Luis Larrad

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.

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.

Liposome-Bound APO2L/TRAIL Is an Effective Treatment in a Rabbit Model of Rheumatoid Arthritis

OBJECTIVE We previously observed that T lymphocytes present in synovial fluid (SF) from patients with rheumatoid arthritis (RA) were sensitive to APO2L/TRAIL. In addition, there was a drastic decrease in the amount of bioactive APO2L/TRAIL associated with exosomes in SF from RA patients. This study was undertaken to evaluate the effectiveness of bioactive APO2L/TRAIL conjugated with artificial lipid vesicles resembling natural exosomes as a treatment in a rabbit model of antigen-induced arthritis (AIA). METHODS We used a novel Ni(2+)-(N-5-amino-1-carboxypentyl)-iminodiacetic acid)-containing liposomal system. APO2L/TRAIL bound to liposomes was intraarticularly injected into the knees of animals with AIA. One week after treatment, rabbits were killed, and arthritic synovial tissue was analyzed. RESULTS Tethering APO2L/TRAIL to the liposome membrane increased its bioactivity and resulted in more effective treatment of AIA compared with soluble, unconjugated APO2L/TRAIL, with substantially reduced synovial hyperplasia and inflammation in rabbit knee joints. The results of biophysical studies suggested that the increased bioactivity of APO2L/TRAIL associated with liposomes was due to the increase in the local concentration of the recombinant protein, augmenting its receptor crosslinking potential, and not to conformational changes in the protein. In spite of this increase in bioactivity, the treatment lacked systemic toxicity and was not hepatotoxic. CONCLUSION Our findings indicate that binding APO2L/TRAIL to the liposome membrane increases its bioactivity and results in effective treatment of AIA.

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.

Immune response and in vivo production of cytokines in patients with liver hydatidosis

Cytokines play an important role in the human immunological response, but the exact role of cytokines in the human immune response against parasites, especially against Echinococcus granulosus, remains unclear. IL‐1, IL‐2, IL‐4 and tumour necrosis factor (TNF) levels in peripheral blood of 21 patients with liver hydatidosis were evaluated before surgical treatment, and the levels of IgA, IgM, IgG, IgE, specific IgE against E. granulosus, C3, C4 and BF complement fractions and CD20, CD3, CD4, CD8 and CD16 cell percentages were also determined, as was the relationship between these variables and cytokine levels. Data from hydatid patients were compared with data obtained from 21 healthy volunteers. Hydatid patients showed increases of IgG, IgE, IgEs and IL‐2 (P < 0.01), and decreases of IL‐1 and TNF levels (P < 0.001), but these variables (respectively) increased in patients showing cysts in the central area of the liver or with a wide opening of cysts in the biliary tract. The increase of IL‐1, IL‐2 and IL‐4 showed a close relationship with the number, characteristics and above all the location of cysts within the liver itself. IgG and IL‐4 levels and also IgG and IgE levels showed a significant correlation (P < 0.05).

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.

CD59 cross-linking induces secretion of APO2 ligand in overactivated human T cells.

Jurkat cells and the derived TCR / CD3‐defective subline, J.RT3.T3.5 undergo activationinduced cell death (AICD) when stimulated with phytohemagglutinin (PHA). Since J.RT3.T3.5 cells do not express antigen receptor, we searched for the molecules that could be ligated by PHA and induce AICD in this cell line. We show here that the glycosylphosphatidylinositollinked CD59 molecule is expressed at the surface of Jurkat and J.RT3.T3.5 cells, and when cross‐linked by specific antibodies can induce cell death. The toxicity of supernatants from PHA‐stimulated Jurkat or J.RT3.T3.5 cells was prevented by a combination of the blocking anti‐Fas mAb SM1 / 23 and anti‐APO2L / TRAIL mAb 5C2. However, toxicity of supernatants from anti‐CD59 stimulated cells was specifically prevented by the anti‐APO2L blocking antibody. Anti‐CD59 cross‐linking induced AICD also in normal human T cell blasts, which secreted toxic molecules into the supernatant. The toxicity of these supernatants on Jurkat cells was fully prevented by the anti‐APO2L blocking antibody, showing that CD59 crosslinking induces the preferential release of APO2L also in normal T cells. The possible physiological and / or pathological consequences of this observation are discussed.

