Trichostrongylus colubriformis extract upregulates TNF-alpha receptor expression and enhances TNF-alpha sensitivity of L929 cells

Many pathogens have developed strategies to avoid the host's immune system and hence improve their long-term survival. These strategies include antigenic variation, mimicry of host regulatory proteins and production of immunoregulatory molecules. The ruminant gastrointestinal nematode Trichostrongylus colubriformis produces several factors with homology to human immunoregulatory proteins. However, direct immunomodulation by T. colubriformis proteins has not yet been unequivocally demonstrated. Results in the present paper demonstrate that soluble T. colubriformis factors promote proliferation of the TNF-susceptible mouse fibrosarcoma cell line L929, while inhibiting proliferation of all other cell types tested. In addition, T. colubriformis homogenate enhanced the susceptibility of L929 cells to the cytotoxic action of ovine TNF-alpha. Within 1 h of exposure, T. colubriformis factors bind L929 cells in a stable fashion, yet it takes up to 24 h for the cells to become sensitised to TNF-alpha. Interestingly, the increase of both TNF-alpha sensitivity and proliferation of treated L929 cells correlated with an upregulation in expression of TNF-alpha p55 and p75 receptors.     

Localization of active form of caspase-8 in mouse L929 cells induced by TNF treatment and polyglutamine aggregates

The relation between activation of caspase-8 and polyglutamine aggregates has been focused. We prepared an antiserum (anti-m8D387) that recognizes the active form but not the proform of mouse caspase-8. We used immunostaining with anti-m8D387 antiserum to compare the localizations of activated mcaspase-8 in L929 (clone 1422) cells induced by TNF and polyglutamine aggregates. Anti-m8D387 was positive throughout cytoplasm of the TUNEL-positive cells induced by TNF treatment, whereas the anti-m8D387 reactivity was not positive throughout cytoplasm of the cells expressing polyglutamine but was restricted to polyglutamine aggregates. In contrast with TNF-treated cells, cells expressing anti-m8D387-positive cytoplasmic polyglutamine aggregates did not undergo TUNEL-positive apoptosis. Thus activated caspase-8 associated with polyglutamine aggregates alone was not sufficient to induce TUNEL-positive apoptosis of L929 (clone 1422) cells. The distribution of activated caspase-8 associated with polyglutamine aggregates may be essential for the polyglutamine-mediated cell death or downstream of caspase-8 may be different in the TNF-treated cells and cells expressing polyglutamine.     

Tumour necrosis factor induced an early release of superoxide and a late mitochondrial membrane depolarization in L929 cells. Increase in the production of superoxide is not sufficient to mimic the action of TNF

Tumour necrosis factor alpha (TNF) cytotoxicity is mediated, at least in part, by oxidative stress. One of the post-receptor events shortly after the addition of TNF is the generation of the superoxide anion (O2-*). In the present study, we attempted to examine the role of O2-* in the regulation of mitochondrial membrane potential (Delta(Psi)m) and the release of cytochrome c (cyto c) in L929 cells after stimulation with TNF. Challenge of cells with TNF (50 ng/ml) resulted in an early (30 min after the addition of TNF) increase in the production of O2-*. The use of mitochondrial electron transport chain inhibitors such as antimycin A and rotenone could, respectively, potentiate or suppress the TNF-mediated release of O2-* and cytotoxicity. TNF also induced a late (>3 h after the addition of TNF) depolarization in the Delta(Psi)m. Reduction in the release of O2-* by rotenone (50 microM) or thenoyltrifluoroacetone (250 microM) suppressed both the TNF-mediated Delta(Psi)m depolarization and cyto c release. However, increase in the production of O2-* by antimycin A (25 microM) only slightly enhanced the TNF effect in altering the Delta(Psi)m and the release of cyto c. Treating cells with antimycin A alone could not induce a reduction in Delta(Psi)m nor a release of cyto c. Taken together, our results indicate that TNF induced damage in mitochondria in L929 cells. Our data also show that an increase in the production of O2-* was important in the TNF cytotoxicity, but was not sufficient to mimic the action of TNF.     

TNFalpha-induced glutathione depletion lies downstream of cPLA(2) in L929 cells.

