Ferenc Boldizsár

Emerging pathways of non-genomic glucocorticoid (GC) signalling in T cells

In the last decade new glucocorticoid (GC)-signalling mechanisms have emerged. The evolving field of non-genomic GC actions was precipitated from two major directions: (i) some rapid/acute clinical GC applications could not be explained based on the relatively slowly appearing genomic GC action and (ii) accumulating evidence came to light about the discrepancy in the apoptosis sensitivity and GR expression of thymocytes and other lymphoid cell types. Herein, we attempt to sample the latest information in the field of non-genomic GC signalling in T cells, and correlate it with results from our laboratory. We discuss some aspects of the regulation of thymocyte apoptosis by GCs, paying special interest to the potential role(s) of mitochondrial GR signalling. The interplay between the T cell receptor (TcR) and glucocorticoid receptor (GR) signalling pathways is described in more detail, focusing on ZAP-70, which is a novel target of rapid GC action.

Oncopharmacological Perspectives of a Plant Lectin (Viscum album Agglutinin-I): Overview of Recent Results from In vitro Experiments and In vivo Animal Models, and Their Possible Relevance for Clinical Applications

An old goal of natural complementary medical therapy has been to aim at a long-term stimulation of natural resistance in order to restrain cancer progression or improve defective immunological conditions without toxic side effects. Mistletoe extracts were applied to a large number of cancer patients because of their modulatory effect on the natural immune system. By carefully removing lectins, an essential group of components, from mistletoe extracts, a significant reduction of their effectiveness on several cellular immune parameters could be observed in vivo (1). That is the reason why, for the last 14 years, biological research of mistletoe extracts has focused on lectins. Meanwhile, the quantitatively dominant lectin, Viscum album agglutinin (VAA)-I has become available in a recombinant form (rVAA). Other constitiuents of plant extracts such as viscotoxins (2,3), poly- and oligosaccharides (4), flavonoids (5,6), chitin-binding mistletoe lectin (7) and arginine have also been investigated in connection with the effects of mistletoe extracts on the host defense. However, little evidence has been found that these substances contribute to the effects of mistletoe in vivo (8).

Wnt-4 protects thymic epithelial cells against dexamethasone-induced senescence

Glucocorticoids are widely used immunosuppressive drugs in treatment of autoimmune diseases and hematological malignancies. Glucocorticoids are particularly effective immune suppressants, because they induce rapid peripheral T cell and thymocyte apoptosis resulting in impaired T cell-dependent immune responses. Although glucocorticoids can induce apoptotic cell death directly in developing thymocytes, how exogenous glucocorticoids affect the thymic epithelial network that provides the microenvironment for T cell development is still largely unknown. In the present work, we show that primary thymic epithelial cells (TECs) express glucocorticoid receptors and that high-dosage dexamethasone induces degeneration of the thymic epithelium within 24 h of treatment. Changes in organ morphology are accompanied by a decrease in the TEC transcription factor FoxN1 and its regulator Wnt-4 parallel with upregulation of lamina-associated polypeptide 2α and peroxisome proliferator activator receptor γ, two characteristic molecular markers for adipose thymic involution. Overexpression of Wnt-4, however, can prevent upregulation of adipose differentiation-related aging markers, suggesting an important role of Wnt-4 in thymic senescence.

Mitochondrial translocation of the glucocorticoid receptor in double-positive thymocytes correlates with their sensitivity to glucocorticoid-induced apoptosis

Glucocorticoid receptor (GR) signaling plays an important role in the selection and apoptosis of thymocytes. Besides nuclear translocation, mitochondrial translocation of the ligand-bound GR in lymphoid cells was also shown, which might determine glucocorticoid (GC)-induced apoptosis sensitivity. In the present work, we followed the ligand-induced GR trafficking in CD4+CD8+ double-positive (DP) thymocytes. Using confocal microscopy, we found that upon short-term in vitro GC analog [dexamethasone (DX)] treatment, the GR translocates into the mitochondria but not into the nucleus in DP cells. We also analyzed the GR redistribution in cytosolic, nuclear and mitochondrial fractions of unseparated thymocytes by western blot and confirmed that in DX-treated cells a significant fraction of the GR translocates into the mitochondria. DX reduced the mitochondrial membrane potential of DP cells within 30 min, measured by flow cytometry, which refers to a direct modulatory activity of mitochondrial GR translocation. The abundant mitochondrial GR found in DP cells well correlates with their high GC-induced apoptosis sensitivity.

