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H Benoist

Publications and source records attributed to H Benoist.

At least 19 recordsLinked to original sources
PubMed1999

Mildly oxidized low-density lipoproteins decrease early production of interleukin 2 and nuclear factor kappaB binding to DNA in activated T-lymphocytes.

Activated T-lymphocytes are found early in atherosclerosis lesions, but little is known about their role. Oxidized low-density lipoproteins (oxLDLs) are considered to be involved in the pathogenesis of the lesions, and we have previously demonstrated that oxLDLs inhibit not only interleukin (IL)-2-receptor expression on the surface of in vitro-activated T-lymphocytes but also their proliferation. We have now investigated the effect of oxLDLs on blast differentiation, on IL-2 synthesis and on the activation of the nuclear factor kappaB (NF-kappaB) system in activated lymphocytes. Mildly oxLDLs (50 and 100 microgram/ml) decreased the number of lymphoblasts and the level of IL-2 concentration in the culture supernatants after activation of lymphocytes by phytohaemagglutinin and PMA+ionomycin. The inhibition of IL-2 production was observed in the CD3(+) T-lymphocyte cytoplasm as early as 4 h after activation by PMA+ionomycin. The study of NF-kappaB showed that oxLDLs led to a decrease of activation-induced p65/p50 NF-kappaB heterodimer binding to DNA, whereas the presence of the constitutive nuclear form of p50 dimer was unchanged. This was correlated with an unchanged level of the active form of the cytosolic inhibitor protein IkappaB-alpha. Taken together, these observations suggest that the immunosuppressive effect of oxLDLs might operate via a dysregulation of the T-lymphocyte activation mechanisms.

Arteriosclerosis↗
PubMed1999

N-linked oligosaccharides can protect target cells from the lysis mediated by NK cells but not by cytotoxic T lymphocytes: role of NKG2-A.

We have previously shown that glycophorin A (GPA), inserted by electropulsation into the membrane of K562 cells, protected them from natural killer (NK) cell-mediated cytotoxicity and the unique N-linked oligosaccharide of GPA was essential for resistance to occur. The present study demonstrates that the protection level conferred by GPA is similar to the resistance induced by HLA-Cw3 expressed by transfected K562 cells. A monoclonal antibody against NKG2-A, an NK inhibitory receptor interacting with HLA class I antigens and belonging to the C-type lectin receptor, was able to restore the ability of NK cells to lyse K562 cells expressing HLA-Cw3 at the cell membrane but not electroinserted-GPA, suggesting that the N-linked oligosaccharide of GPA cannot be a ligand for NKG2-A. GPA was then electroinserted into the membrane of two lymphoblastoid B-cell lines: one was sensitive to NK cell-mediated lysis, the other was susceptible to cytotoxic CD8+ T-lymphocyte (CTL)-mediated cytotoxicity. The electroinserted GPA protected the target cells from NK-mediated cytotoxicity, whereas it did not modify the cell susceptibility to lysis by CTL. Endoglycosidase F treatment abolished the resistance towards NK cell-mediated lysis, suggesting that N-linked glycans could inhibit mechanisms used by NK cells to exert their cytotoxic function in agreement with our previous results.

Carbohydrate Conformation↗
PubMed1999

Oxidized low density lipoproteins induce apoptosis in PHA-activated peripheral blood mononuclear cells and in the Jurkat T-cell line.

Oxidized low density lipoproteins (oxLDLs) and activated T lymphocytes are present in early atherosclerotic plaques. It has been shown that oxLDLs are cytotoxic to cultured vascular cells but their possible toxic action on T lymphocytes has not been described. Peripheral blood lymphocytes from healthy individuals were stimulated in vitro with the polyclonal activator phytohemagglutinin and treated with various doses of native and mildly oxidized LDLs. Low doses of oxLDLs inhibited cell growth and DNA synthesis after 48 h culture and at 200 microg apoB/ml we observed a loss of cell viability. Dead cells did not exhibit significant increase of alteration of membrane integrity (i.e., necrosis) but showed chromatin fragmentation evaluated by DNA staining with 4', 6-diamidino-2-phenylindole and propidium iodide. This fragmentation increased with TBARS and hydroperoxide levels. The expression of early apoptosis marker Apo2.7 rose among the CD3(+) T-cell population. In addition, morphological analysis showed apoptotic features (cell shrinking, nucleus condensation, and fragmentation). Study of phosphatidylserine expression using Annexin V confirmed that oxLDLs induced apoptosis in activated lymphocytes. In the Jurkat T-cell line cultured with oxLDLs, apoptotic morphological changes (condensation and nucleus fragmentation) were observed and they were accompanied by DNA fragmentation visualized by propidium iodide staining and electrophoresis showing apoptotic ladder. These results demonstrate that mildly oxidized LDLs induce apoptosis in a part of activated and proliferating T cells. T-lymphocyte apoptosis induction in atherosclerotic lesions might contribute to the development of an inappropriate local T cell response.

