László Fésüs

Publications of László Fésüs

• The glucocorticoid dexamethasone programs human dendritic cells for enhanced phagocytosis of apoptotic neutrophils and inflammatory response.

  Authors: Judit Hodrea, Gyöngyike Majai, Zoltán Doró, Gábor Zahuczky, Attila Pap, Éva Rajnavölgyi, László Fésüs

  Journal of leukocyte biology. 01/2012; 91(1):127-36.

  GCs are powerful anti-inflammatory compounds inhibiting inflammatory cell recruitment and production of proinflammatory cytokines. We have recently found that DCs, the key players of T cell primingGCs are powerful anti-inflammatory compounds inhibiting inflammatory cell recruitment and production of proinflammatory cytokines. We have recently found that DCs, the key players of T cell priming and polarization, respond to allogeneic apoptotic neutrophils with proinflammatory cytokine release and Th1 cell activation. Here, we show that monocyte-derived human DCs develop their capacity to engulf apoptotic cells by up-regulating a set of apoptophagocytic genes. This gene expression pattern was reprogrammed when differentiation took place in the presence of the synthetic GC Dex, which increased the expression of phagocytosis receptors MERTK and CD14, the bridging molecule C1QA, DNASE2, and ADORA3. The increased phagocytosis was attenuated by the addition of ADORA3 antagonist and could not be observed when bone marrow-derived DCs of ADORA3 KO mice were treated with Dex. The GC-treated human DCs loaded with allogeneic apoptotic neutrophils secreted, in response to LPS and IFN-γ, the inflammatory cytokine TNF-α. Furthermore, the Dex-treated DCs could activate autologous T lymphocytes toward Th1 effector cells, and this was enhanced by their exposure to allogeneic apoptotic neutrophils.

• A single conformational transglutaminase 2 epitope contributed by three domains is critical for celiac antibody binding and effects.

  Authors: Zsófia Simon-Vecsei, Róbert Király, Péter Bagossi, Boglárka Tóth, Ingrid Dahlbom, Sergio Caja, Éva Csosz, Katri Lindfors, Daniele Sblattero, Éva Nemes, Markku Mäki, László Fésüs, Ilma R Korponay-Szabó

  Proceedings of the National Academy of Sciences of the United States of America. 12/2011; 109(2):431-6.

  The multifunctional, protein cross-linking transglutaminase 2 (TG2) is the main autoantigen in celiac disease, an autoimmune disorder with defined etiology. Glutamine-rich gliadin peptides fromThe multifunctional, protein cross-linking transglutaminase 2 (TG2) is the main autoantigen in celiac disease, an autoimmune disorder with defined etiology. Glutamine-rich gliadin peptides from ingested cereals, after their deamidation by TG2, induce T-lymphocyte activation accompanied by autoantibody production against TG2 in 1-2% of the population. The pathogenic role and exact binding properties of these antibodies to TG2 are still unclear. Here we show that antibodies from different celiac patients target the same conformational TG2 epitope formed by spatially close amino acids of adjacent domains. Glu153 and 154 on the first alpha-helix of the core domain and Arg19 on first alpha-helix of the N-terminal domain determine the celiac epitope that is accessible both in the closed and open conformation of TG2 and dependent on the relative position of these helices. Met659 on the C-terminal domain also can cooperate in antibody binding. This composite epitope is disease-specific, recognized by antibodies derived from celiac tissues and associated with biological effects when passively transferred from celiac mothers into their newborns. These findings suggest that celiac antibodies are produced in a surface-specific way for which certain homology of the central glutamic acid residues of the TG2 epitope with deamidated gliadin peptides could be a structural basis. Monoclonal mouse antibodies with partially overlapping epitope specificity released celiac antibodies from patient tissues and antagonized their harmful effects in cell culture experiments. Such antibodies or similar specific competitors will be useful in further functional studies and in exploring whether interference with celiac antibody actions leads to therapeutic benefits.

• Polymorphism of transglutaminase 2: unusually low frequency of genomic variants with deficient functions.

