María José Martínez-Lorenzo

Differential Secretion of Fas Ligand- or APO2 Ligand/TNF-Related Apoptosis-Inducing Ligand-Carrying Microvesicles During Activation-Induced Death of Human T Cells

Preformed Fas ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL) are stored in the cytoplasm of the human Jurkat T cell line and of normal human T cell blasts. The rapid release of these molecules in their bioactive form is involved in activation-induced cell death. In this study, we show by confocal microscopy that FasL and APO2L/TRAIL are mainly localized in lysosomal-like compartments in these cells. We show also by immunoelectron microscopy that FasL and APO2L/TRAIL are stored inside cytoplasmic compartments ∼500 nm in diameter, with characteristics of multivesicular bodies. Most of these compartments share FasL and APO2L/TRAIL, although exclusive APO2L/TRAIL labeling can be also observed in separate compartments. Upon PHA activation, the mobilization of these compartments toward the plasma membrane is evident, resulting in the secretion of the internal microvesicles loaded with FasL and APO2L/TRAIL. In the case of activation with anti-CD59 mAb, the secretion of microvesicles labeled preferentially with APO2L/TRAIL predominates. These data provide the basis of a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation and could modify the interpretation of the role of FasL and APO2L/TRAIL as effector mechanisms in physiological and pathological situations.

Involvement of APO2 ligand/TRAIL in activation- induced death of Jurkat and human peripheral blood T cells

The interaction of Fas with Fas ligand (FasL) mediates activation‐induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti‐APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)‐prestimulated Jurkat or human PBMC on non‐activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti‐APO2L and anti‐Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA‐activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.

Doxorubicin-induced apoptosis in human T-cell leukemia is mediated by caspase-3 activation in a Fas-independent way

It has recently been proposed that doxorubicin (DOX) can induce apoptosis in human T‐leukemia cells via the Fas/FasL system in an autocrine/paracrine way. We show here that treatment of Jurkat cells with either anti‐Fas antibodies, anthracyclin drugs or actinomycin D induces the activation of CPP32 (caspase‐3) and apoptosis. However, DOX treatment did not induce the expression of membrane FasL or the release of soluble FasL and co‐incubation with blocking anti‐Fas antibodies prevented Fas‐induced but not DOX‐induced apoptosis. All the morphological and biochemical signs of apoptosis induced by anti‐Fas or DOX can be prevented by Z‐VAD‐fmk, a general caspase inhibitor. DEVD‐cho, a specific inhibitor of CPP32‐like caspases which completely blocks Fas‐mediated apoptosis, prevented drug‐induced nuclear apoptosis but not cell death. We conclude that: (i) DOX‐induced apoptosis in human T‐leukemia/lymphoma is Fas‐independent and (ii) caspase‐3 is responsible of DOX‐induced nuclear apoptosis but other Z‐VAD‐sensitive caspases are implicated in cell death.

Release of preformed Fas ligand in soluble form is the major factor for activation‐induced death of Jurkat T cells

Interaction of Fas/APO‐1 (CD95) and its ligand (FasL) plays an important role in the activation‐induced cell death (AICD) of T lymphocytes. In the present work, the contribution of soluble FasL to AICD of the human T‐cell line Jurkat has been studied. Jurkat cells prestimulated with phytohaemagglutinin (PHA) induced the death of non‐activated Jurkat cells, and also of L1210Fas, but not that of Fas‐negative L1210 cells. Culture supernatants from prestimulated Jurkat cells were highly toxic to their non‐activated counterparts. Time–course analysis revealed that PHA‐stimulated Jurkat cells quickly release (less than 15 min) to the medium a toxic molecule following a biphasic pattern, with maximal cytotoxic activities at 1 hr and 7 hr after stimulation. The cytotoxic effect of those supernatants was prevented by the addition of a blocking anti‐Fas monoclonal antibody, suggesting that PHA‐stimulated Jurkat cells exert Fas‐based cytotoxicity mainly through the release of soluble FasL. The constitutive intracellular expression of FasL in non‐activated Jurkat cells and its release as a consequence of PHA activation were detected by immunostaining and immunoblotting using an anti‐FasL antibody. These data indicate that, at least in Jurkat cells, AICD is mainly mediated by the rapid release of preformed FasL in soluble form upon stimulation.

