Necroptosis contributes to the cyclosporin A-induced cytotoxicity in NRK-52E cells

Cyclosporin A (CsA) induces renal tubular epithelial cells apoptosis and necrosis following in vitro exposure. The mechanisms of CsA-induced apoptosis have been studied intensively, whereas the mechanisms of necrosis remain to be elucidated. Necroptosis has been described as programmed necrosis. This study investigated the ability of CsA to induce necroptosis in the rat tubular cell line NRK-52E. The NRK-52E cells were incubated with CsA for 24 hours with or without necrostatin-1 (Nec-1). The majority of the NRK-52E cells died of necrosis as indicated by LDH leakage, Hoechst 33342/PI staining, and flow cytometry analysis. Cell death was significantly reduced by Nec-1 pretreated before CsA exposure. CsA-induced apoptosis and necrosis were also compared in NRK-52E cells with or without knockdown of receptor interaction protein 3 (RIP3) expression using small interfering RNA. Moreover, the role of reactive oxygen species (ROS) in CsA-induced cell death was also attempted. The result suggests that necroptosis contributes to the CsA-induced cytotoxicity in NRK-52E cells. Meanwhile, RIP3 and ROS are involved in CsA-induced necroptosis. To our knowledge, this is the first report on necroptosis in CsA-induced renal tubular cell death pathways, which might offer a novel protective target for CsA nephrotoxicity.     

In vitro effect of ethanol on cell-mediated cytotoxicity by murine spleen cells

The effects of ethanol on murine spleen cell-mediated lysis have been studied. Concentrations of 5.5-176 mM ethanol produced progressive inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC). Binding of spleen cells to antibody-sensitized target cells was not inhibited by comparable concentrations of ethanol. Kinetic analysis revealed decreased rates of lysis with increasing concentrations of ethanol. Changes of effector to target cell ratios revealed an inhibition of maximum lysis and decreased lytic efficiency in the presence of 88 mM ethanol. Preincubation experiments showed the inhibitory effect of ethanol to be reversible. Macrophage-depleted spleen cells appeared to be as susceptible to inhibition by ethanol as unfractionated spleen cells. Ethanol also inhibited natural killer and alloimmune cytotoxic T cell activity. The ADCC data were analysed by using a mathematical model which incorporates the kinetics of lysis, dose-response relationships, heterogeneity of the lytic effectors, reversibility of inhibition and ethanol loss during incubation. An inhibition constant (KI) of 373 mM-2 when two ethanol molecules interact with the site of inhibition was calculated. 50% inhibition of lysis is produced by 52 mM (0.24%) ethanol. The results are consistent with a model which assumes that lysis is due to a critical number of interactions which ultimately trigger the lytic event. Alcohol interferes with lysis by reacting with sites which are required for triggering the lytic event. Although the molecular details of the mechanism of inhibition are as yet undefined, we infer that ethanol inhibits ADCC at the programming for lysis or the lethal hit stages.     

Inhibition of antibody-dependent cell-mediated cytotoxicity by ethanol

Ethanol has been shown to inhibit spontaneous cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity in vitro in a dose-dependent manner. The effect was observed under differing assay conditions such as incubation time and effector: target cell ratio. Acetaldehyde, the primary metabolite of ethanol, did not inhibit these functions of the immune system. A mixture of ethanol and acetaldehyde showed no interaction with respect to spontaneous and antibody-dependent cell-mediated cytotoxicity. These observations may help to explain the apparently increased incidence of infection for individuals who chronically ingest large amounts of alcoholic beverages.     

Kupffer cell-mediated IL-2 suppression of CYP3A activity in human hepatocytes.