  Authors: Róbert Király, Endre Barta, László Fésüs

  Amino acids. 12/2011;

  Transglutaminase 2 (TG2) is a multifunctional member of an enzyme family: it modifies glutamine residues by cross-linking proteins and incorporating primary amines into them, has protein disulphideTransglutaminase 2 (TG2) is a multifunctional member of an enzyme family: it modifies glutamine residues by cross-linking proteins and incorporating primary amines into them, has protein disulphide isomerase and protein kinase activities, mediates trans-membrane signal transduction and interactions between cell surface proteins and the extracellular matrix. These unusual multiple roles encoded into one polypeptide chain suggest that genomic variations in the TGM2 gene should be limited. Indeed, the available information in databases shows that unlike in the case of most other transglutaminases there are no common single nucleotide polymorphisms in exons of human TGM2. We collected data on and produced some of the rare genetic variants of TGM2 by site directed mutagenesis and found that some were less stable than the most abundant (wild type) enzyme variant and the majority had deficient transamidating activity. Further studies are required to clarify the pathologic significance of these rare TGM2 alleles in the human population.

• Cellular biochemistry of the multifunctional transglutaminase 2: challenging issues and novel concepts.

  Authors: László Fésüs

  The FEBS journal. 12/2011; 278(24):4703.

  The transglutaminase TG2 can do a lot more than just transamidation: it has multiple biochemical activities, appears in various cell compartments, and has been implicated in a broad range of cellularThe transglutaminase TG2 can do a lot more than just transamidation: it has multiple biochemical activities, appears in various cell compartments, and has been implicated in a broad range of cellular processes and pathologies. Decades after its discovery, this enigmatic protein still intrigues researchers. Some of the most recent and compelling findings concerning TG2 are reviewed in four minireivews in this issue of FEBS Journal.

• Retinoids produced by macrophages engulfing apoptotic cells contribute to the appearance of transglutaminase 2 in apoptotic thymocytes.

  Authors: Eva Garabuczi, Beáta Kiss, Szabolcs Felszeghy, Gregory J Tsay, László Fésüs, Zsuzsa Szondy

  Amino acids. 10/2011;

  Transglutaminase 2 (TG2) has been known for a long time to be associated with the in vivo apoptosis program of various cell types including T cells. Though the expression of the enzyme was stronglyTransglutaminase 2 (TG2) has been known for a long time to be associated with the in vivo apoptosis program of various cell types including T cells. Though the expression of the enzyme was strongly induced in mouse thymocytes following apoptosis induction in vivo, no significant induction of TG2 could be detected, when thymocytes were induced to die by the same stimuli in vitro indicating that signals arriving from the tissue environment are required for the in vivo induction of the enzyme in apoptotic thymocytes. Previous studies have shown that one of these signals is transforming growth factor-β (TGF-β) which is released by macrophages engulfing apoptotic cells. Besides TGF-β, the TG2 promoter contains retinoic acid response elements as well. Here we show that in vitro retinoic acids, or TGF-β and retinoic acids together can significantly enhance the TG2 mRNA expression in dying thymocytes, and the apoptotic signal contributes to the TG2 induction. Inhibition of retinoic acid synthesis either by alcohol or retinaldehyde dehydrogenases significantly attenuates the in vivo induction of TG2 following apoptosis induction indicating that retinoids indeed might contribute in vivo to the apoptosis-related TG2 expression. What is more, the in vivo apoptosis induction in the thymus is accompanied by an enhanced retinoid-dependent transcriptional activity due to the enhanced retinoid synthesis by macrophages engulfing apoptotic cells. Our data reveal a new crosstalk between macrophages and apoptotic cells, in which apoptotic cell uptake-induced retinoid synthesis in macrophages enhances TG2 expression in the dying thymocytes.

• Protein transamidation by transglutaminase 2 in cells: a disputed Ca2+-dependent action of a multifunctional protein.

  Authors: Róbert Király, Mátéá Demény, László Fésüs

  The FEBS journal. 09/2011; 278(24):4717-39.