Resistance to apoptosis correlates with a highly proliferative phenotype and loss of Fas and CPP32 (caspase-3) expression in human leukemia cells

Apoptosis induced by effector cells of the immune system or by cytotoxic drugs is a main mechanism mediating the prevention or elimination of tumoral cells. For instance, the human T‐cell leukemia Jurkat is sensitive to Fas‐induced apoptosis and to activation‐induced cell death (AICD), and the promonocytic leukemia U937 is sensitive to Fas‐ and TNF‐induced apoptosis. In this work, we have analyzed the mechanisms of resistance to physiological or pharmacological apoptosis in human leukemia by generating highly proliferative (hp) sub‐lines derived from Jurkat and U937 cells. These hp sub‐lines were resistant to Fas‐ and TNF‐induced apoptosis, as well as to AICD. This was due to the complete loss of Fas and TNFR surface expression and, in the case of Jurkat‐derived sub‐lines, also of CD3, CD2 and CD59 molecules. The sub‐lines also completely lacked the expression of the apoptotic protease CPP32, present in parental cells. Moreover, these sub‐lines were no longer sensitive to doxorubicin‐induced apoptosis, which was efficiently blocked by the general caspase inhibitor Z‐VAD‐fmk in the parental cell lines. These data suggest a molecular mechanism for the development of resistance of leukemic cells to physiological and pharmacological apoptosis inducers, giving rise to highly proliferative tumoral phenotypes. These results also indicate that Fas and CPP32 could be useful prognostic markers for the progression and/or therapy outcome of human leukemias. Int. J. Cancer 75:473–481, 1998.

Phenotype and chondrogenic differentiation of mesenchymal cells from adipose tissue of different species.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into several mesoderm lineages. They have been isolated from different tissues, such as bone marrow, adult peripheral blood, umbilical cord blood, and adipose tissue. The aim of this study was to analyze the differences in proliferation and phenotype of adipose tissue‐derived MSCs from three different species, and to evaluate their capacity to differentiate into chondrocytes in vitro. A comparative study of cultured human, rabbit, and sheep mesenchymal cells from adipose tissue was carried out, and the main morphological parameters, proliferative activity, and expression of surface markers were characterized. Proliferation and flow cytometry data showed species‐related differences between animal and human MSCs. Histological staining suggested that rabbit and sheep mesenchymal cells were able to differentiate into chondrocytic lineages. Human mesenchymal cells, though they could also differentiate, accomplished it with more difficulty than animal MSCs. These results could help to explain the differences in the chondrogenic capacity of sheep and rabbit MSCs when they are used as animal models compared to human mesenchymal cells in a clinical assay.

Tendon xanthomas in familial hypercholesterolemia are associated with a differential inflammatory response of macrophages to oxidized LDL

Tendon xanthomas (TX) are pathognomonic lipid deposits commonly found in familial hypercholesterolemia (FH) patients. The aim of this study was to determine whether macrophages from FH patients with TX (TX+) have higher predisposition to foam cells formation after oxidized LDL (oxLDL) overload than those from FH patients without TX (TX−), and if their differential gene expression profile could explain these different phenotypes. Total RNA pools from macrophages from FH patients TX+ and TX− were analyzed using Affymetrix oligonucleotide arrays to evaluate the gene expression profile in presence and absence of oxLDL. Also, the intracellular lipid content was measured by fluorescence flow cytometry. Results of these studies suggest that macrophages from FH subjects TX+ compared to those TX− have a differential response to oxLDL, since they show higher intracellular cholesterol ester accumulation and a differential gene expression profile. The gene array data were validated by relative quantitative real‐time RT‐PCR and quantitative ELISA in culture media and plasma samples. FH subjects TX+ showed increased plasma tryptase, TNF‐α, IL‐8 and IL‐6 concentrations. We propose that TX formation are associated with higher intracellular lipid content, and higher inflammatory response of macrophages in response to oxLDL.