Apoptosis↗
PubMed1998

Mildly oxidized low-density lipoproteins suppress the proliferation of activated CD4+ T-lymphocytes and their interleukin 2 receptor expression in vitro.

Activated T-lymphocytes are present in early atherosclerotic lesions where they may interact with oxidized low-density lipoproteins (oxLDLs). In this study the non-specific effect of oxLDLs on the activation of T-cells in vitro was investigated. LDLs were oxidized by UV irradiation and characterized by a low level of lipid peroxidation and only slight apolipoprotein B modification. Peripheral blood lymphocytes from normal individuals were stimulated in vitro with the polyclonal activator phytohaemagglutinin in the presence of various doses of LDLs and oxLDLs. LDLs enhanced the proliferation of peripheral blood lymphocytes at doses up to 100 microg/ml but were inhibitory at 200 microg/ml, whereas low doses of oxLDLs (over 10 microg/ml) inhibited the proliferation. OxLDLs also inhibited the proliferative responses of an alloreactive CD4+ T-cell line immortalized by Herpes virus saimiri and an influenza haemagglutinin-specific CD4+ T-cell clone. Viability tests using Trypan Blue exclusion or expression of Apo2.7, an apoptosis marker, did not indicate any significant cell death at doses up to 100 microg/ml oxLDL. At this concentration, cell-cycle analysis showed an accumulation of cells at the G1/S interface in the CD4+ cell clone, without significant DNA fragmentation. The expression of the activation antigen CD25 on T-lymphocytes (on phytohaemagglutinin-activated T-cells and on CD4+ T-cell clone), requisite to the commitment of activated T-cells from G1 phase to S phase, was also inhibited by oxLDLs whereas expression of other activation antigens such as CD69 and HLA-DR was unchanged. In conclusion, these data show that mildly oxidized LDLs inhibit the proliferation and CD25 expression of activated T-lymphocytes and suggest that oxLDLs may slow down the T-cell response in atherosclerotic lesions.

CD4-Positive T-Lymphocytes↗
PubMed1997

Nucleosome-dependent escape of tumor cells from natural-killer-mediated lysis: nucleosomes are taken up by target cells and act at a postconjugation level.

Our previous data suggested that chromatin fragments released from dead cells into the extracellular medium could be involved in the impairment of natural-killer (NK)-mediated cytotoxicity reported in cancer patients. In the present study, an inhibition of the NK-mediated lysis was obtained in vitro by nucleosome addition to different tumor target cells, independently of their sensitivity to NK-mediated lysis. We observed a rapid endocytosis and degradation of nucleosomes by K562 tumor target cells and (although to a much lesser extent) a binding to a subpopulation of lymphocytes. Nucleosomes impaired neither the conjugation step nor the expression of adhesion molecules at the effector (CD11a, CD18, CD2) or target (CD54, CD58) cell surface. On the contrary, flow-cytometry analysis of the conjugation suggested that nucleosomes might stabilize the conjugates. Investigations of the killing process showed that nucleosomes decreased the NK cytotoxic potential without modifying Ca2+-dependent lethal-hit-delivery kinetics. The cytotoxic potential was not restored by increasing the available magnesium and calcium concentrations in the extracellular medium. Taken together, the results suggest that the inhibition of NK-mediated lysis by nucleosomes may result from alterations of the NK mechanism at the postconjugation level and after lethal-hit delivery. Hence, the inhibition could involve a delay in the recycling of effector cells, or a resistance of tumor target cells to NK cells.

Biological Transport↗
PubMed1995

Glycophorin A protects K562 cells from natural killer cell attack. Role of oligosaccharides.

