Zsolt Sarang

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.

PPARγ‐dependent regulation of human macrophages in phagocytosis of apoptotic cells

Macrophages acquire their capacity for efficient phagocytosis of apoptotic cells during their differentiation from monocytes. The peroxisome proliferator‐activated receptor gamma (PPARγ) is highly up‐regulated during this maturation program. We report that addition of PPARγ antagonist during differentiation of human monocytes to macrophages significantly reduced the capacity of macrophages to engulf apoptotic neutrophils, but did not influence phagocytosis of opsonized bacteria. Macrophage‐specific deletion of PPARγ in mice also resulted in decreased uptake of apoptotic cells. The antagonist acted in a dose‐dependent manner during the differentiation of human macrophages and could also reverse the previously observed augmentation of phagocytosis by glucocorticoids. Blocking activation of PPARγ led to down‐regulation of molecular elements (CD36, AXL, TG2 and PTX3) of the engulfment process. Inhibition of PPARγ‐dependent gene expression did not block the anti‐inflammatory effect of apoptotic neutrophils or synthetic glucocorticoid, but significantly decreased production of IL‐10 induced by LPS. Our results suggest that during differentiation of macrophages natural ligands of PPARγ are formed, regulating the expression of genes responsible for effective clearance of apoptotic cells and macrophage‐mediated inflammatory responses.

Involvement of Adenosine A2A Receptors in Engulfment-Dependent Apoptotic Cell Suppression of Inflammation

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 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 A2A 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.

Tissue transglutaminase (TG2) acting as G protein protects hepatocytes against Fas‐mediated cell death in mice

Tissue transglutaminase (TG2) is a protein cross‐linking enzyme known to be expressed by hepatocytes and to be induced during the in vivo hepatic apoptosis program. TG2 is also a G protein that mediates intracellular signaling by the alpha‐1b‐adrenergic receptor (AR) in liver cells. Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti‐Fas antibodies to mice causes both disseminated endothelial cell apoptosis and fulminant hepatic failure. Here we report that an intraperitoneal dose of anti‐Fas antibodies, which is sublethal for wild‐type mice, kills all the TG2 knock‐out mice within 20 hours. Although TG2−/− thymocytes exposed to anti‐Fas antibodies die at the same rate as wild‐type mice, TG2−/− hepatocytes show increased sensitivity toward anti‐Fas treatment both in vivo and in vitro, with no change in their cell surface expression of Fas, levels of FLIPL (FLICE‐inhibitory protein), or the rate of I‐κBα degradation, but a decrease in the Bcl‐xL expression. We provide evidence that this is the consequence of the impaired AR signaling that normally regulates the levels of Bcl‐xL in the liver. In conclusion, our data suggest the involvement of adrenergic signaling pathways in the hepatic regeneration program, in which Fas ligand‐induced hepatocyte proliferation with a simultaneous inhibition of the Fas‐death pathway plays a determinant role. (HEPATOLOGY 2005.)

Transcript profiling of enzymes involved in detoxification of xenobiotics and reactive oxygen in human normal and simian virus 40 T antigen‐immortalized oral keratinocytes

The metabolic detoxification capacity may critically regulate the susceptibility of human tissues to cancer development. We used standardized and quantitative, reverse transcription‐polymerase chain reaction (StaRT‐PCR) and microarray chip techniques to analyze transcript levels of multiple detoxification enzymes in cultured normal human oral keratinocytes (NOK) and the Siman virus 40 T antigen‐immortalized oral keratinocyte line SVpgC2a, viewing the latter as a model of a benign tumor state. With good agreement between the 2 methodologies, NOK and SVpgC2a were found to express transcripts for cytochrome P450 enzymes (CYPs), factors related to CYP induction and enzymes involved in conjugation reactions or detoxification of reactive oxygen. The cell types expressed similar levels of CYP 2B6/7, CYP 2E1, P450 oxidoreductase, the aryl hydrocarbon receptor nuclear translocator, sulfotransferase 1A1, sulfotransferase 1A3, epoxide hydrolase, glutathione S‐transferase M3, glutathione S‐transferase pi and catalase, superoxide dismutase 1, glutathione peroxidase 1 and glutathione peroxidase 3. In contrast, SVpgC2a exhibited comparatively higher levels of CYP1A1, 1B1, aryl hydrocarbon receptor, glutathione S‐transferase M1, 2, 4, 5, glutathione S‐transferase theta 1 and superoxide dismutase 2 and comparatively lower levels of UDP glycosyltransferase 2 and microsomal glutathione S‐transferase 1. Some transcripts, e.g., CYP 2A6/7, were not detected by either standard, non quantitative RT‐PCR or the above methods, whereas others were barely quantifiable by StaRT‐PCR, i.e., were present at 1–10 molecules/106 molecules of actin. Overall, the expression analysis demonstrated presence of mRNA for multiple enzymes involved in foreign compound metabolism and detoxification pathways, including several enzymes not previously reported for oral epithelium. Generally, the comparison of NOK from 2 individuals indicated relatively similar transcript levels of these enzymes. In contrast, differences between NOK and SVpgC2a, e.g., for CYP1B1, may reflect alteration caused by immortalization and aid identification of early stage tumor markers in oral epithelium.