Interleukin (IL)-2 administration has been shown to decrease CYP3A enzyme activity in vivo. To determine whether IL-2 suppression of human hepatocyte CYP3A activity is direct or whether it is facilitated by the presence of Kupffer cells, primary human hepatocytes were cultured alone or cocultured with primary human Kupffer cells at physiologic hepatocyte/Kupffer cell ratios of 10:1 or 10:4. Using proinflammatory cytokines as positive controls, IL-1 (0.2-20 ng/ml) and IL-6 (2-200 ng/ml) exposure resulted in a 70 to 90% decrease in CYP3A activity after 72 h in hepatocyte cultures. In the hepatocyte/Kupffer cell cocultures, an 80% decrease in CYP3A activity was observed with IL-1 (2 ng/ml) or IL-6 (20 ng/ml), suggesting that direct suppressive effects of proinflammatory cytokines on hepatocyte CYP3A activity are not substantially altered by Kupffer cells. In contrast to the effects of these proinflammatory cytokines, no sustained suppression of CYP3A activity was observed with IL-2 (2-200 ng/ml) in hepatocyte cultures. However, in hepatocyte/Kupffer cell cocultures, a concentration-dependent 50 to 70% suppression of CYP3A activity was observed with IL-2 at 72 h. In summary, these data suggest that Kupffer cells are required to reconstitute the suppressive effects of IL-2 on CYP3A activity that are observed in vivo and that hepatocyte/Kupffer cell cocultures may provide a useful model for investigating mechanisms of CYP3A4 regulation by cytokines. Of particular relevance to certain hepatic diseases, these findings suggest potential mechanisms whereby cytokines released from infiltrating blood mononuclear cells might modulate intercellular signaling and controls on hepatocyte function by various cell types that reside in liver.     

Enhancement of cell-mediated cytotoxicity by Clostridium difficile toxin A: an in vitro study

Cells from the immune system exhibiting cytotoxic activity are able to kill tumor or infected cells in a major histocompatibility complex-restricted (cytotoxic lymphocytes) or non-restricted (natural killer cells) manner. In order to exert such a cytotoxicity they have to bind the target cell and release cytotoxic factors able to induce target cell death. Treatment of human peripheral blood mononuclear cells with toxin A from Clostridium difficile induced an enhancement of the cytotoxic efficiency of these effector cells. Morphological analysis of effector/target cell pairs seems to suggest that this could be related to an increased ability of cytotoxic effectors to establish close and intertwined contacts with target cells. These contacts involve adhesion molecules and lead to the formation of a "closed chamber" which probably improves the efficacy of lytic factors and results in an increased cytotoxicity.     

Cytoskeletal changes induced in HEp-2 cells by the cytotoxic necrotizing factor of Escherichia coli

The effect of the cytotoxic necrotizing factor of Escherichia coli on HEp-2 cells was studied by fluorescence and scanning electron microscopy. This cytotoxin, known for inducing the formation of giant multinucleated cells in several cell lines, caused changes in actin and tubulin organization. The presence of membrane ruffles at the cell border and of numerous thick bundles of actin crossing the cell body, suggests that the factor promotes cell spreading; this probably interferes with cytokinesis, ultimately leading to the formation of very large flattened multinucleated cells. Moreover, the nuclear segmentation observed in treated cells seems to be associated with a rearrangement of actin in the perinuclear region and with the presence of tubulin bundles in proximity to nuclear clefts. Although the primary target is still unknown, these findings suggest that the cytoskeleton is affected accounting for the multinucleation process induced by the factor.     

Altered subcellular distribution of nucleolar protein fibrillarin by actinomycin D in HEp-2 cells

INTRODUCTION The nucleolus of eukaryotic cells is the site of ri-bosome assembly where synthesis and processing ofthe rRNA precursor molecules (pre-rRNAs) as well astheir coordinate assembly with specific ribosomal andnonribosomal proteins to form preribosomal particlestook place[1]. Besides the small nucleolar RNAs(snRNAs), several proteins, includingfibrillarin andB23,are found in the nucleolus[2]. Fibrillarin (B36, NOP1) is an abundant nucleolarprotein which plays a r…     

Altered subcellular distribution of nucleolar protein fibrillarin by actinomycin D in HEp-2 cells

