Andrés Piñeiro

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.

The effect of trypsin on bovine transferrin and lactoferrin.

The iron-saturated and iron-free (apo) forms of bovine transferrin and lactoferrin were digested with trypsin and the digests analysed by column chromatography and electrophoresis. Both of the iron-saturated proteins were more resistant to proteolysis than the corresponding apoproteins, and iron-transferrin was more resistant than iron-lactoferrin. Digestion of iron-transferrin yielded two iron-binding fragments with molecular weights of 32 000 and 38 500 whereas apotransferrin yielded only the larger fragment. In digests of lactoferrin, up to five different fragments with molecular weights ranging from 25 000 to 52 700 were detected, there being no obvious qualitative difference between digests of iron-lactoferrin and apolactoferrin. The susceptibility of apolactoferrin to tryptic digestion was only slightly reduced when apolactoferrin was complexed with beta-lactoglobulin, suggesting that complex-formation is not a mechanism for protecting lactoferrin against intestinal degradation. There was no immunological cross reaction between bovine transferrin or its digestion products against anti-lactoferrin antiserum, or vice-versa.

CPP32 inhibition prevents Fas-induced ceramide generation and apoptosis in human cells

Intracellular activation of sphingomyelinase, leading to ceramide generation, and ICE‐like proteases have been implicated in TNF and Fas‐induced apoptosis, but the links between these intracellular apoptotic mediators remain undefined. We show here that a specific peptide inhibitor of the ICE‐like protease CPP32/Yama (DEVD‐cho) blocks anti‐Fasinduced apoptosis in Jurkat and U937 cells, while having no effect on TNF‐induced apoptosis in U937 cells. This peptide also prevents ceramide accumulation induced by Fas engagement. Jurkat and U937 cells, as well as their mtDNA‐depleted derived lines (π° cells), were sensitive to ceramide toxicity, which was not prevented by ICE‐like protease inhibitors. These results, taken together, suggest that ICE‐like protease activation is a prerequisite for ceramide generation and subsequent apoptosis, at least in the case of Fas‐induced cell death.

The major acute phase serum protein in pigs is homologous to human plasma kallikrein sensitive PK-120.

A major acute phase protein (pig‐MAP) has been isolated from the sera of pigs after turpentine injection. The protein is the pig counterpart of a recently cloned human serum protein denominated PK‐120, which is a putative substrate for kallikrein [Nishimura et al., 1995 FEBS Lett. 357, 207–211]. The protein exists in other mammalian species and it is also an acute phase protein, at least in the rat. Pig‐MAP shows homology, as PK‐120, with the heavy chain 2 (HC‐2) of the inter‐α‐trypsin inhibitor superfamily but does not possess trypsin inhibitory activity.

Fatty acid metabolism in human lymphocytes. I. Time-course changes in fatty acid composition and membrane fluidity during blastic transformation of peripheral blood lymphocytes

The time-course changes in fatty acid composition of human T-lymphocytes during blastic transformation were analysed, as well as the variations in membrane fluidity determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), using a fluorescence-activated cell sorter. The more important changes observed, in activated relative to quiescent cells, started after 24 h and consisted in an increase in the proportion of oleic (18:1(n - 9)), docosapentaenoic (22:5(n - 3)) and docosahexaenoic (22:6(n - 3)) acids and a decrease in that of linoleic (18:2(n - 6)) and arachidonic (20:4(n - 6)) acids. This represented a relative increase of 26% for 18:1, 56% for 22:5 and 84% for 22:6 in peripheral blood mononuclear cells (PBMC) and 35%, 182% and 94%, respectively, in purified T-lymphocytes, both activated for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and 19%, respectively, for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and phosphatidylethanolamine) rather than neutral lipids. The 18:1/18:0 ratio increased greatly in major cell phospholipids. The proportion of 20:4, 22:5 and 22:6 in phosphatidylinositol was not significantly altered after 72 h of activation. The molar ratio cholesterol/phospholipids was reduced in 72-h-activated lymphocytes (0.29) compared to quiescent cells (0.5). On the other hand, the stimulation of human T-lymphocytes caused a significant decrease in the order parameter (S) of DPH, according to the observed changes in lipid composition. After 72 h in culture, the S value for quiescent and stimulated T-lymphocytes was 0.530 and 0.326, respectively. In conclusion, the blastic transformation of human T-lymphocytes is associated with changes in lipid composition which modify the physical properties of their membranes. These modifications could modulate, in turn, the activity of membrane proteins implicated in the process of blastic transformation.

Interaction of bovine lactoferrin with other proteins of milk whey

The association between bovine lactoferrin and the major bovine whey proteins, beta-lactoglobulin, alpha-lactalbumin and albumin has been studied by immunochemical techniques, gel filtration and affinity chromatography in lacteal secretions and using purified proteins. Bovine lactoferrin is able to form non-covalent complexes with beta-lactoglobulin or albumin, with lactoferrin-protein molar ratios of 2:1 and 1:1 respectively. No association was detected with alpha-lactalbumin. Lactoferrin interacts with beta-lactoglobulin-Sepharose at low ionic strength, but not in the presence of 0.3 M NaCl, indicating that ionic interactions are important. The lack of association with alpha-lactalbumin suggests however a certain degree of specificity in this electrostatic interaction.

Biosynthesis of docosahexaenoic acid in human cells: evidence that two different Δ6-desaturase activities may exist

It has been proposed that synthesis of docosahexaenoic acid (22:6(n-3) in rat hepatocytes occurs by a route independent of delta 4-desaturase, which involves delta 6-desaturation and retroconversion (Voss A., Reinhart M., Sankarappa S. and Sprecher H. (1991) J. Biol. Chem. 266, 19995-20000). However, most cells exhibit these enzymatic activities and nevertheless synthesize low to undectectable amounts of 22:6(n-3). Moreover, there are few data on the occurrence of this pathway in human cells. In the present work, we have analysed the biosynthetic pathway of 22:6(n-3) in human Y-79 retinoblastoma and Jurkat T-cells. Y-79 cells were supplemented with 18:3(n-3) and 20:5(n-3) or incubated with [1-14C]18:3(n-3) and [1-14C]20:5(n-3) and lipids analysed by argentation TLC, reverse-phase TLC and GLC-mass spectrometry. Pulse-chase experiments revealed that synthesis of 22:6(n-3) from 20:5(n-3) in Y-79 cells occurred through two successive elongations, followed by a delta 6-desaturation of 24:5(n-3) to 24:6(n-3) and retroconversion to 22:6(n-3). Incubation of Y-79 cells with [1-14C]18:3(n-3) in medium containing 50 microM trans-9,12-18:2, a potent inhibitor of delta 6-desaturase, caused a reduction of 22:6(n-3) synthesis mainly by interfering with the desaturation of 18:3(n-3). However, when [1-14C]20:5(n-3) was used as precursor, synthesis of 22:6(n-3) was depressed to a lesser extent and mainly by reduction of 24:6(n-3) retroconversion. Neuronal differentiation of Y-79 cells caused a great increase in delta 6-desaturase activity on 18:3(n-3), though the amount of 22:6(n-3) synthesized did not change or diminish, suggesting the existence of a particular delta 6-desaturase involved in the synthesis of 22:6(n-3). The existence of a distinctive delta 6-desaturase activity could also explain why Jurkat cells growing in serum-free medium showed a near 3-fold increase in the synthesis of pentaenes from 18:3(n-3) and, at the same time, a large decrease in the synthesis of 22:6(n-3). The verification of the involvement of two delta 6-desaturase activities in 22:6(n-3) synthesis would have important implications for the formulation of the nutritional requirements of this fatty acid during development.