Both glutathione (GSH) depletion and arachidonic acid (AA) generation have been shown to regulate sphingomyelin (SM) hydrolysis and are known components in tumor necrosis factor alpha (TNFalpha)-induced cell death. In addition, both have hypothesized direct roles in activation of N-sphingomyelinase (SMase); however, it is not known whether these are independent pathways of N-SMase regulation or linked components of a single ordered pathway. This study was aimed at differentiating these possibilities using L929 cells. Depletion of GSH with L-buthionin-(S,R)-sulfoximine (BSO) induced 50% hydrolysis of SM at 12 h. In addition, TNF induced a depletion of GSH, and exogenous addition of GSH blocked TNF-induced SM hydrolysis as well as TNF-induced cell death. Together, these results establish GSH upstream of SM hydrolysis and ceramide generation in L929 cells. We next analyzed the L929 variant, C12, which lacks both cytosolic phospholipase A(2) (cPLA(2)) mRNA and protein, in order to determine the relationship of cPLA(2) and GSH. TNF did not induce a significant drop in GSH levels in the C12 line. On the other hand, AA alone was capable of inducing a 60% depletion of GSH in C12 cells, suggesting that these cells remain responsive to AA distal to the site of cPLA(2). Furthermore, depleting GSH with BSO failed to effect AA release, but caused a drop in SM levels, showing that the defect in these cells was upstream of the GSH drop and SMase activation. When cPLA(2) was restored to the C12 line by expression of the cDNA, the resulting CPL4 cells regained sensitivity to TNF. Treatment of the CPL4 cells with TNF resulted in GSH levels dropping to levels near those of the wild-type L929 cells. These results demonstrate that GSH depletion following TNF treatment in L929 cells is dependent on intact cPLA(2) activity, and suggest a pathway in which activation of cPLA(2) is required for the oxidation and reduction of GSH levels followed by activation of SMases.     

Retinoid x receptor agonists increase bcl2a1 expression and decrease apoptosis of naive T lymphocytes

Vitamin A affects many aspects of T lymphocyte development and function. The vitamin A metabolites all-trans- and 9-cis-retinoic acid regulate gene expression by binding to the retinoic acid receptor (RAR), while 9-cis-retinoic acid also binds to the retinoid X receptor (RXR). Naive DO11.10 T lymphocytes expressed mRNA and protein for RAR-alpha, RXR-alpha, and RXR-beta. DNA microarray analysis was used to identify RXR-responsive genes in naive DO11.10 T lymphocytes treated with the RXR agonist AGN194204. A total of 128 genes was differentially expressed, including 16 (15%) involved in cell growth or apoptosis. Among these was Bcl2a1, an antiapoptotic Bcl2 family member. Quantitative real-time PCR analysis confirmed this finding and demonstrated that Bcl2a1 mRNA expression was significantly greater in nonapoptotic than in apoptotic T lymphocytes. The RXR agonist 9-cis-retinoic acid also increased Bcl2a1 expression, although all-trans-retinoic acid and ligands for other RXR partner receptors did not. Treatment with AGN194204 and 9-cis-retinoic acid significantly decreased apoptosis measured by annexin V staining but did not affect expression of Bcl2 and Bcl-xL. Bcl2a1 promoter activity was examined using a luciferase promoter construct. Both AGN194204 and 9-cis-retinoic acid significantly increased luciferase activity. In summary, these data demonstrate that RXR agonists increase Bcl2a1 promoter activity and increase expression of Bcl2a1 in naive T lymphocytes but do not affect Bcl2 and Bcl-xL expression in naive T lymphocytes. Thus, this effect on Bcl2a1 expression may account for the decreased apoptosis seen in naive T lymphocytes treated with RXR agonists.     

Role of modulation on the effect of microwaves on ornithine decarboxylase activity in L929 cells

