptor activation may offer a new therapeutic approach for the management of sepsis.

Adenosine Augments IL-10 Production by Microglial Cells through an A2B Adenosine Receptor-Mediated Process

Microglia are activated by pathogen-associated molecular patterns and produce proinflammatory cytokines, such as TNF-α, IL-6, and IL-12, and the anti-inflammatory cytokine IL-10. Adenosine is an endogenous purine nucleoside and a ligand of four G protein-coupled adenosine receptors (ARs), which are the A1AR, A2AAR, A2BAR, and A3AR. ARs have been shown to suppress TNF-α production by microglia, but their role in regulating IL-10 production has not been studied. In this study, we demonstrate that adenosine augments IL-10 production by activated murine microglia while suppressing the production of proinflammatory cytokines. Because the order of potency of selective AR agonists in inducing IL-10 production was NECA > IB-MECA > CCPA ≥ CGS21680, and the A2BAR antagonist MRS1754 prevented the effect of NECA, we conclude that the stimulatory effect of adenosine on IL-10 production is mediated by the A2BAR. Mechanistically, adenosine augmented IL-10 mRNA accumulation by a transcriptional process. Using mutant IL-10 promoter constructs we showed that a CREB-binding region in the promoter mediated the augmenting effect of adenosine on IL-10 transcription. Chromatin immunoprecipitation analysis demonstrated that adenosine induced CREB phosphorylation at the IL-10 promoter. Silencing CREB using lentivirally delivered short hairpin RNA blocked the enhancing effect of adenosine on IL-10 production, confirming a role for CREB in mediating the stimulatory effect of adenosine on IL-10 production. In addition, adenosine augmented IL-10 production by stimulating p38 MAPK. Collectively, our results establish that A2BARs augment IL-10 production by activated murine microglia.

Adenosine A2A receptor activation protects CD4+ T lymphocytes against activation-induced cell death

Activation‐induced cell death (AICD) is initiated by T‐cell receptor (TCR) restimulation of already activated and expanded peripheral T cells and is mediated through Fas/Fas ligand (FasL) interactions. Adenosine is a purine nucleoside signaling molecule, and its immunomodulatory effects are mediated by 4 G‐protein‐coupled receptors: A1,A2A, A2B, and A3. In this study, we investigated the role of A2A receptors in regulating CD4+ T lymphocyte AICD. Our results showed that the selective A2A receptor agonist CGS21680 (EC50 = 15.2–32.6 nM) rescued mouse CD4+ hybridomas and human Jurkat cells from AICD and that this effect was reversed by the selective A2A receptor antagonist ZM241385 (EC50 = 2.3 nM). CGS21680 decreased phosphatidylserine exposure on the membrane, as well as the cleavage of caspase‐3, caspase‐8 and poly(ADP‐ribose) polymerase indicating that A2A receptor stimulation blocks the extrinsic apoptotic pathway. In addition, CGS21680 attenuated both Fas and FasL mRNA expression. This decrease in FasL expression was associated with decreased activation of the transcription factor systems NF‐κB, NF‐ATp, early growth response (Egr)‐1, and Egr‐3. The antiapoptotic effect of A2A receptor stimulation was mediated by protein kinase A. Together, these results demonstrate that A2A receptor activation suppresses the AICD of peripheral T cells.—Himer, L., Csóka, B., Selmeczy, Z., Koscsó, B., Pócza, T., Pacher, P., Németh, Z. H., Deitch, E. A., Vizi, E. S., Cronstein, B. N., Haskó, G. Adenosine A2A receptor activation protects CD4+ T lymphocytes against activation‐induced cell death. FASEB J. 24, 2631–2640 (2010). www.fasebj.org

Dual β-adrenergic modulation in the immune system : Stimulus-dependent effect of isoproterenol on MAPK activation and inflammatory mediator production in macrophages

This is the first study to demonstrate that the interaction between beta-adrenoceptor activation, and the production of inflammatory mediators can be modulated in opposite ways by two inflammatory stimuli, namely, protein kinase C (PKC)-activating phorbol myristyl acetate (PMA) and lipopolysaccharide (LPS). We provided evidence that isoproterenol treatment, when combined with phorbol ester increased the production of tumor necrosis factor-alpha, interleukin-12, and nitric oxide in murine macrophages, as well as in human monocytes and differentiated PLB-985 cells, while in agreement with earlier findings, it decreased inflammatory mediator production in combination with LPS stimulation. The contrasting effect on inflammatory mediator production, shown for the PMA and LPS activated cells was accompanied by parallel changes in activation of ERK1/2 and p38 MAPKs. Thus, isoproterenol significantly increased MAPK activation (phosphorylation) in PMA-treated cells and, conversely, it decreased the activation of extracellular signal regulated kinase 1/2 (ERK1/2) and p38 in LPS-stimulated cells. The opposing effects of isoproterenol on LPS-induced versus PMA-induced mediator production and the concurrent changes in MAPK activation highlight the role of this kinase pathway in macrophage activation and provide new insights regarding the flexible ways through which beta-adrenoceptor stimulation can modulate the inflammatory response in macrophages. Our results challenge the dogma that beta-adrenoceptor signaling is only immunosuppressive, and offer potential opportunities for new therapeutic approaches in the treatment of inflammatory and autoimmune diseases.

