Ildikó Kiss

Transglutaminase 2-/- mice reveal a phagocytosis-associated crosstalk between macrophages and apoptotic cells

Tissue transglutaminase (TGase2) is a protein-crosslinking enzyme known to be associated with the in vivo apoptosis program. Here we report that apoptosis could be induced in TGase2-/- mice; however, the clearance of apoptotic cells was defective during the involution of thymus elicited by dexamethasone, anti-CD3 antibody, or γ-irradiation, and in the liver after induced hyperplasia. The lack of TGase2 prevented the production of active transforming growth factor-β1 in macrophages exposed to apoptotic cells, which is required for the up-regulation of TGase2 in the thymus in vivo, for accelerating deletion of CD4+CD8+ cells and for efficient phagocytosis of apoptotic bodies. The deficiency is associated with the development of splenomegaly, autoantibodies, and immune complex glomerulonephritis in TGase2-/- mice. These findings have broad implications not only for diseases linked to inflammation and autoimmunity but also for understanding the interrelationship between the apoptosis and phagocytosis process.

Ligation of Retinoic Acid Receptor α Regulates Negative Selection of Thymocytes by Inhibiting Both DNA Binding of nur77 and Synthesis of Bim

Negative selection refers to the selective deletion of autoreactive thymocytes. Its molecular mechanisms have not been well defined. Previous studies in our laboratory have demonstrated that retinoic acids, physiological ligands for the nuclear retinoid receptors, selectively inhibit TCR-mediated death under in vitro conditions, and the inhibition is mediated via the retinoic acid receptor (RAR) α. The present studies were undertaken to investigate whether ligation of RARα leads to inhibition of TCR-mediated death in vivo and to identify the molecular mechanisms involved. Three models of TCR-mediated death were studied: anti-CD3-mediated death of thymocytes in wild-type mice, and Ag- and bacterial superantigen-driven thymocyte death in TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c in the context of the Ek (class II MHC) molecule. Our data demonstrate that the molecular program of both anti-CD3- and Ag-driven, but not that of superantigen-mediated apoptosis involves up-regulation of nur77, an orphan nuclear receptor, and bim, a BH3-only member of the proapoptotic bcl-2 protein family, proteins previously implicated to participate in the negative selection. Ligation of RARα by the synthetic agonist CD336 inhibited apoptosis, DNA binding of nur77, and synthesis of bim induced by anti-CD3 or the specific Ag, but had no effect on the superantigen-driven cell death. Our data imply that retinoids are able to inhibit negative selection in vivo as well, and they interfere with multiple steps of the T cell selection signal pathway.

Retinoid receptor-activating ligands are produced within the mouse thymus during postnatal development

Vitamin A deficiency is known to be accompanied with immune deficiency and susceptibility to a wide range of infectious diseases. Experimental evidence suggests that the active metabolites of vitamin A that mediate its effects on the immune system are the retinoic acids (RA), which are ligands for the nuclear RA receptor (RAR) family. RA were previously shown both to promote proliferation and to regulate apoptosis of thymocytes. In this study we detected the age‐dependent mRNA expression of retinaldehyde dehydrogenases (RALDH1 and 2), cellular RA binding protein‐II and CYP26A, proteins responsible for the synthesis, nuclear transport and degradation of RA in the postnatally developing thymus. RALDH1 was located in thymic epithelial cells. However, the amount of all‐trans RA in thymic homogenates was close to the detection limit, suggesting that in this tissue all‐trans RA is not the main RAR‐regulating product of retinol metabolism. At the same time, by measuring the induction of a RAR‐responsive transgene in two independent transgenic mouse strains, we demonstrated the production of an RAR‐activating ligand, which was age and RALDH dependent. Our data provide evidence for the existence of endogenous retinoid synthesis in the thymus and suggest that retinoids similar to glucocorticoids might indeed be involved in the regulation of thymic proliferation and selection processes by being present in the thymus in functionally effective amounts.

Prevalence of Respiratory Symptoms and Diseases Associated with Gastroesophageal Reflux Disease

Aim: Investigation of the prevalence of respiratory symptoms and diseases associated with gastroesophageal reflux disease (GERD). Patients and Methods: 299 subjects with GERD were submitted to upper gastrointestinal endoscopy and 24-hour esophageal pH monitoring and a symptom analysis. Results: Chronic respiratory symptoms or diseases were present in 18% (56/299). Chronic cough was observed in 42/56 patients, while typical reflux symptoms such as heartburn and acid regurgitation were observed in 30/56 and 24/56 cases, respectively. The prevalence of airway diseases was chronic bronchitis 12/56, asthma 10/56, recurrent pneumonia 10/56, chronic sinusitis 7/56 and chronic laryngitis 1/56. In patients with respiratory complications pathologic acid reflux was established in 29/51 cases on the basis of the DeMeester score, while 17/51 had pathologic postprandial, nocturnal or diurnal reflux events. Upper gastrointestinal endoscopy revealed a normal esophageal mucosa in 6/56, Savary-Miller stage I esophagitis in 23/56, stage II in 15/56, stage III in 5/56 and stage IV in 6/56 patients. Conclusions: These investigations have demonstrated an abnormal 24-hour pH score in about half of the patients with GERD-associated respiratory complications, and indicated that short reflux events are characteristic of the reflux activity in one third of this population.