AIM: To study the effects of actinomycin D on subcellular distribution of nucleolar protein fibrillarin in HEp-2 (human esophageal epithelial type 2) cells, and molecular mechanisms for maintenance of fibrillarin in nucleolus.METHODS: Indirect immunofluorescence assay was employed to investigate subcellular distribution of nucleolarprotein fibrillarin and immunoblotting analysis was used to detect the total cellular amount of fibrillarin. RESULTS:Control cells with no drug treatment showed bright clumpy nucleolar staining, which indicated that fibrillarin decorated the nucleolus only. Treatment with actinomycin D caused dislocation of fibrillarin from nucleoli to nucleoplasm with numerous stained small nucleoplasmic entities. Immunoblotting showed that neither total cellular amount of fibrillarin nor the integrity of fibrillarin was changed upon the treatment. The dislocation of fibrillarin incells treated at a lower concentration (0.05 mg/L) of actinomycin D, was totally reversible after removal of the drug from the medium. However, this reversion was not observed at a high drug concentration (1 mg/L). CONCLUSION:Actinomycin D induced dislocation of fibrillarin from nucleoli to nucleoplasm in HEp-2 cells. The retention of fibrillarin within the nucleolus was related to active RNA synthesis.     

Requirement of expression of P-glycoprotein on human natural killer leukemia cells for cell-mediated cytotoxicity

The requirement of P-glycoprotein, a product of the multidrug resistance (MDR)1 gene, for natural killer (NK) cell-mediated cytotoxicity was examined by using a human NK-like cell line, YTN, which is cytotoxic toward JY cells. YTN cells express P-glycoprotein, a judged by flow cytometry and polymerase chain reaction of reverse-transcribed mRNA. YTN cell-mediated cytotoxicity was inhibited by MDR-reversing reagents as well as the F(ab')2 fragment of a monoclonal antibody against P-glycoprotein. Furthermore, antisense oligonucleotides for MDR1 mRNA inhibited expression of P-glycoprotein as well as YTN cell-mediated cytotoxicity. Thus, this study provides firm evidence that P-glycoprotein plays an essential role in cell-mediated cytotoxicity.     

Inhibition of cytotoxicity by sulfasalazine. I. Sulfasalazine inhibits spontaneous cell-mediated cytotoxicity by peripheral blood and intestinal mononuclear cells from control and inflammatory bowel disease patients

We have studied the effects of sulfasalazine and its metabolites on cell-mediated cytotoxicity by peripheral blood and intestinal mononuclear cells from both control and inflammatory bowel disease (IBD) patients. Sulfasalazine and sulfapyridine, as well as hydrocortisone and nordihydroguaiaretic acid inhibited spontaneous cell-mediated cytotoxicity by control and IBD peripheral blood cells. Sulfasalazine and nordihydroguaiaretic acid inhibited spontaneous cell-mediated cytotoxicity by control and IBD intestinal mononuclear cells cultured for 72 h in media alone. In contrast, 5-aminosalicylate, indomethacin and benzylimidazole had no effect on cytotoxicity by any cell population. Lectin-induced, antibody-dependent and interleukin-2-induced cell-mediated cytotoxicity, as well as lymphokine-activated killing were not inhibited by the drugs: inhibitory effects in these assays were primarily upon the underlying spontaneous cell-mediated cytotoxicity. The inhibition induced by sulfasalazine, sulfapyridine and nordihydroguaiaretic acid could not be reversed by adding the lipoxygenase metabolites leukotriene B4 or 12-hydroxyeicosatetraenoic acid. These findings demonstrate that spontaneous cell-mediated cytotoxicity by control and IBD mononuclear cells can be inhibited by sulfasalazine.     