Glycophorin A is a protein with an abundant glycosylation (60% carbohydrate by weight), and studies have suggested that resistance of target cells to natural killing may be correlated with the level of glycophorin A expression. To assess the role of glycophorin A and of its carbohydrates in sensitivity to lysis by natural killer (NK) cells, the glycoprotein was inserted into the membrane of K562 target cells using electropulsation. Peripheral blood lymphocytes were used as effector cells. When glycophorin A was inserted into the membrane, the level of resistance to NK cell attack increased with the number of glycophorin A molecules electroinserted. The resistance to lysis was not due to a defect in target cell-effector cell binding. Electroassociation of glycophorin A did not cause a decrease in the expression of either "positive signals" for NK cells (such as CD71, CD15, and CD32 antigens) or cellular adhesion molecules (CD18, CD29, CD54, and CD58). Furthermore, electroinsertion of glycophorin A did not trigger any "negative signals," such as class I HLA antigen expression. Finally, it was shown that the sialic acid and O-linked oligosaccharides of glycophorin A did not play any role in its effect against NK cells. Conversely, the unique N-linked oligosaccharide was shown to be essential for resistance to occur.

Antigens, Surface↗
PubMed1995

Study of LDL and acetylated LDL endocytosis by mononuclear cells in HIV infection.

Activated lymphocytes have a high level of low density lipoprotein (LDL) uptake as compared to resting lymphocytes, whereas scavenger receptors for acetylated LDL (Ac-LDL) are expressed on limited number of immune cells, i.e., monocytes/macrophages. The endocytosis of LDL and Ac-LDL by mononuclear cells was studied during in vitro and in vivo HIV infection, in order to use LDL and Ac-LDL as carriers of antiviral and/or immunomodulatory drugs towards lymphocytes and monocytes. The uptake of LDL and Ac-LDL was analyzed by cytofluorimetry. LDL endocytosis in PHA/IL2-activated lymphocytes was higher than in resting lymphocytes. In vitro HIV infection of PHA/IL2-activated lymphocytes did not alter the high LDL endocytosis in lymphocytes. CD4+ and CD8+ cells. In a group of 12 symptomatic patients there was no alteration of LDL endocytosis in lymphocytes, CD4 and CD8 lymphocytes. In another group of 23 individuals, the Ac-LDL endocytosis mediated by CD14+ monocytes was unaltered in asymptomatic patients (n = 6) and in some symptomatic patients (n = 6, CD14+ cells > 100/mm3). On the contrary, in other symptomatic patients (n = 11, CD14+ cells < 100/mm3), the number of Ac-LDL+ CD14+ cells decreased, whereas their efficiency of Ac-LDL endocytosis increased as compared to those of other HIV+ patients. In conclusion, the use of lipoproteins as carriers to increase the drug delivery to CD4+ lymphocytes and to CD14+ monocytes can be envisaged, since: (i) the LDL endocytosis was not impaired in CD4 lymphocytes of HIV+ patients, and (ii) the Ac-LDL uptake by monocytes was altered only in some patients of stage IV.

Antigens, CD↗
PubMed1995

Evidence for a dual effect of acebutolol, a beta blocker, on the mouse humoral immune response.

Acebutolol induces transient polyclonal B cell activation in C57B1/6 mice but down-modulates the spontaneous polyclonal activation of NZBxNZW lupus mice. The immunomodulatory effects of this beta-blocker were studied in C57l/6 mice injected with LPS or immunized with sheep red blood cells. The effect of acebutolol on the polyclonal activation of lymphocytes induced by LPS was also investigated in heterozygous and nu/nu C57BL/6 mice. Finally, the direct effect of acebutolol on spleen cells was studied in vitro. Acebutolol treatment for 15 days (50mg/kg/day) inhibited the polyclonal activation of lymphocytes induced by LPS in C57BL/6 and in C57BI/6 nu/nu mice, but increased the humoral response to sheep red blood cells in C57Bl/6 mice. Moreover, spleen cells from C57Bl/6 mice treated for 15 days with acebutolol showed an increased number of CD5+ and CD4+ lymphocytes, as well as an increased reactivity to concanavalin A but not to LPS. In vitro, acebutolol at 10(-5)-10(-7) M induced an increased reactivity of spleen cells from naive mice to concanavalin A, whereas it did not affect the B cell responsiveness to LPS. These results indicate that acebutolol modulates both T-cells and non T-cells in the immune system.