  Transglutaminase 2 (TG2) is the first described cellular member of an enzyme family catalyzing Ca(2+)-dependent transamidation of proteins. During the last two decades its additional enzymatic (GTPTransglutaminase 2 (TG2) is the first described cellular member of an enzyme family catalyzing Ca(2+)-dependent transamidation of proteins. During the last two decades its additional enzymatic (GTP binding and hydrolysis, protein disulfide isomerase, protein kinase) and non-enzymatic (multiple interactions in protein scaffolds) activities, which do not require Ca(2+) , have been recognized. It became a prevailing view that TG2 is silent as a transamidase, except in extreme stress conditions, in the intracellular environment characterized by low Ca(2+) and high GTP concentrations. To counter this presumption a critical review of the experimental evidence supporting the role of this enzymatic activity in cellular processes is provided. It includes the structural basis of TG2 regulation through non-canonical Ca(2+) binding sites, mechanisms making it sensitive to low Ca(2+) concentrations, techniques developed for the detection of protein transamidation in cells and examples of basic cellular phenomena as well as pathological conditions influenced by this irreversible post-translational protein modification.

• Involvement of adenosine A2A receptors in engulfment-dependent apoptotic cell suppression of inflammation.

  Authors: Krisztina Köröskényi, Edina Duró, Anna Pallai, Zsolt Sarang, Doris Kloor, David S Ucker, Susana Beceiro, Antonio Castrillo, Ajay Chawla, Catherine A Ledent, László Fésüs, Zsuzsa Szondy

  Journal of immunology (Baltimore, Md. : 1950). 06/2011; 186(12):7144-55.

  Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cellsEfficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble "find me" signals, 2) recognition and phagocytosis via cell surface-presenting "eat me" signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A(2A) receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.

• In vitro and in vivo activity of 4-thio-uridylate against JY cells, a model for human acute lymphoid leukemia.

  Authors: Erika Berényi, Ilona Benko, György Vámosi, Krisztina Géresi, Ilona Tárkányi, István Szegedi, Levente Lukács, István Juhász, Csongor Kiss, László Fésüs, János Aradi

  Biochemical and biophysical research communications. 06/2011; 410(3):682-7.

  We have previously reported the in vitro anti-proliferative effect of 4-thio-uridylate (s(4)UMP) on OCM-1 uveal melanoma cells. Here, we assessed the efficacy of s(4)UMP on JY cells. Treatment of JYWe have previously reported the in vitro anti-proliferative effect of 4-thio-uridylate (s(4)UMP) on OCM-1 uveal melanoma cells. Here, we assessed the efficacy of s(4)UMP on JY cells. Treatment of JY cells with s(4)UMP suppressed their colony forming activity and induced apoptosis; healthy human bone marrow granulocyte-macrophage progenitor cells were 14-fold less sensitive to the nucleotide. In vivo effectiveness of s(4)UMP was determined using xenograft SCID mouse model. s(4)UMP decreased the cell number and colony forming activity of the total cell content of the femur of SCID mice transplanted with JY cells without affecting the bone marrow of healthy mice. These results suggest that s(4)UMP alone or in combination with other clinically approved anti-leukemic remedies should be further explored as a potential novel therapeutic agent.

• Autophagy shapes inflammation.

  Authors: László Fésüs, Máté Á Demény, Goran Petrovski

  Antioxidants & redox signaling. 06/2011; 14(11):2233-43.

  Autophagy is a basic cell biological process ongoing under physiologic circumstances in almost all cell types of the human organism and upregulated by various stress conditions including thoseAutophagy is a basic cell biological process ongoing under physiologic circumstances in almost all cell types of the human organism and upregulated by various stress conditions including those leading to inflammation. Since autophagy affects the effector cells of innate and adaptive immunity mediating the inflammatory response, its activity in these cells influences the antimicrobial response, the development of an effective cognate immune defense, and the course of the normal sterile inflammatory reactions. The level of autophagic activity may determine whether tissue cells die by apoptosis, necrosis, or through autophagy, and, as a consequence, whether the clearance of these dying cells is a silent process or results in an inflammatory response. Loss or decreased autophagy may lead to necrotic death that can initiate an inflammatory reaction in phagocytes through their surface and cytosolic receptors. Engulfment of certain cells dying through autophagy can activate the inflammasome. The intertwining regulatory connections between inflammation and immunity extend to pathologic conditions including chronic inflammatory diseases, autoimmunity and cancer.