Adenosine A2A receptor-mediated cell death of mouse thymocytes involves adenylate cyclase and Bim and is negatively regulated by Nur77.

Adenosine is generated in the microenvironment of emerging thymocytes through normal mechanisms of lymphocyte selection. In a normal thymus, most of the adenosine is catabolized by adenosine deaminase; however, in an environment where up to 95% of the cells undergo programmed cell death, a sufficient amount of adenosine is accumulated to trigger cell surface adenosine receptors. Here we show that accumulated adenosine can induce apoptosis in immature mouse thymocytes, mostly via adenosine A2A receptors. The signaling pathway is coupled to adenylate cyclase activation, induction of the Nur77 transcription factor, Nur77‐dependent genes, such as Fas ligand and TRAIL, and the pro‐apoptotic BH3‐only protein Bim. We analyzed several knockout and transgenic mouse lines and found that adenosine‐induced killing of mouse thymocytes requires Bim, occurs independently of “death receptor” signaling and is inhibited by Bcl‐2 and Nur77. Collectively our data demonstrate that adenosine‐induced cell death involves signaling pathways originally found in negative selection of thymocytes and suggest a determining role of Bim and a regulatory role for Nur77.

Relationship between Serum Nickel and Homocysteine Concentration in Hemodialysis Patients

Severe hyperhomocysteinemia (HHC) is associated with atherosclerosis. In hemodialysis (HD) patients, one of the main causes of death is cardiovascular disease. In animals, trace elements such as cobalt, copper, iron, and nickel ameliorated vitamin B12 deficiency-induced HHC. However, correlations between plasma total homocysteine (tHcy) and trace elements in HD patients have not been investigated. Therefore, tHcy, folate, vitamin B12, trace elements (cobalt, copper, iron, and nickel), and some laboratory parameters such as serum total protein, albumin, transferrin, ferritin, C-reactive protein (CRP), and interleukin-6 concentrations were determined in 122 hemodialysis patients. When patients were divided into groups according to their tHcy, we found no significant differences in concentrations of cobalt, copper, and total protein, while nickel was higher, and folate, vitamin B12, and iron were lower in patients with lower than higher tHcy. In univariate regression analysis, tHcy negatively correlated with concentrations of folate (r = −0.302, p < 0.006), vitamin B12 (r = −0.347, p < 0.0001), nickel (r = −0.289, p < 0.006), and CRP (r = −0.230, p < 0.02) and positively with serum albumin (r = 0.316, p < 0.0004) and hemoglobin (r = 0.329, p < 0.0001) values. No relationship between tHcy and serum concentrations of cobalt, copper, iron, or other laboratory parameters was found in HD patients. The effect of cobalt and nickel on homocysteine production was assessed in human peripheral mononuclear cells (PBMCs). Nickel but not cobalt at concentrations found in HD patients significantly inhibited homocysteine, cysteine, and S-adenosylhomocysteine production in human PBMCs. These results suggest that nickel might also be involved in the regulation of the methionine–folate cycle in humans, as was demonstrated in animal experiments.

Retinoids induce Fas(CD95) ligand cell surface expression via RARγ and nur77 in T cells

Cells from the CD4+ murine T hybridoma line IP‐12‐7 enter the apoptotic suicide program via the Fas ligand (FasL)/Fas‐mediated pathway upon TCR stimulation. This stimulus regulates the sensitization of the Fas death pathway and the cell surface appearance of preformed FasL. The apoptosis is dependent on new mRNA and protein synthesis and involves up‐regulation of nur77.Two groups of nuclear receptors for retinoic acids (RA) have been identified: retinoic acid receptors (RAR) and retinoid X receptors. IP‐12‐7 cells express RARα and RARγ. Here we show that,in the IP‐12‐7 T cells, RA also induced the expression and DNA binding of nur77, and the cell surface appearance of FasL. The induction was mediated via RARγ. Despite the induced expression of cell surface FasL, only two structurally related RARγ‐selective compounds, CD437 and CD2325, initiated apoptosis in these cells. The lack of apoptosis induction by natural RA was related to the inability of RARγ to sensitize the Fas death‐pathway. Cell surface FasL, however, was able to induce cell death in Fas‐bearing target cells. Natural RA also induced the expression of FasL in phytohemagglutinin‐activated peripheral murine T cells. It is proposed that therapeutically administered RA might induce apoptosis in Fas‐sensitive cells via induction of FasL expression in activated Tcells.