Acebutolol↗
PubMed1994

LDL and acetyl-LDL inhibit the NK activity and are taken up by CD56+ lymphocytes.

The effect of LDL and modified LDL (acetyl-LDL) was studied on human natural killer cell-mediated cytotoxicity against K562 cells. Incubation for 24 h of peripheral blood lymphocytes (PBL) with a high concentration (200 micrograms/ml) of LDL decreased the NK activity in some donors. After acetylation of the LDL protein (apoB), the modified-LDL systematically inhibited the NK function of PBL in a time- and dose-dependent manner. Inhibition mediated by acetyl-LDL (AcLDL) was significantly greater than that of LDL, indicating that the apoB modification can mediate the inhibition of the NK function. AcLDL also inhibited the NK activity of peripheral blood mononuclear cells, suggesting that, under our experimental conditions, monocytes are not efficient enough to protect NK cells against the adverse effects of modified-LDL. With a cytofluorimetric analysis, the internalization of acetyl-LDL by PBL was demonstrated and was only 3-4 times lower than LDL internalization in lymphocytes. It appeared to be time, temperature and dose dependent, saturable and different from the internalization mediated by the known scavenger receptors. Finally, CD14- CD3+ lymphocytes and CD14- CD56+ lymphocytes were able to internalize AcLDL in the same way. Our results suggest that in some in vivo circumstances, when the LDL concentration and/or the modified-LDL/LDL ratio increase in tissues, lipoproteins are internalized by NK cells and also can induce adverse effects on the NK function.

Antigens, CD↗
PubMed1994

Electropermeabilization mediates a stable insertion of glycophorin A with Chinese hamster ovary cell membranes.

Electropulsation allowed us to incorporate glycophorin A, an integral membrane protein, into mammalian nucleated cell membranes (Chinese hamster ovary cells). The induction of stable protein association is effective only when the field intensity is higher than its threshold value, creating membrane permeabilization to small molecules. Under controlled conditions, cell viability was only slightly altered by this treatment. Pulse number and duration controlled both the number of modified cells and incorporated molecules. The phenomena was temperature dependent. An average of 5 x 10(4) molecules/cell was bound. About 80% of cells in the pulsed population were observed to incorporate glycophorin. The protein incorporation was shown to be stable 48 h after electroassociation. Electrically bound proteins were shared between the cells after each division. As enhanced binding is detected if glycophorin is added after the pulses, it is the long-lived alteration of the membrane mediated by the pulses which supports the association.

Animals↗
PubMed1994

In vitro inhibition of natural-killer-mediated lysis by chromatin fragments.

A qualitative impairment of natural killer (NK) function and the presence of circulating DNA have been independently reported in clinical situations such as cancer and lupus. The existence of receptors for chromatin fragments at the leukocyte membrane raised the question of the relation between the presence of chromatin fragments in the extracellular medium and the impairment of NK function. The present study shows that plasmas from patients with metastatic cancer and with pathological DNA concentrations inhibited significantly the NK activity of normal lymphocytes as compared to cancer plasmas with DNA concentrations in the normal range. In vitro, it was demonstrated that chromatin fragments inhibited the NK-mediated cytotoxicity in a dose-dependent manner. Inhibitory concentrations of nucleosomes (2.5-10 micrograms/ml) were lower than those of DNA and histones alone (100 micrograms/ml). Inhibitory effects of nucleosomes, DNA and histones differed also according to the effector population used: nucleosomes were effective whatever the CD56+ cell enrichment of the effector population, while DNA inhibition needed T cells, and histone inhibition probably resulted from a subtoxic effect, prevented by the presence of adherent cells. Finally we found that nucleosomes could inhibit the NK function only when they were present in the extracellular medium. Taken together, these data suggest that the persistence of nucleosomal DNA at sites of cell death or in the blood might be responsible, at least partly, for the NK activity impairment observed in pathological circumstances characterized by a high rate of cell death phenomena such as cancer.

Cell Adhesion↗
PubMed1994

Autoimmunity to histones, ubiquitin, and ubiquitinated histone H2A in NZB x NZW and MRL-lpr/lpr mice. Anti-histone antibodies are concentrated in glomerular eluates of lupus mice.