The effect of 835 MHz microwaves on the activity of ornithine decarboxylase (ODC) in L929 murine cells was investigated at an SAR of ∼2.5 W/kg. The results depended upon the type of modulation employed. AM frequencies of 16 Hz and 60 Hz produced a transient increase in ODC activity that reached a peak at 8 h of exposure and returned to control levels after 24 h of exposure. In this case, ODC was increased by a maximum of 90% relative to control levels. A 40% increase in ODC activity was also observed after 8 h of exposure with a typical signal from a TDMA digital cellular telephone operating in the middle of its transmission frequency range (∼840 MHz). This signal was burst modulated at 50 Hz, with approximately 30% duty cycle. By contrast, 8 h exposure with 835 MHz microwaves amplitude modulated with speech produced no significant change in ODC activity. Further investigations, with 8 h of exposure to AM microwaves, as a function of modulation frequency, revealed that the response is frequency dependent, decreasing sharply at 6 Hz and 600 Hz. Exposure with 835 MHz microwaves, frequency modulated with a 60 Hz sinusoid, yielded no significant enhancement in ODC activity for exposure times ranging between 2 and 24 h. Similarly, exposure with a typical signal from an AMPS analog cellular telephone, which uses a form of frequency modulation, produced no significant enhancement in ODC activity. Exposure with 835 MHz continuous wave microwaves produced no effects for exposure times between 2 and 24 h, except for a small but statistically significant enhancement in ODC activity after 6 h of exposure. Comparison of these results suggests that effects are much more robust when the modulation causes low-frequency periodic changes in the amplitude of the microwave carrier. Bioelectromagnetics 18:132–141, 1997. © 1997 Wiley-Liss, Inc.     

Role of P2z purinergic receptors in ATP-mediated killing of tumor necrosis factor (TNF)-sensitive and TNF-resistant L929 fibroblasts.

Two closely related cell lines were characterized in their responses to extracellular ATP (ATPo): the fibroblast cell line L929 and a TNF-resistant variant L929/R. Both lines showed ATPo-activated increases in intracellular Ca2+, inward current, and sustained depolarization of the plasma membrane, cell responses compatible with activation of purinergic receptors of the P2y, P2x, or P2z subtype; however, only the L929/R variant was susceptible to ATPo-dependent early permeabilization of the plasma membrane to hydrophilic solutes of M(r) below 900, a response uniquely caused by the activation of P2z receptors. Both cell types were susceptible to the cytotoxic effect of ATPo, but killing of the L929/R variant required much shorter incubations in the presence of this nucleotide. Morphologic examination of ATPo-challenged L929 and L929/R cells showed that cell death occurred by two alternative mechanisms: colloido-osmotic lysis or apoptosis. Occurrence of apoptosis was confirmed by agarose gel analysis of cellular DNA. Although ATPo caused a fast mobilization of intracellular Ca2+, neither colloido-osmotic lysis nor apoptosis were Ca2+ dependent. Our results show that the L929/R variant, but not the L929 parental fibroblast cell line, expresses functional purinergic receptors of the P2z subtype. The presence of P2z receptors confers to L929/R cells enhanced susceptibility to ATPo-mediated cytotoxicity.     

Tumor necrosis factor-induced interleukin-6 expression and cytotoxicity follow a common signal transduction pathway in L929 cells.

Interleukin (IL)-6 gene induction was studied in response to treatment with Tumor Necrosis Factor (TNF) in the sensitive murine L929 cell line. Under conditions where TNF-mediated cytotoxicity was either increased or decreased, depending on addition of activators or inhibitors, we found that the TNF-induced IL6 gene expression was likewise enhanced or repressed. We conclude that the signal (or part of the signals) going to the nucleus and responsible for gene activation is conducted along the reaction mechanism leading to cellular toxicity.     

Adhesion and the cell cycle in cultured L929 and CHO cells

The adhesiveness of L929 and CHO-K1 cells was monitored throughout their respective cell cycles. The cell cycle stages were identified by a combination of measuring, histochemical and autoradiographic techniques. Adhesiveness was shown to be maximal during the S and late G2 phases, and minimal at mitosis. S cells adhered selectively to other S cells, while late G2 phase cells adhered selectively to other late G2 cells. Mixing of the two cell lines, L929 and CHO-K1, resulted in the S phase cells adhering to each other irrespective of the cell line to which they belonged. The phases of increased adhesiveness and selectivity coincided with a decrease in cell surface negative charge, corresponding to well documented changes in the morphology of the cells.     

tRNA体外抑制L929细胞生长的作用观察

目的:探讨tRNA对不同哺乳动物细胞生长的影响.方法:L929细胞、NIH3T3细胞、MCF-7细胞和PC12细胞接种96孔板,37℃,5% CO2细胞培养箱中培养4 h后,直接加入酵母tRNA,继续培养一定时间后采用MTT法检测细胞生长情况;将200 μg/ml酵母tRNA加入至L929细胞中,在不同时间点观察细胞形态并收集细胞进行细胞流式分析.结果:tRNA对L929细胞有特异的抑制作用,并且表现出一定的剂量依赖性;tRNA处理后L929细胞与正常细胞相比,形态明显变大,而且突起增长;流式细胞仪分析进一步发现tRNA使细胞阻滞于S期.结论:tRNA的这种细胞生长的抑制效应可能是通过其一些小的降解片段发挥的,提示tRNA或者其降解片段可能具有调控L929细胞增殖的重要作用.     