茚并吡啶类衍生物诱导HEp-2细胞凋亡

目的:探讨1-甲基-5-(4-二甲氨基苄烯)-5H-茚[1,2b]吡啶三氟甲磺酸盐(受试物)对人喉表皮样肿瘤细胞HEp-2的增殖抑制和凋亡诱导作用。方法:采用MTT法观察受试物对HEp-2增殖的抑制作用,Hoechst 33258荧光染色观察HEp-2细胞凋亡形态学变化,流式细胞仪PI单染法检测细胞周期及凋亡率,采用Western Blot分析检测促凋亡蛋白Bid的表达。结果:1-甲基-5-(4-二甲氨基苄烯)-5H-茚[1,2b]吡啶三氟甲磺酸盐可抑制HEp-2细胞的增殖,并呈时间和剂量依赖性;用受试物(4μmol.L-1)处理细胞(24和48 h),经Hoechst 33258荧光染色,可见细胞凋亡形态学改变;流式细胞仪检测显示,HEp-2细胞周期发生改变,凋亡率增加;Western Blot分析结果显示,受试物可使促凋亡蛋白Bid表达水平上升。结论:1-甲基-5-(4-二甲氨基苄烯)-5H-茚[1,2b]吡啶三氟甲磺酸盐可抑制HEp-2细胞增殖,诱导其凋亡。     

Calcineurin-independent inhibition of mitochondrial Ca2+ uptake by cyclosporin A

1. Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, derived mainly from the inhibition of calcineurin, and also because of its ability to block the mitochondrial permeability transition pore (PTP). This second effect has been involved in the protection against apoptosis mediated by release of mitochondrial factors. We show here that CsA (1-10 microm) has an additional effect on Ca(2+) homeostasis in mitochondria that cannot be attributed to inhibition of PTP. 2. By measuring specifically mitochondrial [Ca(2+)] with targeted aequorin, we show that CsA inhibited Ca(2+) entry into mitochondria both in intact and in permeabilized cells, and this effect was stronger when Ca(2+) entry was triggered by low cytosolic [Ca(2+)], below 5 microm. 3. Inhibition of mitochondrial Ca(2+) uptake required micromolar concentrations of CsA and was not mimicked by other inhibitors of calcineurin such as FK-506 or cypermethrin, nor by a different inhibitor of the PTP, bongkrekic acid. 4. CsA blocked the increase in mitochondrial Ca(2+) uptake rate induced by the mitochondrial Ca(2+) uniporter activator SB202190. 5. Our results suggest that CsA inhibits Ca(2+) entry through the Ca(2+) uniporter by a mechanism independent of the inhibition of PTP or calcineurin. This effect may contribute to reduce depolarization and Ca(2+) overloading in mitochondria after cell stimulation, and thus cooperate with the direct inhibition of PTP to prevent apoptosis.     

Dynamic detection of natural killer cell-mediated cytotoxicity and cell adhesion by electrical impedance measurements

Measurement of electrical impedance is a relatively new real-time and label-free method for monitoring cell adhesive properties. Impedance measurements are performed in tissue culture wells in which the bottom is equipped with gold electrodes. The extent of electrode coverage by living cells as well as the strength of the bond between the cell membrane and the electrode surface determines the impedance, which in real-time cell electrical sensing (RT-CES, ACEA Biosciences, San Diego, CA) is measured as the cell index (CI). We showed for carcinoma cells that CI was linearly correlated to the number of cells and that CI also was related to the amount of coating (laminin-5) of the wells. When natural killer (NK) cells were added to adherent carcinoma cells (target cells) CI declined rapidly dependent on the NK cell:target cell ratio. The initial decrease of CI was much more pronounced than target cell death as measured by [(3)H]thymidine incorporation assay. Such a rapid fall of CI was due to changes in the adhesion and morphology of target cell undergoing apoptosis. It took more than 6 h before the extent of cell death and fall of CI were comparable. We also showed using A431 cells and an antibody specific for the human epidermal growth factor receptor (Erbitux, manufactured by Merck KGaA, Darmstadt, Germany) that RT-CES could be used to monitor antibody-dependent cellular cytotoxicity. Thus RT-CES is a convenient way to continuously determine cell number and cell adhesion and may offer early detection of NK cell-mediated cytotoxic effects.     