In lupus diseases products of chromatin catabolism released from dead cells might be involved in the induction of autoantibody and in the development of glomerulonephritis. While the pathogenic role of anti-DNA antibodies is recognized, the role of antibodies directed against structural proteins of chromatin is still questioned. IgG antibodies to histones, ubiquitin, and ubiquitinated histone H2A (UH2A) have been investigated both in plasma and in glomerular eluates of NZB x NZW and MRL-lpr/lpr mice. In NZB x NZW mice, anti-ubiquitin and anti-UH2A antibodies were detected at 8 weeks of age, simultaneously with anti-double-stranded DNA antibodies, whereas anti-histone antibodies appeared later. In MRL-lpr/lpr mice, anti-DNA antibodies were detected at 4 weeks, whereas anti-histone, anti-ubiquitin, and anti-UH2A antibodies were not detected at that age but appeared in plasma rapidly thereafter. In both strains, increased anti-histone activity was found in IgG eluted from glomeruli. These results support the suggestion that anti-histone antibodies are likely to play a pathogenic role in lupus nephritis. They also indicate that, like human lupus, murine lupus is characterized by the production of anti-ubiquitin and anti-UH2A antibodies.

Animals↗
PubMed1993

Electric field-mediated glycophorin insertion in cell membrane is a localized event.

Purified soluble glycophorin, an intrinsic protein, can be back 'electroinserted' in the membrane of Chinese hamster ovary cells by submitting the cell/protein mixture to short electric field pulses. Previous studies showed that this complex between pulsed cells and proteins, which is detected only when the cell membrane is electropermeabilized, was very stable. This strongly suggested that the protein was indeed inserted in the membrane. The basic processes involved in this phenomena are studied in the present work. The association is observed at the single cell level by means of videoimmunofluorescence. Electric field-mediated insertion occurs firstly in a limited patch of the cell surface, which size is in agreement with the prediction of Electropermeabilization theory. A free diffusion of the inserted proteins then follows on the cell surface. The diffusion coefficient is computed to be less than 10(-10) cm2/s as observed for transmembranous proteins. This slow process gives an homogeneous distribution of the inserted protein.

Animals↗
PubMed1993

In vitro stimulation of polymorphonuclear cell adhesion by ribomunyl and antibiotic + ribomunyl combinations: effects on CD18, CD35 and CD16 expression.

Several functions of polymorphonuclear cells (PMNs) require adhesion to occur. Various membrane proteins' functions such as CD18 (beta 2 chain of integrin), CD35 (CR1) and CD16 (F c gamma Receptor III) participate in adhesion. In vivo treatment with Ribomunyl (R), an immunomodulating agent, was shown to enhance adhesion and migration of PMNs. To explore the direct effect of R on PMNs, cells from healthy subjects were treated in vitro with R. A significant increase of PMN adhesion and expression of CD18 and CD35 molecules were observed with 50 and 100 micrograms/ml of R after 2 h incubation. However, R-treatment decreased the PMN reactivity towards anti-CD16 (F c gamma RIII) monoclonal antibody. The effect of R on adhesion and membrane molecule expression was independent of the presence of serum and of polymixin B. Thus, this effect cannot be due to lipopolysaccharide (LPS) contaminants and does not require interactions with serum components. In previous studies, it was shown that in vitro amoxicillin increased some PMN functions whereas josamycin decreased them. The in vitro incubation of PMNs with R and amoxicillin (100 micrograms/ml) potentiated the positive effect of amoxicillin on adhesion and the antibiotic counterbalanced the negative effect of R on CD16 expression. In addition, R compensated the negative effect of josamycin (100 micrograms/ml) on PMN adhesion and on CD18 and CD35 expression. This study indicates: (1) the direct effect of R on PMN adhesion and on expression of molecules involved in adhesive-mediated functions, and (2) the beneficial effect of the association of R with antibiotics which can stimulate PMN activity.

Adjuvants, Immunologic↗
PubMed1992

Studies on the susceptibility to NK-mediated lysis and the simultaneous expression of various surface molecules in anthracyclin-treated K562 cells and in four K562 cell clones.

