Influence of lactic acid bacteria and their spent culture supernatant on hydrogen peroxide-induced interleukin-8 synthesis and necrosis of Caco-2 cells

Intestinal epithelial cells are confronted with many noxious stimuli which play an important role in the mucosal immune response. In the present study, Caco-2 cells treated with hydrogen peroxide were used as a model system for studying inflammatory responses and induction of cell death. Live lactobacilli and their concentrated spent culture supernatant (SCS) were applied for studying possible short- and long-term protective effects against hydrogen peroxide-mediated oxidative stress in Caco-2 cells. The secretion of pro-inflammatory cytokine interleukin-8 (IL-8) was investigated in non-filter grown Caco-2 cells, while transepithelial electrical resistance and cell death was studied in filter grown Caco-2 cells. Pre-incubation of Caco-2 cells with Lactobacillus plantarum 2142 did not decrease IL-8 levels induced by 1 mM hydrogen peroxide, nor did lactobacilli suppress IL-8 levels induced by hydrogen peroxide when Caco-2 cells were treated simultaneously with 1 mM hydrogen peroxide and L. plantarum 2142. Thus, lactobacilli did not exert a long-term protective effect against hydrogen peroxide in non-filter grown Caco-2 cells. However, the concentrated SCS of lactobacilli was able to reduce IL-8 levels by more than 6-fold, as determined 24 h after treatment. A short-term effect of lactobacilli was observed in filter grown Caco-2 cells, as they inhibited cell death induced by hydrogen peroxide in the concentration range 10-40 mM. Pre-incubation of epithelial cells with L. plantarum 2142 or simultaneous exposure to hydrogen peroxide and lactobacilli protected Caco-2 cells against cell death. In spite of the presence of lactobacilli, the permeability of membrane increased (transepithelial electrical resistance decreased), and exhibited a similar characteristic pattern as under treatment with hydrogen peroxide alone.     

Protective effect of Hypericum perforatum Linn (St. John's wort) against hydrogen peroxide-induced apoptosis on human neuroblastoma cells

The medicinal plant Hypericum perforatum Linn, commonly known as St. John's wort, has been used as an antidepressant. To investigate whether St. John's wort possesses a protective effect against hydrogen peroxide (H(2)O(2))-induced cytotoxicity in neuronal cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, 4,6-diamidino-2-phenylindole staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, flow cytometry analysis, DNA fragmentation assay, and caspase-3 enzyme assay were performed on SK-N-MC human neuroblastoma cells. Cells treated with H(2)O(2) exhibited several apoptotic features, while those pre-treated with St. John's wort prior to H(2)O(2) exposure showed a decreased occurrence of apoptotic features. In addition, pre-treatment with St. John's wort inhibited H(2)O(2)-induced increase in caspase-3 enzyme activity. These results suggest that St. John's wort may exert a protective effect against H(2)O(2)-induced apoptosis in human neuroblastoma cells.     

Secretion of IL-1 and H2O2 by human mononuclear cells in vitro

The secretion of hydrogen peroxide (H2O2) and interleukin-1alpha (IL-1alpha) was evaluated during in vitro culturing of human monocytes. The oxidative metabolism and cytokine secretion were correlated to the cell distribution (number of surface-associated cells), the DNA content and their integrity, evaluated by lactate dehydrogenase (LDH) assay. The differentiation of cultured monocytes was determined by the expression of CD14, 27E10 and RM3/1. After 24 h cultivation, unstimulated cells had a low production of H2O2 and IL-1alpha. A four-fold increase in the production of H2O2 was detected with 5 and 10 microg/ml of lipopolysaccharide (LPS) and polystyrene (PS) particles. PS particles induced a concentration-dependent increase in IL-1alpha after 24 h. In contrast, cultivation for 48 h, did not result in any measurable production of H2O2, irrespective of the type of stimulus. A decreased viability of monocytes was shown after stimulation with PS particles in high concentrations. Our results indicate that the phenotype expression, adhesion, integrity and secretory pattern of human monocytes is dependent on the culture time and the type and concentration of stimulus.     