• Phagocytosis of cells dying through autophagy induces inflammasome activation and IL-1β release in human macrophages.

  Authors: Goran Petrovski, Gizem Ayna, Gyöngyike Majai, Judit Hodrea, Szilvia Benko, András Mádi, László Fésüs

  Autophagy. 03/2011; 7(3):321-30.

  Phagocytosis of naturally dying cells usually blocks inflammatory reactions in host cells. We have recently observed that clearance of cells dying through autophagy leads to a pro-inflammatoryPhagocytosis of naturally dying cells usually blocks inflammatory reactions in host cells. We have recently observed that clearance of cells dying through autophagy leads to a pro-inflammatory response in human macrophages. Investigating this response further, we found that during engulfment of MCF-7 or 293T cells undergoing autophagic death, but not apoptotic or anoikic ones, caspase-1 was activated and IL-1β was processed, then secreted in a MyD88-independent manner. Autophagic dying cells were capable of preventing some LPS-induced pro-inflammatory responses, such as TNFα, IL-6 and IL-8 induction, but synergized with LPS for IL-1β production. Caspase-1 inhibition prevented macrophage IL-1β release triggered by the dying cells and also other pro-inflammatory cytokines which were not formed in the presence of IL-1 receptor antagonist anakinra either. IL-1β secretion was also observed using calreticulin knock down or necrostatin treated autophagic MCF-7 cells and it required phagocytosis of the dying cells which led to ATP secretion from macrophages. Blocking K (+) efflux during phagocytosis, the presence of apyrase, adding an antagonist of the P2X7 receptor or silencing the NOD-like receptor protein NALP3 inhibited IL-1β secretion. These data suggest that during phagocytosis of autophagic dying cells ATP, acting through its receptor, initiates K (+) efflux, inflammasome activation and secretion of IL-1β, which initiates further pro-inflammatory events. Thus, autophagic death of malignant cells and their clearance may lead to immunogenic response.

• Transglutaminase 2 null macrophages respond to lipopolysaccharide stimulation by elevated proinflammatory cytokine production due to an enhanced αvβ3 integrin-induced Src tyrosine kinase signaling.

  Authors: Zsolt Sarang, Krisztina Köröskényi, Anna Pallai, Edina Duró, Gerry Melino, Martin Griffin, László Fésüs, Zsuzsa Szondy

  Immunology letters. 03/2011; 138(1):71-8.

  Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and alteredTransglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin β(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFβ activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFα production. Though TGFβ has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFβ production. Instead, in the absence of TG2 integrin β(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the IκBα. Low basal levels of IκBα explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-κB. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.

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• Clearance of dying ARPE-19 cells by professional and nonprofessional phagocytes in vitro- implications for age-related macular degeneration (AMD).

  Authors: Goran Petrovski, Erika Berényi, Morten C Moe, Attila Vajas, László Fésüs, András Berta, Andrea Facskó

  Acta ophthalmologica. 02/2011; 89(1):e30-4.

  Failure of retinal pigment epithelial (RPE) cells and macrophages to engulf different dying cells in the retina may result in accumulation of debris and development of age-related macularFailure of retinal pigment epithelial (RPE) cells and macrophages to engulf different dying cells in the retina may result in accumulation of debris and development of age-related macular degeneration (AMD). The dynamics and influence of different treatments on this clearance process can be studied in vitro using human ARPE-19 cells and macrophages as phagocytes modelling dry and wet type of AMD, respectively. Death through extracellular matrix detachment using polyHEMA-coated surfaces (anoikis) and UV irradiation (apoptosis) was induced in ARPE-19 cells. Two-coloured phagocytic assays were performed to quantify the amount of dying cells phagocytes engulfed (flow cytometry) and for visualization (fluorescent and scanning electron microscopy). The effect of phosphatidylserine inhibition with recombinant annexin-V and glucocorticoid (triamcinolone) treatment on the phagocytic process was tested. The clearance of anoikic and apoptotic cells by nondying ARPE-19 cells over 8 hr of co-incubation increased over time (at 8 hr, over 53% and 35% of the phagocytes contained engulfed dying cells, respectively). The human macrophages engulfed the anoikic and apoptotic ARPE-19 cells with seven and four times lower capacity, respectively. Phosphatidylserine appearance on the dying cells did not affect, but triamcinolone treatment enhanced the phagocytosis of the dying cells by macrophages. ARPE-19 cells are more efficient in clearing anoikic than UV-induced apoptotic cells. Macrophages are less efficient in the clearance process than ARPE-19 cells. The present model can be used for studying both dry and wet type of AMD in vitro and for testing different pharmacological aspects affecting this disease.