Impairment of reovirus mRNA 'cap' methylation in interferon-treated mouse L929 cells.

Reovirus mRNAs synthesized in vitro by the virionassociated enzyme have a 5' 'cap 1' structure (m7G(5')ppp(5')GmpCp...). However, about one third to one half of the reovirus mRNAs formed in mouse L929 cells have a 5' 'cap 2' structure (m7G(5')ppp(5')GmpCmp...) and the rest have a 5' 'cap 1' structure. The finding that virus mRNA 'cap' methylation is impaired in extracts of interferon-treated cells prompted us to study the effect of interferon on virus mRNA 'cap' methylation in vivo. Using labeling with [3H]-guanosine and dual labeling with [3H]methionine and [14C]uridine we compared the 5' structures of reovirus mRNAs accumulating between 5 and 11 h after infection in: L929 cells treated with 390 to 2600 U/ml of a partially purified mouse interferon preparation and untreated L929 cells. The treatment resulted in a 70 to 98% decrease in the 24 h virus yield and in a 50 to 55% decrease in the label accumulated in virus mRNAs. The 'capping' of virus mRNAs and the methylation of their 5' terminal and adjacent G residues were not diminished in interferon-treated cells. However, the percent of 'cap 2' termini was 36 to 47% lower in virus mRNAs from interferon-treated cells than in virus mRNAs from control cells. The interferon treatment did not result in the appearance of additional methylated nucleotides in the virus mRNAs.     

Effect of tumor necrosis factor on GTP binding and GTPase activity in HL-60 and L929 cells

Tumor necrosis factor (TNF) is a monokine that induces pleiotropic events in both transformed and normal cells. These effects are initiated by the binding of TNF to high affinity cell surface receptors. The post-receptor events and signaling mechanisms induced by TNF, however, have remained unknown. The present studies demonstrate the presence of a single class of high affinity receptors on membranes prepared from HL-60 promyelocytic leukemic cells. The interaction of TNF with these membrane receptors was associated with a 3.8-fold increase in specific binding of the GTP analogue, GTP gamma S. Scatchard analysis of GTP gamma S binding data demonstrated that TNF stimulates GTP binding by increasing the affinity of available sites. The TNF-induced stimulation of GTP binding was also associated with an increase in GTPase activity. Moreover, the increase in GTPase activity induced by TNF was sensitive to pertussis toxin. The results also demonstrate that TNF similarly increased GTP binding and pertussis toxin-sensitive GTPase activity in membranes from mouse L929 fibroblasts, thus indicating that these effects are not limited to hematopoietic cells. Analysis of HL-60 membranes after treatment with pertussis toxin in the presence of [32P]NAD revealed three substrates with relative molecular masses of approximately Mr 41,000, 40,000, and 30,000. In contrast, L929 cell membranes had only two detectable pertussis toxin substrates of approximately Mr 41,000 and 40,000. Although the Mr 41,000 pertussis toxin substrate represents the guanine nucleotide-binding inhibitory protein Gi, the identities of the Mr 40,000 and Mr 30,000 substrates remain unclear. In any event, inhibition of the TNF-induced increase in GTPase activity and ADP-ribosylation of Gi by pertussis toxin suggested that TNF might act by increasing GTPase activity of the Gi protein. However, the results further indicate that TNF has no detectable effect on basal or prostaglandin E2-stimulated cAMP levels in HL-60 cells. Taken together, these findings indicate that a pertussis toxin-sensitive GTP-binding protein other than Gi, and possibly the Mr 40,000 substrate, is involved in the action of TNF. Finally, the demonstration that pertussis toxin inhibited TNF-induced cytotoxicity in L929 cells supports the presence of a GTP-binding protein which couples TNF-induced signaling to a biologic effect.     