Impaired clearance of apoptotic cells in chronic inflammatory diseases: therapeutic implications

In healthy individuals, billions of cells die by apoptosis every day. Removal of the dead cells by phagocytosis (a process called efferocytosis) must be efficient to prevent secondary necrosis and the consequent release of pro-inflammatory cell contents that damages the tissue environment and provokes autoimmunity. In addition, detection and removal of apoptotic cells generally induces an anti-inflammatory response. As a consequence improper clearance of apoptotic cells, being the result of either genetic anomalies and/or a persistent disease state, contributes to the establishment and progression of a number of human chronic inflammatory diseases such as autoimmune and neurological disorders, inflammatory lung diseases, obesity, type 2 diabetes, or atherosclerosis. During the past decade, our knowledge about the mechanism of efferocytosis has significantly increased, providing therapeutic targets through which impaired phagocytosis of apoptotic cells and the consequent inflammation could be influenced in these diseases.

Transglutaminase 2 is expressed and active on the surface of human monocyte-derived dendritic cells and macrophages

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-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 catalytically 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.

Some lessons from the tissue transglutaminase knockout mouse.

Transglutaminase 2 (TG2) is an inducible transamidating acyltransferase that catalyzes Ca2+-dependent protein modifications. It acts as a G protein in transmembrane signaling and as a cell surface adhesion mediator, this distinguishes it from other members of the transglutaminase family. The sequence motifs and domains revealed in the TG2 structure, can each be assigned distinct cellular functions, including the regulation of cytoskeleton, cell adhesion, and cell death. Though many biological functions of the enzyme have already been described or proposed previously, studies of TG2 null mice by our laboratory during the past years revealed several novel in vivo roles of the protein. In this review we will discuss these novel roles in their biological context.

Tissue transglutaminase (TG2) facilitates phosphatidylserine exposure and calpain activity in calcium-induced death of erythrocytes

Tissue transglutaminase (TG2) facilitates phosphatidylserine exposure and calpain activity in calcium-induced death of erythrocytes

Retinoids enhance glucocorticoid-induced apoptosis of T cells by facilitating glucocorticoid receptor-mediated transcription

Glucocorticoid-induced apoptosis of thymocytes is one of the first recognized forms of programmed cell death. It was shown to require gene activation induced by the glucocorticoid receptor (GR) translocated into the nucleus following ligand binding. In addition, the necessity of the glucocorticoid-induced, but transcription-independent phosphorylation of phosphatidylinositol-specific phospholipase C (PI-PLC) has also been shown. Here we report that retinoic acids, physiological ligands for the nuclear retinoid receptors, enhance glucocorticoid-induced death of mouse thymocytes both in vitro and in vivo. The effect is mediated by retinoic acid receptor (RAR) alpha/retinoid X receptor (RXR) heterodimers, and occurs when both RARα and RXR are ligated by retinoic acids. We show that the ligated RARα/RXR interacts with the ligated GR, resulting in an enhanced transcriptional activity of the GR. The mechanism through which this interaction promotes GR-mediated transcription does not require DNA binding of the retinoid receptors and does not alter the phosphorylation status of Ser232, known to regulate the transcriptional activity of GR. Phosphorylation of PI-PLC was not affected. Besides thymocytes, retinoids also promoted glucocorticoid-induced apoptosis of various T-cell lines, suggesting that they could be used in the therapy of glucocorticoid-sensitive T-cell malignancies.