Liposomes decorated with Apo2L/TRAIL overcome chemoresistance of human hematologic tumor cells.

Human Apo2-ligand/TRAIL is a member of the TNF cytokine superfamily capable of inducing apoptosis on tumor cells while sparing normal cells. Besides its antitumor activity, Apo2L/TRAIL is also implicated in immune regulation. Apo2L/TRAIL is stored inside activated T cells in cytoplasmic multivesicular bodies and is physiologically released to the extracellular medium inserted in the internal membrane vesicles, known as exosomes. In this study we have generated artificial lipid vesicles coated with bioactive Apo2L/TRAIL, which resemble natural exosomes, to analyze their apoptosis-inducing ability on cell lines from hematological tumors. We have tethered Apo2L/TRAIL to lipid vesicles by using a novel Ni(2+)-(N-5-amino-1-carboxylpentyl)-iminodiacetic acid, NTA)-containing liposomal system. This lipidic framework (LUVs-Apo2L/TRAIL) greatly improves Apo2L/TRAIL activity, decreasing by around 14-fold the LC50 on the T-cell leukemia Jurkat. This increase in bioactivity correlated with the greater ability of LUVs-Apo2L/TRAIL to induce caspase-3 activation and is probably due to the increase in local concentration of Apo2L/TRAIL, improving its receptor cross-linking efficiency. More important, liposome-bound Apo2L/TRAIL overcame the resistance to soluble recombinant Apo2L/TRAIL exhibited by tumor cell mutants overexpressing Bcl-xL or by a Bax and Bak-defective Jurkat cell mutant (Jurkat-shBak) and are also effective against other hematologic tumor cells. Jurkat-Bcl-xL and Jurkat-shBak cells are resistant to most chemotherapeutic drugs currently used in cancer treatment, and their sensitivity to LUVs-Apo2L/TRAIL could have potential clinical applications.

Dendritic cell uptake of iron-based magnetic nanoparticles

We have investigated the internalization of magnetic nanoparticles (NPs) into dendritic cells (DCs) in order to assess both the final location of the particles and the viability of the cultured cells. The particles, consisting of a metallic iron core covered with carbon, showed no toxic effects on the DCs and had no effect in their viability. We found that mature DCs are able to incorporate magnetic nanoparticles in a range of size from 10 nm to ca. 200 nm, after 24 h of incubation. We describe a method to separate cells loaded with NPs, and analyze the resulting material by electron microscopy and magnetic measurements. It is found that NPs are internalized in lysosomes, providing a large magnetic signal. Our results suggest that loading DCs with properly functionalized magnetic NPs could be a promising strategy for improved vectorization in cancer diagnosis and treatment.

Human CD8+ T cell blasts are more sensitive than CD4+ T cell blasts to regulation by APO2L/TRAIL

The mechanisms responsible for the down‐modulation of the activation of separated CD4+ or CD8+ human T cell blasts were studied using cells obtained from healthy donors. In the presence of IL‐2, human CD8+ T cell blasts were more sensitive than CD4+ T cell blasts to regulation by APO2 ligand/TNF‐related apoptosis‐inducing ligand (APO2L/TRAIL), while both T cell subsets were equally sensitive to Fas/CD95 regulation. This regulation was defined as inhibition of IL‐2‐dependent T cell growth in the absence of cell death induction, characterized by cell cycle arrest in G2/M. The physiological validity of these observations was corroborated by the demonstration of intracellular FasL and APO2L/TRAIL expression in CD4+ and CD8+ T cell blasts, which were secreted in their bioactive form into the supernatant upon PHA, CD3 or CD59 reactivation. Additionally, the inhibition of IL‐2‐dependent CD4+ or CD8+ T cell blast growth upon CD3 or CD59 ligation was dependent, at least partially, on FasL and/or APO2L/TRAIL. These data precisely define the role of APO2L/TRAIL in the regulation of human T cell activation.