• Transglutaminase 2 dysfunctions in the development of autoimmune disorders: celiac disease and TG2-/- mouse.

  Authors: Zsuzsa Szondy, Ilma Korponay-Szabó, Róbert Király, László Fésüs

  Advances in enzymology and related areas of molecular biology. 01/2011; 78:295-345.

• Differentiation and glucocorticoid regulated apopto-phagocytic gene expression patterns in human macrophages. Role of Mertk in enhanced phagocytosis.

  Authors: Gábor Zahuczky, Endre Kristóf, Gyöngyike Majai, László Fésüs

  PloS one. 01/2011; 6(6):e21349.

  The daily clearance of physiologically dying cells is performed safely mainly by cells in the mononuclear phagocyte system. They can recognize and engulf dying cells utilizing several cooperativeThe daily clearance of physiologically dying cells is performed safely mainly by cells in the mononuclear phagocyte system. They can recognize and engulf dying cells utilizing several cooperative mechanisms. In our study we show that the expression of a broad range of apopto-phagocytic genes is strongly up-regulated during differentiation of human monocytes to macrophages with different donor variability. The glucocorticoid dexamethasone has a profound effect on this process by selectively up-regulating six genes and down-regulating several others. The key role of the up-regulated mer tyrosine kinase (Mertk) in dexamethasone induced enhancement of phagocytosis could be demonstrated in human monocyte derived macrophages by gene silencing as well as blocking antibodies, and also in a monocyte-macrophage like cell line. However, the additional role of other glucocorticoid induced elements must be also considered since the presence of autologous serum during phagocytosis could almost completely compensate for the blocked function of Mertk.

• Tissue transglutaminase contributes to the all-trans-retinoic acid-induced differentiation syndrome phenotype in the NB4 model of acute promyelocytic leukemia.

  Authors: Krisztián Csomós, István Német, László Fésüs, Zoltán Balajthy

  Blood. 11/2010; 116(19):3933-43.

  Treatment of acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA) results in terminal differentiation of leukemic cells toward neutrophil granulocytes. Administration of ATRA leadsTreatment of acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA) results in terminal differentiation of leukemic cells toward neutrophil granulocytes. Administration of ATRA leads to massive changes in gene expression, including down-regulation of cell proliferation-related genes and induction of genes involved in immune function. One of the most induced genes in APL NB4 cells is transglutaminase 2 (TG2). RNA interference-mediated stable silencing of TG2 in NB4 cells (TG2-KD NB4) coupled with whole genome microarray analysis revealed that TG2 is involved in the expression of a large number of ATRA-regulated genes. The affected genes participate in granulocyte functions, and their silencing lead to reduced adhesive, migratory, and phagocytic capacity of neutrophils and less superoxide production. The expression of genes related to cell-cycle control also changed, suggesting that TG2 regulates myeloid cell differentiation. CC chemokines CCL2, CCL3, CCL22, CCL24, and cytokines IL1B and IL8 involved in the development of differentiation syndrome are expressed at significantly lower level in TG2-KD NB4 than in wild-type NB4 cells upon ATRA treatment. Based on our results, we propose that reduced expression of TG2 in differentiating APL cells may suppress effector functions of neutrophil granulocytes and attenuate the ATRA-induced inflammatory phenotype of differentiation syndrome.

• PPARγ modulated inflammatory response of human dendritic cell subsets to engulfed apoptotic neutrophils.

  Authors: Gyöngyike Majai, Péter Gogolák, Csilla Ambrus, György Vereb, Judit Hodrea, László Fésüs, Eva Rajnavölgyi

  Journal of leukocyte biology. 11/2010; 88(5):981-91.