Enigma of IL-17 and Th17 Cells in Rheumatoid Arthritis and in Autoimmune Animal Models of Arthritis

Rheumatoid arthritis (RA) is one of the most common autoimmune disorders characterized by the chronic and progressive inflammation of various organs, most notably the synovia of joints leading to joint destruction, a shorter life expectancy, and reduced quality of life. Although we have substantial information about the pathophysiology of the disease with various groups of immune cells and soluble mediators identified to participate in the pathogenesis, several aspects of the altered immune functions and regulation in RA remain controversial. Animal models are especially useful in such scenarios. Recently research focused on IL-17 and IL-17 producing cells in various inflammatory diseases such as in RA and in different rodent models of RA. These studies provided occasionally contradictory results with IL-17 being more prominent in some of the models than in others; the findings of such experimental setups were sometimes inconclusive compared to the human data. The aim of this review is to summarize briefly the recent advancements on the role of IL-17, particularly in the different rodent models of RA.

Th1/Th17 polarization and acquisition of an arthritogenic phenotype in arthritis-susceptible BALB/c, but not in MHC-matched, arthritis-resistant DBA/2 mice

Proteoglycan (PG) aggrecan-induced arthritis (PGIA) is a murine model of rheumatoid arthritis (RA). Although BALB/c and DBA/2 mice share the same MHC (H-2d) haplotype, the BALB/c strain is susceptible to PGIA, while DBA/2 mice are resistant. Therefore, these two inbred mouse strains provide an opportunity to study arthritis susceptibility factors excluding the effects of MHC-associated genetic components. The goal of this study was to monitor changes in the cellular composition and activation state following intra-peritoneal (i.p.) immunization to induce PGIA; additionally, we sought to identify new susceptibility factors by comparing PG-induced immune responses in BALB/c and DBA/2 mice. Upon i.p. PG injection, resident naive B1 cells are replaced by both T cells and conventional B cells in the peritoneum of BALB/c mice. These peritoneal T cells produce IFNγ and IL-17, cytokines shown to be important in RA and corresponding arthritis models. Moreover, peritoneal cells can adoptively transfer PGIA to SCID mice, demonstrating their arthritogenic properties. Our results indicate that repeatedly injected antigen leads to the recruitment and activation of immune cells in the peritoneum; these cells then trigger the effector phase of the disease. The migration and activation of Th1/Th17 cells in the peritoneal cavity in response to PG immunization, which did not occur in the arthritis-resistant DBA/2 strain, may be critical factors of arthritis susceptibility in BALB/c mice.

Dexamethasone induces rapid tyrosine-phosphorylation of ZAP-70 in Jurkat cells

Steroid hormones are known to mediate rapid non-genomic effects occurring within minutes, besides the classical genomic actions mediated by the nuclear translocation of the cytoplasmic glucocorticoid receptor (GR). The glucocorticoid hormone (GC) has significant role in the regulation of T-cell activation; however, the cross-talk between the GC and T-cell receptor (TcR) signal transducing pathways are still to be elucidated. We examined the rapid effects of GC exposure on in vitro cultured human T-cells. Our results showed that Dexamethasone (DX), a GC analogue, when applied at high dose (10 microM), induced rapid (within 5 min) tyrosine-phosphorylation events in Jurkat cells. Short DX pre-treatment strongly inhibited the tyrosine-phosphorylation stimulated by CD3 cross-linking. Furthermore, we also investigated the phosphorylation status of ZAP-70, an important member of tyrosine kinase mediated signalling pathway of TcR-elicited T-cell activation. Here, we demonstrate that high dose DX induced a rapid ZAP-70 tyrosine-phosphorylation in Jurkat T-cells. DX-induced ZAP-70 phosphorylation could be inhibited by RU486 (GR antagonist), suggesting that this process was GR mediated. DX-induced ZAP-70 phosphorylation did not occur in the absence of active p56-lck as examined in the p56-lck kinase-deficient Jurkat cell line JCaM1.6. Our results show that DX, at a high dose, can rapidly influence the initial tyrosine-phosphorylation events of the CD3 signalling pathway in Jurkat cells, thereby modifying TcR-derived signals. Lck and ZAP-70 represent an important molecular link between the TcR and GC signalling pathways.