Opposite role of α2- and β-adrenoceptors in the modulation of interleukin-10 production in endotoxaemic mice

Our aim was to investigate the role of adrenoceptors in the modulation of in vivo interleukin-10 (IL-10) production in lipopolysaccharide (LPS)-treated mice. The effect of different adrenergic drugs on plasma concentration of IL-10 was measured by ELISA 90 min after LPS injection. Our results confirmed the involvement of β-adrenoceptors since the β-agonist isoproterenol significantly increased the IL-10 production in response to LPS stimulation, whereas the β-antagonists propranolol decreased it. In contrast, the β2-agonists UK-14304, clonidine and xylazine significantly decreased the IL-10 plasma level, whereas the α2-antagonists CH-38083, prazosine and WB-4101 increased it. Our results provide the first in vivo evidence that, in addition to β-adrenoceptors, α2-adrenoceptors play also a very important role in the regulation of IL-10 production under endotoxaemic conditions.

Immunomodulatory effect of antidepressants

Basic and clinical studies have provided evidence that the biophase level of monoamines, determined by the balance of their release and uptake, is involved in the pathophysiology and treatment of depression, whereas other arguments cite the role of inflammatory mediators in the etiology of psychiatric disorders. A bidirectional interaction between the monoamine system and the inflammatory system might explain the concurrent therapeutical efficacy and immunomodulatory features of antidepressants.

Multiple G‐protein‐coupling specificity of β‐adrenoceptor in macrophages

Adrenergic signalling of the immune system is one of the important modulator pathways of the inflammatory immune response realized via G protein‐mediated pathways. The resulted signal depends on the type of the receptor‐coupled G‐protein (GPCR) that, according to the classical paradigm in the case of β‐adrenergic receptor (β‐AR), is Gs‐type. Recently, alternate and/or multiple G protein coupling specificity of GPCRs have been demonstrated including a switch from Gs to Gi binding. The possibility of a Gs/Gi switch and its role in the immune response of macrophages has not been investigated yet. In this study, we demonstrate that β‐adrenergic stimulation itself is able to induce a transient mitogen‐activated protein kinase phosphorylation in murine peritoneal macrophages in a pertussis toxin‐sensitive manner, suggesting that the Gs/Gi switch also occurs in the immune system. Although this process is very rapid, it can influence different signalling pathways and can reprogramme effector functions suggesting that sympathetic modulation of the defence mechanism of the innate immune system has an additional, Gs/Gi switch‐dependent component.

Intact noradrenaline transporter is needed for the sympathetic fine-tuning of cytokine balance.

Earlier studies demonstrated that cytokine production is under the tonic control of noradrenaline. As the level and/or the duration of noradrenaline action is regulated by the noradrenaline transporter (NET), which is also a target of antidepressant treatment, we studied its role in the regulation of the cytokine response during inflammation. The endotoxin-evoked tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 response was studied in genetically produced noradrenaline transporter-deficient (NET-KO) mice and by treatment with desipramine, a monoamine uptake-blocker antidepressant. NET-KO mice responded to endotoxin with significantly lower TNF-alpha and interleukin-10 production in comparison to their wild-type counterparts. Functional involvement of both alpha- and beta-adrenoceptors could be demonstrated in our model systems, using 7,8-methylenedioxy-14 alpha-hydroxy-alloberbane.HCl (CH-38083) and propranolol; however, the differences between the two phenotypes remained, suggesting a limited role of alpha-adrenoceptors in the observed changes. Acute treatment of both wild-type and NET-KO mice with desipramine significantly decreased the TNF-alpha response and significantly increased interleukin-10 production, indicating the role of an intact noradrenaline transporter in anti-inflammatory responses.

The adenosine A2A receptor agonist CGS 21680 fails to ameliorate the course of dextran sulphate-induced colitis in mice

Abstract.Objective:In this study we investigated the effect of CGS 21680 (2-p-(2-Carboxyethyl)phenethylamino-5-N-ethylcarboxamidoadenosine hydrochloride), an adenosine A2A receptor agonist, in a model of dextran sulphate sodium (DSS)-induced colitis.Methods:NMRI mice were fed 5 % (w/v) DSS, and were treated intraperitoneally with 0.5 mg/kg CGS 21680 or vehicle for 10 days. Changes of bodyweight, colon length, the incidence of rectal bleeding, levels of macrophage inflammatory protein (MIP)-1α, MIP-2, interferon γ, interleukin (IL)-1β, IL-12 and tumour necrosis factor-α from homogenates of colon biopsies, and the release of [3H]acetylcholine (ACh) from longitudinal muscle strip were determined.Results:DSS significantly decreased bodyweight, colon length, and it increased the incidence of rectal bleeding and levels of MIP-1α, MIP-2 and IL-1β compared to DSS-untreated animals. CGS 21680 had no effect on these changes. No change could be observed in release of ACh in DSS-induced colitis with or without CGS 21680.Conclusion:In summary, CGS 21680 is ineffective in ameliorating DSS-induced colitis in mice.