  The means of how phagocytes handle apoptotic cells has a great impact on the outcome of immune responses. Here, we show that phagocytosis of allogeneic, apoptotic neutrophils by humanThe means of how phagocytes handle apoptotic cells has a great impact on the outcome of immune responses. Here, we show that phagocytosis of allogeneic, apoptotic neutrophils by human monocyte-derived DCs is slow and less efficient than that of macrophages, and CD1a(-) DCs are more active in the engulfment of apoptotic neutrophils than CD1a(+) DCs. Blocking DC-SIGN function partially interferes with the uptake of apoptotic cells, and long-term interaction of apoptotic neutrophils with DCs makes them prone to proinflammatory cytokine responses. Engulfment of apoptotic cells sensitizes CD1a(-) DCs for high IL-8, TNF-α, IL-6, and CD1a(+) cells for IL-12 and IL-10 cytokine secretion elicited by additional inflammatory stimuli, which also result in the polarization of autologous T lymphocytes to Th1 effector cells. Ligand-induced activation of PPARγ by RSG results in enhanced phagocytosis, but the proinflammatory response and the capacity to trigger Th1 cell activation of CD1a(-) DCs are not enhanced. These results demonstrate that DCs are able to respond to allogeneic, apoptotic neutrophils with inflammatory cytokines and T cell responses in a subtype-specific manner that is modulated by the anti-inflammatory effects of PPARγ.

• Altered sialylation on the cell-surface proteins of dexamethasone-treated human macrophages contributes to augmented uptake of apoptotic neutrophils.

  Authors: András Mádi, Gyöngyike Majai, Cornelia Koy, György Vámosi, Attila Szántó, Michael O Glocker, László Fésüs

  Immunology letters. 10/2010; 135(1-2):88-95.

  Macrophages eliminate apoptotic granulocytes before their secondary necrosis during resolution of inflammation. A well-known glucocorticoid, the anti-inflammatory dexamethasone augments phagocytosisMacrophages eliminate apoptotic granulocytes before their secondary necrosis during resolution of inflammation. A well-known glucocorticoid, the anti-inflammatory dexamethasone augments phagocytosis capacity of macrophages with a so far not fully clarified mechanism. We have found that sialylation of cell-surface proteins on human macrophages is markedly altered by dexamethasone. Compared to non-treated cells, dexamethasone-treated macrophages can bind significantly less Sambucus nigra lectin specific for sialic acids on their surfaces as a result of undersialylation of annexin-II and an HLA-II protein. Non-treated macrophages covered by S. nigra lectin had increased uptake of apoptotic cells; however, the significantly higher phagocytosis capacity of dexamethasone-treated macrophages could not be stimulated further this way. Our results suggest that dexamethasone treatment leads to decreased number of sialic acids on the surfaces of human macrophages promoting recognition and uptake of apoptotic cells.

• Transglutaminase 2 is Expressed and Active on the Surface of Human Monocyte-derived Dendritic Cells and Macrophages.

  Authors: Judit Hodrea, Máté Á Demény, Gyöngyike Majai, Zsolt Sarang, Ilma Rita Korponay- Szabób, László Fésüs

  Immunology letters. 12/2009;

  The multifunctional enzyme, transglutaminase 2 (TG2), can be found intracellularly, in the extracellular matrix and on the cell surface. Cell surface TG2 (csTG2) could not be detected by TG2-specificThe multifunctional enzyme, transglutaminase 2 (TG2), can be found intracellularly, in the extracellular matrix and on the cell surface. Cell surface TG2 (csTG2) could not be detected by TG2-specific antibodies or autoantibodies on immunocompetent cells. A supposedly csTG2 specific antibody, 6B9, was recently shown to actually react with CD44. Though the importance of TG2-mediated deamidation of gluten in the pathogenesis of celiac disease has been well recognized, it is not known in which intestinal cells or cell compartment the deamidation occurs. Duodenal dendritic cells (DCs) can be directly involved in gluten reactive T-cell activation. Here we use blood monocyte-derived dendritic cells (iDC) and macrophages (MPhi) as a model for intestinal antigen presenting cells (APCs) and show that they contain large amounts of TG2. We found that TG100, a commercial TG2-specific monoclonal antibody can recognize TG2 on the surface of these cells, that is monocyte-derived APCs express surface-associated TG2. TG2 expression was found on the surface of individual tunica propria cells in frozen small bowel tissue sections from both normal and celiac subjects. We also demonstrate that the pool of TG2 on the surface of iDCs can be catalitically active, hence it might directly be involved in the deamidation of gliadin peptides. Bacterial lipopolysaccharide (LPS) increased the level of TG2 on the surface of maturing DCs, supporting the hypothesis that an unspecific inflammatory process in the gut may expose more transglutaminase activity.