Biosynthesis of unsaturated fatty acids in the main cell lineages of human leukemia and lymphoma

Unsaturated fatty acids are essential for the proliferation of many haematopoietic cells, but little is known about their biosynthetic pathways in these cells. We have studied the activity of the main desaturation-elongation enzymes in human B-(Reh-6, Raji, Ramos) and T-(CEM, Jurkat) lymphocytic, promonocytic (U937), promyelocytic (HL-60) and pluripotent myeloid (K562) cell lineages, as well as the changes induced by cell differentiation. Cells were incubated with 14C-labelled 18:0, 18:2(n - 6) and 18:3(n - 3) or supplemented with the corresponding unlabelled fatty acid and synthesis of polyunsaturated fatty acids (PUFA) was evaluated by argentation-TLC and GLC. The main activity present in most cells was delta 9-desaturase (range between 200-1000 pmol/24 h per 10(6) cells) that was regulated by the type of free fatty acids in culture media. A great variability in the activities of delta 6- and delta 5-desaturase was observed. They were virtually absent in B-cells and only one (Jurkat) T-cell line synthesized significant amounts of (n - 6) and (n - 3) PUFA. The main PUFA formed by Jurkat cells were 20:3 and 20:4(n - 6) (30 and 40%, respectively, of cell lipid radioactivity) and 20:5, 22:5 and 22:6(n - 3) (60, 20 and 10%, respectively, of cell radioactivity). Cell differentiation caused complex changes in desaturase activities. The activity of delta 9-desaturase increased with the degree of differentiation of B-cells. Differentiation of U937 cells to macrophages with PMA caused a 2-3-fold increase in the activity of (delta 6 + delta 5)- and delta 9-desaturases and no changes and a 2-fold decrease, respectively, if the inducer was DMSO. Differentiation of HL-60 cells to granulocytes with DMSO virtually abolished delta 9-desaturase activity and greatly reduced that of delta 6- and delta 5-desaturases. delta 9-Desaturase activity increased (2.5-fold) in myeloid K562 cells differentiated to erythroblasts with hemin. No induction of delta 6-desaturase, absent in K562 cells, occurred after differentiation to erythroblasts or megakaryoblasts and they synthesized alternative PUFA through sequential elongation and delta 5-desaturation of 18:2(n - 6) and 18:3(n - 3). The activities of delta 6- and delta 5-desaturase in HL-60 and U937 cells increased when differentiation also stimulated the synthesis of eicosanoids and extracellular release of PUFA.

Apo2L/TRAIL and immune regulation

Apo2L/TRAIL is a member of the TNF family, with its receptors DR4 and DR5 containing a death domain. Multiple tumors are sensitive to Apo2L/TRAIL-induced apoptosis, while normal cells are not, so it constitutes a promising new antitumoral therapy. In this review we deal rather with the physiological role of Apo2L/TRAIL, which, in one hand, is clearly related with immune antitumoral surveillance. However, a role of Apo2L/TRAIL as a fine-tuning regulator of the immune system, especially in the regulation of CD8+ T cell activation and memory, has been also demonstrated. In fact, Apo2L/TRAIL can be considered as an additional mechanism needed to prevent the development of autoimmune disease. Indeed, recent developments indicate that Apo2L/TRAIL can be also useful as a treatment against certain chronic autoimmune diseases.

Tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase correlates with high proliferation rates in sublines derived from the Jurkat leukemia

A prominent tyrosine phosphorylated protein of 85 kDa (p85) was detected in highly proliferative sublines derived from the Jurkat T cell leukemia. We undertook a study to characterize the identity of this protein and its possible role in the hyperproliferative phenotypes observed. Using immunoblot and immunoprecipitation techniques, this protein was characterized as the p85 regulatory subunit of phosphatidylinositol 3-kinase. Cell proliferation and p85 tyrosine phosphorylation was not affected by tyrphostin AG-490, an inhibitor of Jak kinases, wortmannin or LY294002, inhibitors of the activity of the catalytic phosphatidylinositol 3-kinase subunit. Herbimycin-A and PPI, inhibitors of src-like protein tyrosine kinases, and genistein, a general tyrosine kinase inhibitor, inhibited p85 tyrosine phosphorylation and induced cell death in the sublines. PD98059, an inhibitor of Mek, inhibited cell growth of the sublines, but not that of the parental cells. It was concluded that tyrosine phosphorylation of p85 is associated with highly proliferative tumoral phenotypes, at least in T cell leukemias, independent of the phosphatidylinositol 3-kinase activity of the catalytic subunit.