Age-related changes in arthritis susceptibility and severity in a murine model of rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) most often begins in females in the fourth-fifth decade of their life, suggesting that the aging of the immune system (immunosenescence) has a major role in this disease. Therefore, in the present study, we sought to investigate the effect of age on arthritis susceptibility in BALB/c mice using the proteoglycan (PG)-induced arthritis (PGIA) model of RA.ResultsWe have found that young, 1-month-old female BALB/c mice are resistant to the induction of PGIA, but with aging they become susceptible. PG-induced T cell responses decline with age, whereas there is a shift toward Th1 cytokines. An age-dependent decrease in T cell number is associated with an increased ratio of the memory phenotype, and lower CD28 expression. Antigen-presenting cells shifted from macrophages and myeloid dendritic cells in young mice toward B cells in older mice. The regulatory/activated T cell ratio decreases in older mice after PG injections indicating impaired regulation of the immune response.ConclusionWe conclude that immunosenescence could alter arthritis susceptibility in a very complex manner including both adaptive and innate immunities, and it cannot be determined by a single trait. Cumulative alterations in immunoregulatory functions closely resemble human disease, which makes this systemic autoimmune arthritis model of RA even more valuable.

Developmental shift in TcR-mediated rescue of thymocytes from glucocorticoid-induced apoptosis

Glucocorticoid hormone (GC) production by thymic epithelial cells influences TcR signalling in DP thymocytes and modifies their survival. In the present work, we focused on exploring details of GC effects on DP thymocyte apoptosis with or without parallel TcR activation in AND transgenic mice, carrying TcR specific for pigeon cytochrome C, in vivo. Here we show that the glucocorticoid receptor (GR) protein level was the lowest in DP thymocytes, and it was slightly down-regulated by GC analogue, anti-CD3, PCC and combined treatments as well. Exogenous GC analogue treatment or TcR stimulation alone lead to marked DP cell depletion, coupled with a significant increase of early apoptotic cell ratio (AnnexinV staining), marked abrogation of the mitochondrial function in DP cells (CMXRos staining), and significant decrease in the Bcl-2(high) DP thymocyte numbers, respectively. On the other hand, the simultaneous exposure to these two proapototic signals effectively reversed all the above-described changes. The parallel analysis of CD4 SP cell numbers, AnnexinV, CMXRos, Bcl-2 and GR stainings revealed, that the GR and TcR signals were not antagonistic on the mature thymocytes. These data provide experimental evidence in TcR transgenic mice, in vivo, that when TcR activation and GR signals are present simultaneously, they rescue double positive thymocytes from programmed cell death. The two separate signalling pathways merge in DP thymocytes at such important apoptosis regulating points as the Bcl-2 and GR, showing that their balanced interplay is essential in DP cell survival.

Galactoside-specific plant lectin, Viscum album agglutinin-I induces enhanced proliferation and apoptosis of murine thymocytes in vivo

Galactoside-specific plant lectin, Viscum album agglutinin-I (VAA-I) has been shown to act as a biomodulator with proinflammatory and apoptosis-inducing effects, however its cellular targets and mechanism of immunobiological action in vivo are less well understood. Therefore, in the present work the short- and long-term in vivo effects of VAA-I on thymocyte subpopulations and peripheral T cells were tested using a murine (Balb/c) model. Cell surface CD4/CD8 staining and flow cytometry allowed us to follow the changes of thymocyte subpopulations: CD4-CD8- double negative (DN), CD4+CD8+ double positive (DP), CD4+ or CD8+ single positive (SP) and mature peripheral T cells after single or repeated injections with low doses of VAA-I. The apoptosis of the cells was detected by flow cytometry using propidium iodide (PI) and Annexin V staining. To detect the short-term effects of the lectin, the animals were investigated 24 h after a single injection of 1 or 30 ng/kg body weight (BW) VAA-I+/-1 mg/kg Dexamethasone (DX). The total number of mature CD8+ SP thymocytes increased significantly with an enhancement of the ratio of apoptotic cells. In contrast, in the blood samples an elevated CD4/CD8 ratio was found. In the next trial, Balb/c mice were treated twice weekly with 1 or 30 ng/kg VAA-I+/-1 mg/kg DX for 3 weeks. The total cell count of thymocytes showed significant increases after both doses of VAA-I, but an elevated percentage of apoptotic cells was found only after treatment with 30 ng/kg VAA-I. SP thymocytes revealed higher increases in lectin-induced apoptosis than DN or DP cells. In addition, both lectin doses significantly inhibited the DX-induced reduction of all thymocyte subpopulations investigated. In conclusion, our data suggest that VAA-I is able to modulate the maturation of thymocytes in vivo.