Target molecules for NK cells are unknown. Numerous studies have proposed putative target molecules, but have examined their role in the modulation of sensitivity to NK-mediated lysis one independently of each other. We examined the simultaneous expression of various surface molecules and the susceptibility of K562 cells to NK attack. We have previously shown that adriamycin (40 nM) and aclacinomycin (15 nM) can induce, in vitro, an increase of glycophorin A (GPA) on K562 cells, a modulation of transferrin receptor (TfR) and CD15 antigen expression and a significant resistance of cells to NK-mediated lysis. In the present work, Fc gamma receptor II (CD32) expression at the K562 cell membrane was clearly decreased after aclacinomycin-treatment but was unaltered by adriamycin-treatment. Four K562 cell clones were studied. Two clones (F and G) expressed a higher level of CD32 at the membrane (62% and 70% of erythrocyte antibody (EA) rosettes respectively) and two clones (9 and 19) expressed lower a level (18% and 7% EA rosettes respectively) than the original population (43%). The sensitivity to lysis by NK cells was increased in clones F, G and 9 but decreased in clone 19 (without alteration in the binding capacity). Relationships between the sensitivity to NK attack and the levels of simultaneous expression of CD32, TfR, CD15, glycophorin A (GPA) and MHC class I monomorphic antigens were studied. In addition, the presence at the membrane of some cellular adhesion molecules (CD54, CD58, CD29, CD18, CD56) was examined in anthracyclin-treated cells and in the four clones. The difference in the sensitivity of target cells to NK attack is not strictly related to variation of one or other of these molecules. Our previous and present data suggest that the resistance of K562 cells to NK cells may correlate with the level of erythroid maturation at the cell membrane, involving simultaneous variations in expression of several molecules such as a decrease of TfR, CD15 and CD32 and an increase of GPA.

Aclarubicin↗
PubMed1992

An in vivo model for the experimental selection of drugs able to prevent immune complex glomerulonephritis.

Polyclonal activation of lymphocytes and immune complex-mediated glomerular lesions were induced in C57Bl/6 mice by injecting bacterial lipopolysaccharide (LPS) twice a week for 2 weeks. The usefulness of such a model for in vivo evaluation of immunomodulatory and therapeutic effects of drugs, was investigated by treating mice with DIAM4, a cyclophosphazenic compound known to modulate polyclonal activation of lymphocytes and to prevent mouse lupus nephritis. Prevention of LPS-triggered lymphocyte polyclonal activation and glomerular lesions was observed in the DIAM4-treated mice. Such a model can be used conveniently to select compounds effective in the treatment of immune glomerulonephritis.

Animals↗
PubMed1989

Comparative effects of fagaronine, adriamycin and aclacinomycin on K562 cell sensitivity to natural-killer-mediated lysis. Lack of agreement between alteration of transferrin receptor and CD15 antigen expressions and induction of resistance to natural killer.

Little is known about membrane target antigens for natural killer (NK) cells. Transferrin receptor and CD15 antigen might be two of these target structures. A novel antileukemic alkaloid, fagaronine, is able to induce hemoglobin synthesis in the K562 cell line. Numerous reports suggest relations between the expression of natural killer target structures and the differentiation stage of malignant cells. Effects of fagaronine on the expression of glycophorin A, transferrin receptor and CD15 antigen and susceptibility to NK-mediated lysis have been investigated in K562 cells and compared to those of two anthracyclines (Adriamycin and aclacinomycin A) known to be erythroid-differentiation inducers. When comparing the balance of differentiating effect and toxicity, the dose and time-dependent effects of the drugs, fagaronine and aclacinomycin, are equivalent on K562 cells. In experimental conditions where fagaronine (3500 nM), Adriamycin (40 nM) and aclacinomycin (15 nM) recruit the same percentage of hemoglobin-containing cells (40%-50%), glycophorin A expression increases and transferrin receptor expression decreases. Only Adriamycin treatment decreases CD15 antigen expression. In addition, Adriamycin and aclacinomycin, but not fagaronine, induce resistance to NK-mediated lysis. These data suggest that (a) it is unlikely that CD15 antigen and transferrin receptor, separately considered, can be unique target structures for NK cells; and (b) fagaronine is a potent erythroid inducer which, in our system, has similar effects to aclacinomycin without induced resistance to NK attack.

Aclarubicin↗