• Functional significance of five noncanonical Ca-binding sites of human transglutaminase 2 characterized by site-directed mutagenesis.

  Authors: Róbert Király, Eva Csősz, Tibor Kurtán, Sándor Antus, Krisztián Szigeti, Zsófia Simon-Vecsei, Ilma Rita Korponay-Szabó, Zsolt Keresztessy, László Fésüs

  The FEBS journal. 10/2009;

  The multifunctional tissue transglutaminase 2 (TG2) has a four-domain structure with several Ca(2+)-regulated biochemical activities, including transglutamylation and GTP hydrolysis. The structure ofThe multifunctional tissue transglutaminase 2 (TG2) has a four-domain structure with several Ca(2+)-regulated biochemical activities, including transglutamylation and GTP hydrolysis. The structure of the Ca(2+)-binding form of the human enzyme is not known, and its Ca(2+)-binding sites have not been fully characterized. By mutagenesis, we have targeted its active site Cys, three sites based on homology to Ca(2+)-binding residues of epidermal transglutaminase and factor XIIIa (S1-S3), and two regions with negative surface potentials (S4 and S5). CD spectroscopy, antibody-binding assay and GTPase activity measurements indicated that the amino acid substitutions did not cause major structural alterations. Calcium-45 equilibrium dialysis and isothermal calorimetric titration showed that both wild-type and active site-deleted enzymes (C277S) bind six Ca(2+). Each of the S1-S5 mutants binds fewer than six Ca(2+), S1 is a strong Ca(2+)-binding site, and mutation of one site resulted in the loss of more than one bound Ca(2+), suggesting cooperativity among sites. All mutants were deficient in transglutaminase activity, and GTP inhibited remnant activities. Like those of the wild-type enzyme, the GTPase activities of the mutants were inhibited by Ca(2+), except in the case of the S4 and S5 mutants, which exhibited increased activity. TG2 is the major autoantigen in celiac disease, and testing the reactivity of mutants with autoantibodies from celiac disease patients revealed that S4 strongly determines antigenicity. It can be concluded that five of the Ca(2+)-binding sites of TG2 influence its transglutaminase activity, two sites are involved in the regulation of GTPase activity, and one determines antigenicity for autoantibodies in celiac patients.

• Over-expression of integrin beta3 can partially overcome the defect of integrin beta3 signaling in transglutaminase 2 null macrophages.

  Authors: Beáta Tóth, Zsolt Sarang, György Vereb, Ailiang Zhang, Sakae Tanaka, Gerry Melino, László Fésüs, Zsuzsa Szondy

  Immunology letters. 08/2009;

  Transglutaminase 2 (TG2) is a protein crosslinking enzyme with many additional biological functions. We have previously shown that in TG2(-/-) mice the in vivo clearance of apoptotic cells isTransglutaminase 2 (TG2) is a protein crosslinking enzyme with many additional biological functions. We have previously shown that in TG2(-/-) mice the in vivo clearance of apoptotic cells is defective leading to autoimmunity. TG2 contributes to the formation of phagocytic portals by binding to both integrin beta(3), a known phagocytic receptor, and its bridging molecule, MFG-E8. In TG2 null macrophages integrin beta(3) cannot accumulate around the apoptotic cells and its signaling is impaired. In the present study we describe a subline of TG2 null mice, in which a compensatory increase in integrin beta(3) expression, which resulted alone in a high receptor concentration around the apoptotic cells without the requirement for accumulation, partially corrected the defect in integrin beta(3) signaling. Our data provide a proof for the concept that the function of TG2 is to stabilize accumulated integrin beta(3) concentration in the phagocytic cup.

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