Hypochlorite-modified low-density lipoprotein induces the apoptotic machinery in Jurkat T-cell lines.

Myeloperoxidase is abundantly present in inflammatory diseases where activation of monocytes/macrophages and T-cell-mediated immune response occurs. The potent oxidant hypochlorous acid (HOCl), generated by the myeloperoxidase-H(2)O(2)-chloride system of activated phagocytes, converts low-density lipoprotein (LDL) into a proinflammatory lipoprotein particle. Here, we investigated the apoptotic effect of HOCl-LDL, an in vivo occurring LDL modification, on human T-cell lymphoblast-like Jurkat cells. Experiments revealed that HOCl-LDL, depending on the oxidant:lipoprotein molar ratio, induces apoptosis via activation of caspase-3, PARP cleavage and accumulation of reactive oxygen species. The absence of Fas-associated protein with death domain or caspase-8 in mutant cells did not prevent HOCl-LDL induced apoptosis. In contrast, overexpression of the anti-apoptotic Bcl-2 protein protects Jurkat cells against HOCl-LDL-induced apoptosis and prevents accumulation of reactive oxygen species. We conclude that HOCl-LDL-mediated apoptosis in Jurkat cells follows predominantly the intrinsic, mitochondrial pathway. Insitu experiments revealed that an antibody raised against HOCl-LDL recognized epitopes that colocalize both with myeloperoxidase and CD3-positive T-cells in human decidual tissue where local stimulation of the immune system occurs. We provide convincing evidence that formation of HOCl-modified (lipo)proteins generated by the myeloperoxidase-H(2)O(2)-chloride system contributes to apoptosis in T-cells.

Hypochlorite-modified albumin colocalizes with RAGE in the artery wall and promotes MCP-1 expression via the RAGE-Erk1/2 MAP-kinase pathway.

Signal transduction via the endothelial receptor for advanced glycation end products (RAGE) plays a key role in vascular inflammation. Recent observations have shown that the myeloperoxidase-H2O2-chloride system of activated phagocytes is highly up-regulated under inflammatory conditions where hypochlorous acid (HOCl) is formed as the major oxidant. Albumin, an in vivo carrier for myeloperoxidase is highly vulnerable to oxidation and a major representative of circulating advanced oxidized proteins during inflammatory diseases. Immunohistochemical studies performed in the present study revealed marked colocalization of HOCl-modified epitopes with RAGE and albumin in sections of human atheroma, mainly at the endothelial lining. We show that albumin modified with physiologically relevant concentrations of HOCl, added as reagent or generated by the myeloperoxidase-H2O2-chloride system, is a high affinity ligand for RAGE. Albumin, modified by HOCl in the absence of free amino acids/carbohydrates/lipids to exclude formation of AGE-like structures, induced a rapid, RAGE-dependent activation of extracellular signal-regulated kinase 1/2 and up-regulation of the proinflammatory mediator monocyte chemoattractant protein-1. Cellular activation could be blocked either by a specific polyclonal anti-RAGE IgG and/or a specific mitogen-activated protein-kinase kinase inhibitor. The present study demonstrates that HOCl-modified albumin acts as a ligand for RAGE and promotes RAGE-mediated inflammatory complications.

Expression of indoleamine 2,3-dioxygenase in carcinoma of human endometrium and uterine cervix.

Expression of indoleamine 2,3-dioxygenase (IDO) in epithelium of the endometrium and the cervix is not restricted to normal but also present in carcinomatous tissue. The enzyme was found in the majority of cases studied, pioneer cells at the invasion front of the tumors being especially strongly reactive in immunohistology. In addition, also cells in the peritumoral infiltrate of the stroma expressed IDO. Taken together, these findings together with previous data on the immunosuppressive impact of tryptophan depletion suggest IDO-induced suppression of antitumoral immune response in both adenocarcinoma and squamous cell carcinoma of endometrium and cervix. On the other hand, IDO as also known to inhibit tumor cell proliferation by tryptophan depletion.

LPA-induced suppression of periostin in human osteosarcoma cells is mediated by the LPA(1)/Egr-1 axis.

Lysophosphatidic acid (LPA), a naturally occurring bioactive phospholipid, mediates a multitude of (patho)physiological events including activation of mitogen-activated protein kinases (MAPKs). As LPA may induce cellular reponses in human osteosarcoma, the present study aimed at investigating expression of various LPA receptors, LPA-mediated activation of MAPK via G-protein coupling, and expression of early response genes in a cellular model for human osteosarcoma. We show that MG-63 cells express three members of the endothelial differentiation gene (Edg) family of G-protein coupled receptor transcripts (LPA(1-3)) but only two (LPA(4/5)) out of three members of the non-Edg family LPA receptor transcripts. Stimulation of MG-63 cells with LPA or synthetic LPA receptor agonists resulted in p42/44 MAPK phosphorylation via LPA(1)-LPA(3) receptors. Using pharmacological inhibitors, we show that LPA-mediated phosphorylation of p42/44 MAPK by LPA receptor engagement is transmitted by G(αi)-dependent pathways through the Src family of tyrosine kinases. As a consequence, a rapid and transient upregulation of the zinc finger transcription factor early growth response-1 (Egr-1) was observed. Egr-1 expression was strictly mediated via G(αi)/Src/p42/44 MAPK pathway; no involvement of the G(αq/11)/PLC/PKC or the PLD/PI3 kinase/Akt pathways was found. LPA-induced expression of functional Egr-1 in MG-63 cells could be confirmed by electrophoretic mobility shift assay. LPA-induced Egr-1 upregulation was accompanied by a time-dependent decrease of periostin (previously called osteoblast-specific factor 2), a cell adhesion protein for pre-osteoblasts. Silencing of LPA(1) and/or Egr-1 in MG-63 cells reversed LPA-mediated suppression of periostin. We here demonstrate a crosslink between Egr-1 and periostin in cancer cells, in particular in human osteosarcoma.

ATM protects against oxidative stress induced by oxidized low-density lipoprotein.

Chronic oxidative stress is involved in the pathogenesis of multiple inflammatory diseases, including cardiovascular disease and atherosclerosis. The rare autosomal recessive disorder Ataxia-telangiectasia (A-T) is characterized by progressive cerebellar ataxia secondary to Purkinje cell death, immunodeficiency, and increased cancer incidence. ATM, the protein mutated in A-T, plays a key role in cellular DNA-damage responses. A-T cells show poor cellular anti-oxidant defences and increased oxidant sensitivity compared to normal cells, and ATM functions, in part, as an oxidative stress sensor. The oxidation of low-density lipoprotein (oxLDL) and its uptake by macrophages is an initiating step in the development of atherosclerosis. We demonstrate that oxLDL activates ATM and downstream p21 expression in normal fibroblasts and endothelial cells. In ATM-deficient fibroblasts oxLDL induces DNA double-strand breaks, micronuclei formation and causes chromosome breaks. Furthermore, oxLDL decreases cell viability and inhibits colony formation in A-T fibroblasts more effectively as compared to normal controls. Formation of oxLDL-induced reactive oxygen species is significantly higher in A-T, than normal fibroblasts. Last, pre-treatment of cells with ammonium pyrrolidine dithiocarbamate, a potent antioxidant and inhibitor of transcription factor nuclear factor κB, reduces oxLDL-induced reactive oxygen species formation. Our data indicates that ATM functions in the defence against oxLDL-mediated cytotoxicity.