Sex differences in renin response and changes of capillary diameters after renal ischemia/reperfusion injury.

Activation of the RAS has a crucial role in the progression of ischemia/reperfusion-associated CAD. The regulation of RAS differs in the two genders. However, the extent of gender differences and locations of renin production have not been revealed yet. We investigated in vivo the local renin production in the two genders during ischemia/reperfusion injury. In male and female Wistar rats, renal ischemia was induced followed by a reperfusion period of two, eight, 16, 24, or 48 h. We applied flow cytometry to measure renin content and multiphoton imaging to visualize renin granules and changes of peritubular diameters in vivo during ischemia/reperfusion. Renin content decreased in CD in the first eight h of reperfusion; however, after 16 h, its amount increased. In males, the production of renin was more pronounced, and the duration of vasoconstriction was longer with a subsequent phase of vessel hyperdilation compared to females. Renal ischemia/reperfusion injury induces renin response not only in the JGA, but also in the CD segment. Renin production is more explicit in males than in females which, via increased angiotensin II production, might explain the different dynamism of renal vessel regulation between the two genders.

Calcineurin-inhibition Results in Upregulation of Local Renin and Subsequent Vascular Endothelial Growth Factor Production in Renal Collecting Ducts.

BACKGROUND: Tacrolimus (Tac) and Cyclosporine A (CyA) calcineurin inhibitors (CNIs) are 2 effective immunosuppressants which are essential to prevent allograft rejection. Calcineurin inhibitors are known to be nephrotoxic. However, the precise mechanism of nephrotoxicity is not fully understood. In this study, we investigated the in vivo effects of CNIs on the local renal renin-angiotensin system in the collecting duct (CD). METHODS: Three-week-old mice were treated with either vehicle, CyA (2 mg/kg per day), Tac (0.075 mg/kg per day), CyA + Aliskiren (25 mg/kg per day), or Tac + Aliskiren for 3 weeks. Serum creatinine was measured. Renin and vascular endothelial growth factor (VEGF) contents in CD were evaluated with flow cytometry and multiphoton microscopy. The diameter of vessels was assessed with multiphoton microscopy, and the amount of renal collagen was determined by real-time polymerase chain reaction and Masson staining. RESULTS: The elevated level of serum creatinine in CNI groups was abolished by Aliskiren. Flow cytometric analysis found elevated renin content in principal cells, which was prevented by Aliskiren. This result was further confirmed with multiphoton microscopy. The VEGF content in CD correlated with reduced capillary diameter and with the formation of fibrotic islands. CONCLUSIONS: Calcineurin inhibitors induce production of renin in the CD that may contribute to decreased renal blood flow. In turn, CD responds with increased VEGF production, resulting in disproportional vessel growth, further worsening the local hypoxia and striped fibrosis surrounding the CDs. Aliskiren, a direct renin inhibitor blocks these effects and improves CNI-induced nephropathy by decreasing renin production in the CDs. Our data suggest that Aliskiren may be used for the prevention of CNI nephrotoxicity.

Expression of PARK7 is increased in celiac disease.

Recently, it has been suggested that the gene called Parkinson's disease 7 (PARK7) might be an upstream activator of hypoxia-inducible factor (HIF)-1α, which plays a major role in sustaining intestinal barrier integrity. Furthermore, PARK7 has been proposed to participate in the Toll-like receptor (TLR)-dependent regulation of the innate immune system. Our aim was to investigate the involvement of PARK7 in the pathogenesis of coeliac disease (CD). Duodenal biopsy specimens were collected from 19 children with untreated CD, five children with treated CD (maintained on gluten-free diet), and ten children with histologically normal duodenal biopsies. PARK7 mRNA expression and protein level were determined by real-time polymerase chain reaction (PCR) and Western blot, respectively. Localization of PARK7 was visualized by immunofluorescence staining. Protein level of PARK7 increased in the duodenal mucosa of children with untreated CD compared to children with treated CD or to control biopsies (p <0.03). We detected intensive PARK7 staining in the epithelial cells and lamina propria of the duodenal mucosa of children with untreated CD compared with that in control biopsies. Our finding that mucosal expression of PARK7 is increased suggests that PARK7 is involved in the pathogenesis of gastrointestinal diseases, notably CD. Our results suggest that PARK7 may alter processes mediated by HIF-1α and TLR4, which supports a role for PARK7 in the maintenance of epithelial barrier integrity, immune homeostasis, or apoptosis.

Membrane microdomain organization, calcium signal, and NFAT activation as an important axis in polarized Th cell function.

T helper lymphocytes become polarized upon antigen and cytokine stimuli received after their maturation in the thymus. Since the balance of Th1 and Th2 responses is critical in healthy and pathological immune responses, understanding the molecular base of T cell polarization still remained an important question. Using our Th0/Th1/Th2 hybridoma model system, we performed a comparative study on polarized Th1 and Th2 cells in terms of their membrane raft expression/composition, their TCR mediated activation signaling, and sensitivity to activation-induced cell death (AICD) using flow and image cytometric methods. We show here that the TCR stimulation induced more intense and sustained Ca(2+) -response in Th1 cells compared to Th2 ones correlates well with a shorter nuclear residence time of the Ca(2+) -dependent NFAT transcription factor in Th2 cells. In addition, NFAT translocation directly depended on lipid raft integrity/membrane cholesterol level. Expression pattern of raftophilic accessory proteins (CD4, CD59, and CD48) and lipids (GM1, cholesterol) were also different in the Th1 and Th2 hybridomas, similarly to differentiated spleen Th cells. The activation-induced, remarkably clustered and polarized membrane distribution of TCR/CD3 complex in Th1, but not in Th2 cells, together with an increased raft localization of Kv1.3 ion channels regulating the Ca(2+) -response, are consistent with the above properties of NFAT. Finally, the polarized Th cells, especially Th1, were more sensitive to AICD than their unpolarized Th0 precursor. These results suggest that the membrane microdomain organization-Ca(2+) -signaling-NFAT activation axis is an important determinant of polarized Th cell effector function and fate.