Effect of hydrogen peroxide on interleukin-8 synthesis and death of Caco-2 cells

Intestinal epithelial cells can secrete interleukin-8 (IL-8), among other substances in response to different stimuli, which plays an important role in mucosal immune response. Above a certain concentration range, hydrogen peroxide causes cell death by necrosis or apoptosis. We investigated the time- and dose-dependent induction of IL-8 by hydrogen peroxide in the human colon adenocarcinoma cell line Caco-2. In addition, the changes of transepithelial electrical resistance and cell death induction in response to hydrogen peroxide were studied. Nonfilter-grown and filter-grown Caco-2 cells were employed in our experiments. Interleukin-8 synthesis was measured by ELISA. Necrosis was determined by DAPI staining of cells, apoptosis by measuring caspase-3 enzyme activity or annexin V staining. In nonfilter-grown Caco-2 cells, 1 mM of hydrogen peroxide induced the highest level of IL-8 production 24 hr after treatment. In filter-grown Caco-2 cells, IL-8 was produced only on the apical side in response to 1 mM of hydrogen peroxide. This level was 10-fold lower than that measured in nonfilter-grown Caco-2 cells 24 hr after the treatment. In filter-grown Caco-2 cells 10 mM hydrogen peroxide induced the highest IL-8 level on the apical as well as basolateral side. Transepithelial electrical resistance decreased markedly upon application of 40 mM hydrogen peroxide. Late effect of hydrogen peroxide was observed in nonfilter-grown Caco-2 cells, as 1 mM hydrogen peroxide caused necrosis after 24 hr while early-necrosis induction occurred in filter-grown cells exposed to 40 mM of hydrogen peroxide after 1 hr. Filter-grown Caco-2 cells were less sensitive to hydrogen peroxide than the nonfilter-grown ones.     

Effect of Hydrogen Peroxide on Interleukin-8 Synthesis and Death of Caco-2 Cells

Intestinal epithelial cells can secrete interleukin-8 (IL-8), among other substances in response to different stimuli, which plays an important role in mucosal immune response. Above a certain concentration range, hydrogen peroxide causes cell death by necrosis or apoptosis. We investigated the time- and dose-dependent induction of IL-8 by hydrogen peroxide in the human colon adenocarcinoma cell line Caco-2. In addition, the changes of transepithelial electrical resistance and cell death induction in response to hydrogen peroxide were studied. Nonfilter-grown and filter-grown Caco-2 cells were employed in our experiments. Interleukin-8 synthesis was measured by ELISA. Necrosis was determined by DAPI staining of cells, apoptosis by measuring caspase-3 enzyme activity or annexin V staining. In nonfilter-grown Caco-2 cells, 1 mM of hydrogen peroxide induced the highest level of IL-8 production 24 hr after treatment. In filter-grown Caco-2 cells, IL-8 was produced only on the apical side in response to 1 mM of hydrogen peroxide. This level was 10-fold lower than that measured in nonfilter-grown Caco-2 cells 24 hr after the treatment. In filter-grown Caco-2 cells 10 mM hydrogen peroxide induced the highest IL-8 level on the apical as well as basolateral side. Transepithelial electrical resistance decreased markedly upon application of 40 mM hydrogen peroxide. Late effect of hydrogen peroxide was observed in nonfilter-grown Caco-2 cells, as 1 mM hydrogen peroxide caused necrosis after 24 hr while early-necrosis induction occurred in filter-grown cells exposed to 40 mM of hydrogen peroxide after 1 hr. Filter-grown Caco-2 cells were less sensitive to hydrogen peroxide than the nonfilter-grown ones.     

The Arctic Alzheimer mutation enhances sensitivity to toxic stress in human neuroblastoma cells

The E693G (Arctic) mutation of the amyloid precursor protein was recently found to lead to early-onset Alzheimer's disease in a Swedish family. In the present study, we report that the Arctic mutation decreases cell viability in human neuroblastoma cells. The cell viability, as measured by the MTT assay and propidium iodide staining, was further compromised following exposure to calcium ionophore A23187, microtubule-binding colchicine or oxidative stress inducer hydrogen peroxide. The manner of cell death was found to be apoptotic. During apoptosis, cells with the Arctic mutation also decreased their secretion of beta-secretase cleaved amyloid precursor protein. The enhanced sensitivity to toxic stress in cells with the Arctic mutation most likely contributes to the pathogenic pathway leading to Alzheimer's disease.     