Defects in TLR3 expression and RNase L activation lead to decreased MnSOD expression and insulin resistance in muscle cells of obese people

Obesity is associated with chronic low-grade inflammation and oxidative stress that blunt insulin response in its target tissues, leading to insulin resistance (IR). IR is a characteristic feature of type 2 diabetes. Skeletal muscle is responsible for 75% of total insulin-dependent glucose uptake; consequently, skeletal muscle IR is considered to be the primary defect of systemic IR development. Interestingly, some obese people stay insulin-sensitive and metabolically healthy. With the aim of understanding this difference and identifying the mechanisms responsible for insulin sensitivity maintenance/IR development during obesity, we explored the role of the latent endoribonuclease (RNase L) in skeletal muscle cells. RNase L is a regulator of innate immunity, of double-stranded RNA sensors and of toll-like receptor (TLR) 4 signaling. It is regulated during inflammation by interferons and its activity is dependent on its binding to 2-5A, an oligoadenylate synthesized by oligoadenylate synthetases (OAS). Increased expression of RNase L or downregulation of its inhibitor (RLI) improved insulin response in mouse myogenic C2C12 cells and in primary human myotubes from normal-weight subjects treated with palmitate, a saturated free fatty acid (FFA) known to induce inflammation and oxidative stress via TLR4 activation. While RNase L and RLI levels remained unchanged, OAS level was decreased in primary myotubes from insulin-resistant obese subjects (OB-IR) compared with myotubes from insulin-sensitive obese subjects (OB-IS). TLR3 and mitochondrial manganese superoxide dismutase (MnSOD) were also underexpressed in OB-IR myotubes. Activation of RNase L by 2-5A transfection allowed to restore insulin response, OAS, MnSOD and TLR3 expression in OB-IR myotubes. Due to low expression of OAS, OB-IR myotubes present a defect in RNase L activation and TLR3 regulation. Consequently, MnSOD level is low and insulin sensitivity is reduced. These results support that RNase L activity limits FFA/obesity-induced impairment of insulin response in muscle cells via TLR3 and MnSOD expression.     

锰超氧化物歧化酶(MnSOD)表达与活性调控及其与肿瘤的关系

线粒体锰超氧化物歧化酶(MnSOD)是细胞中主要的抗氧化酶,其在清除细胞中活性氧自由基(ROS),应对细胞氧化应激及维持细胞正常生理代谢过程中起着重要作用。锰超氧化物歧化酶(MnSOD)基因表达以及蛋白活性受到多种途径调控,并与多种疾病的发生密切相关。本文介绍了锰超氧化物歧化酶(MnSOD)的生物学特性,并从锰超氧化物歧化酶(MnSOD)的转录调控、表观遗传调控以及翻译后修饰等方面,对锰超氧化物歧化酶(MnSOD)基因表达及活性调控机制研究进展进行了综述。     

IkappaBalpha is essential for maintaining basal c-Jun N-terminal kinase (JNK) activation and regulating JNK-mediated resistance to tumor necrosis factor cytotoxicity in L929 cells.

Early activation of c-Jun N-terminal kinase (JNK) is believed to block apoptosis in response to death signals such as tumor necrosis factor (TNF). Brief exposure of murine L929 fibroblasts to anisomycin for 1 hr to activate JNK resulted in resistance to TNF killing. TNF rapidly induced cytoplasmic shrinkage in control cells, but not in the anisomycin-pretreated L929 cells. However, the induced TNF resistance was suppressed in the L929 cells which were engineered to stably inhibit IkappaBalpha protein expression by antisense mRNA ( approximately 80% reduction in protein expression). No constitutive NF-kappaB nuclear translocation and increased TNF resistance were found in these IkappaBalpha antisense cells. Notably, these cells had a significantly reduced basal level of JNK activation (50-70%), compared to vector control cells. Furthermore, brief exposure of L929 cells to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), resulted in resistance to TNF killing, probably due to preconsumption of caspases by wortmannin. Nonetheless, wortmannin-induced TNF resistance was suppressed in the IkappaBalpha antisense cells. Thus, these observations indicate that IkappaBalpha is essential for maintaining the basal level of JNK activation and regulating the JNK-induced TNF resistance.