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 A(1)AR, A(2A)AR, A(2B)AR, and A(3)AR. 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 A(2B)AR antagonist MRS1754 prevented the effect of NECA, we conclude that the stimulatory effect of adenosine on IL-10 production is mediated by the A(2B)AR. 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 A(2B)ARs augment IL-10 production by activated murine microglia.

[Role of Th17 cells in rheumatoid arthritis]. / A Th17 sejtek szerepe rheumatoid arthritisben.

Th17 cells are the newly described subset of the CD4(+) T lymphocytes. Activated Th17 cells are characterized by their ability to produce IL-17A and other pro-inflammatory cytokines. IL-17A regulates immune function through its cell-surface receptor expressed on epithelial-and endothelial cells, fibroblasts and leukocytes by promoting neutrophil recruitment and releasing further pro-inflammatory mediators. Failures of the susceptible balance of the immunoregulation may lead to unchecked immune response and autoimmune diseases. The central role of Th17 cells and cytokines produced by Th17 cells were confirmed in a wide variety of human autoimmune diseases, including rheumatoid arthritis. Recently Th17 cells and its cytokines come into the focus of immunological research as potential therapeutic targets.

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 A(2B) adenosine receptors in regulating the mortality and inflammatory response of mice following polymicrobial sepsis. Genetic deficiency of A(2B) receptors increased the mortality of mice suffering from cecal ligation and puncture-induced sepsis. The increased mortality of A(2B) knockout mice was associated with increased levels of inflammatory cytokines and chemokines and augmented NF-kappaB and p38 activation in the spleen, heart, and plasma in comparison with wild-type animals. In addition, A(2B) 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 A(2B) receptors on nonhematopoietic cells that is primarily responsible for the increased inflammation of septic A(2B) receptor-deficient mice. These results indicate that A(2B) receptor activation may offer a new therapeutic approach for the management of sepsis.

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: A(1), A(2A), A(2B), and A(3). In this study, we investigated the role of A(2A) receptors in regulating CD4(+) T lymphocyte AICD. Our results showed that the selective A(2A) receptor agonist CGS21680 (EC(50)=15.2-32.6 nM) rescued mouse CD4(+) hybridomas and human Jurkat cells from AICD and that this effect was reversed by the selective A(2A) receptor antagonist ZM241385 (EC(50)=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 A(2A) 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-kappaB, NF-ATp, early growth response (Egr)-1, and Egr-3. The antiapoptotic effect of A(2A) receptor stimulation was mediated by protein kinase A. Together, these results demonstrate that A(2A) receptor activation suppresses the AICD of peripheral T cells.

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

Adenosine is an immunosuppressive nucleoside, and adenosine A(2A) receptors inhibit T-cell activation. We investigated the role of A(2A) receptors in regulating T helper (Th)1- and Th2-cell development and effector function. A(2A)-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-gamma production by cells developed under Th1-skewing conditions and decreased interleukin (IL) -4, IL-5, and IL-10 production by cells developed under Th2-skewing conditions. Using A(2A) receptor-deficient mice, we demonstrate that A(2A) receptor activation inhibits Th1- and Th2-cell development by decreasing the proliferation and IL-2 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 A(2A) receptor-mediated immunosuppressive effects, because A(2A) receptor activation decreased IFN-gamma and IL-4 secretion and mRNA level of TCR-stimulated effector Th1 and Th2 cells, respectively. A(2A) receptor mRNA expression in both Th1 and Th2 effector cells increased following TCR stimulation. In summary, these data demonstrate that A(2A) receptor activation has strong inhibitory actions during early developmental, as well as late effector, stages of Th1- and Th2-cell responses.

Adenosine receptor activation ameliorates type 1 diabetes.

Growing evidence indicates that adenosine receptors could be promising therapeutic targets in autoimmune diseases. Here we studied the role of adenosine receptors in controlling the course of type 1 diabetes. Diabetes in CD-1 mice was induced by multiple-low-dose-streptozotocin (MLDS) treatment and in nonobese diabetic (NOD) mice by cyclophosphamide injection. The nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) prevented diabetes development in both MLDS-challenged mice and in cyclophosphamide-treated NOD mice. The effect of NECA was reversed by the selective A2B receptor antagonist N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS 1754). The selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were less efficacious in ameliorating the course of diabetes. NECA inhibited diabetes in A2A receptor KO mice and the selective A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethyl-carboxamidoadenosine (CGS21680) had no effect in normal mice, indicating a lack of role of A2A receptors. NECA failed to prevent cytokine-induced beta-cell death in vitro, but NECA strongly suppressed expression of the proinflammatory cytokines TNF-alpha, MIP-1alpha, IL-12, and IFN-gamma in pancreata, endotoxin, or anti-CD3-stimulated splenic cells, and T helper 1 lymphocytes, indicating that the beneficial effect of NECA was due to immunomodulation. These results demonstrate that adenosine receptor ligands are potential candidates for the treatment of type 1 diabetes.