Regulation of in vitro generation of cell-mediated cytotoxicity. II. Characterization of thymosin-induced suppressor T cells

Thymosin fraction 5 can induce the appearance of suppressor T cells capable of regulating the in vitro generation of cytotoxic T lymphocytes (CTL) in a 5-day allogeneic or syngeneic mixed lymphocyte-tumor culture (MLTC). Characterization of the suppressor cell population demonstrated it to be a nonadherent, cyclophosphamide-sensitive T cell. In addition, adult thymectomy (Tx) eliminated suppressor cell precursors in spleen, pointing to a direct thymic dependence of the population. Kinetic studies showed that the thymosin influence was necessary for at least the first 12 hr of culture in order for optimal suppression to occur. The in vitro-generated suppressor T cells were antigen specific and could be generated with spleen, thymus, or lymph node cell preparations, suggesting the widespread existence of a regulatory T-cell population under direct humoral thymic control.     

2种静脉铁剂对肾小管上皮细胞及脐静脉内皮细胞的毒性作用

目的：接受血液透析治疗的终末期肾衰患者常接受多种静脉用铁剂，这些铁剂具有潜在的毒性，本实验比较了右旋糖酐铁及蔗糖铁对肾小管上皮细胞（HK-2）及脐静脉内皮细胞的细胞毒性。方法：使用不同剂量的静脉铁剂（0～1．2g·L^-1），分别加入体外培养的人肾小管上皮细胞及脐静脉内皮细胞，孵育0．5～72h。通过检测HDL释放率、MTT吸收率，观察两种铁荆对HK-2细胞及脐静脉内皮细胞的细胞毒性。结果：2种静脉铁剂均能引起细胞致死性损伤；蔗糖铁组的乳酸脱氧酶（LDH）释放率显著高于对照组及低分子右旋糖酐铁组，而低分子右旋糖酐铁组与对照组之间无明显差异。MTT法测细胞生长抑制率结果发现：两种铁剂刺激72h后活细胞数均下降，蔗糖铁组质量浓度不小于0．12g·L^-1时MTT吸收率显著下降，而低分子右旋糖酐铁组浓度为1．2g·L^-1时才出现显著下降。结论：蔗糖铁及低分子右旋糖酐铁均引起剂量依赖性细胞致死性损伤，不同的复合铁剂对肾小管上皮细胞及脐静脉内皮细胞发挥致死性作用的剂量不同，而蔗糖铁的细胞毒性大于低分子右旋糖酐铁。     

Modulation of energy status and cytotoxicity induced by FK506 and cyclosporin A in a renal epithelial cell line

FK506 and cyclosporin A (CsA) are two potent immunosupressants with similar toxicity profile. Nephrotoxicity is the main adverse effect of both compounds. The aim of this study is to compare the in vitro nephrotoxic effects on renal epithelial cell line LLC-PK1 by measuring cell viability and energy status as evaluated by concentrations of ATP and ATP metabolites. Cell viability (expressed as IC₅₀ was assessed via thiazolyl blue (MTT) assay after incubation for 4–24 h with FK506 or CsA. ATP and its metabolites were determined by HPLC after 4 and 6 h incubation with FK506 or CsA alone at the respective IC₅₀. Both FK506 and CsA decreased cell viability to similar extents, in a dose- and time-dependent manner. After 4 h incubation, both drugs decreased ATP levels (−25%) and increased uric acid levels. However, the latter percentage increase was twofold higher with CsA (18%) than with FK506 (9%). The energy charge, calculated according to levels of adenine nucleotides, was decreased by 10% in FK506-treated cells and by 27% in CsA-treated cells. At the end of 6-h incubation, FK506-treated cells maintained ATP levels coupled with energy charge at near control levels whereas the levels were 32% lower in CsA treated cells. Compared to the 4 h-incubation, the increase in uric acid was similar for FK506 but was doubled with CsA. The decrease in cell integrity and ATP depletion induced by CsA in LLC-PK1 cells was only transiently observed with FK506. By preserving energy status, FK506 leads to fewer metabolic disturbances than CsA in the renal epithelial cell line LLC-PK1, demonstrating a minor potential nephrotoxicity. Received: 8 January 1997 / Accepted: 6 March 1997