Combined effect of hydrogen peroxide induced oxidative stress and IL-1 alpha on IL-8 production in CaCo-2 cells (a human colon carcinoma cell line) and normal intestinal epithelial cells

Oxidative stress, IL-1, and IL-8 are known to contribute to mucosal inflammation of the gastrointestinal tract. We examined the IL-8 response after brief exposure to hydrogen peroxide induced oxidative stress in CaCo-2 cells (a human colon carcinoma cell line) and in human intestinal epithelial cells. In addition, we examined whether exposure to oxidative stress, followed by IL-1, could modulate IL-8 production. A transient up-regulation of IL-8 mRNA expression was observed after hydrogen peroxide treatment. Hydrogen peroxide induced oxidative stress was also observed to promote IL-8 secretion. Exposure to hydrogen peroxide, followed by IL-1, enhanced IL-8 production over that achieved with IL-1 alone. Thus, oxidative stress and IL-1 were observed to cooperatively enhance IL-8 production.     

Modulation of human microglia and THP-1 cell toxicity by cytokines endogenous to the nervous system

Neuroinflammatory processes are thought to be a significant factor in the pathology of a number of degenerative neurological diseases. A variety of cytokines influence inflammatory levels. Here we show that a cooperative action of two or more cytokines is required to induce significantly human microglial and monocytic THP-1 cell toxicity towards SH-SY5Y neuroblastoma cells. Such toxicity was induced by the following combinations: interferon-gamma (IFN-gamma) with tumor necrosis factor-alpha (TNF-alpha); IFN-gamma with interleukin (IL) 1alpha or IL-1beta in the presence of TNF-alpha; and IL-6 with TNF-alpha. Toxicity induced by the various stimulatory combinations was not accompanied by an increased nitrite production. Of the potential inhibitors tested, IL-4 downregulated the toxic action of microglia when applied to THP-1 cells either before stimulation or 24 h after stimulation. Toxicity was not inhibited by IL-10, and was even enhanced by transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF). These data suggest that antagonists of cytokine receptors, as well as inhibitors of their intracellular pathways may be effective anti-inflammatory agents.     

肺巨细胞癌不同转移亚克隆生长因子表达及反应性差异的探讨

目的研究肺巨细胞癌高转移亚系ＰＧｂＥ１和中度转移亚系ＰＧＬＨ７细胞之间部分生长因子的表达及反应性差异。方法利用ＲＴ－ＰＣＲ技术检测了生长因子ＴＧＦα、ＴＧＦβ１、ＩＬ－６、ＩＬ－８、ｂＦＧＦ和ＡＮＧ及受体ＥＧＦＲ、ＩＬ－６Ｒ和ＩＬ－８Ｒ的表达状况;其次采用３Ｈ－ＴｄＲ掺入法观察了重组ＴＧＦα、ＴＧＦβ１和ＩＬ－６对此两个细胞生长的影响。结果ＰＧｂＥ１细胞中ＴＧＦα、ＥＧＦＲ、ＩＬ－６和ＩＬ－６Ｒ的表达水平明显高于ＰＧＨ７细胞,而ＴＧＦβ１、ｂＦＧＦ、ＩＬ－８、ＩＬ－８Ｒ和ＡＮＧ的表达在两个细胞间无明显差别;重组ＴＧＦα和ＩＬ－６对两个细胞均具有生长刺激作用;ＴＧＦβ１具有双重效应。结论ＴＧＦα、ＴＧＦβ１、ｂＦＧＦ、ＩＬ－６、ＩＬ－８、和ＡＮＧ等生长因子可能以自分泌方式参与肺巨细胞癌的生长增殖调控,而ＴＧＦα和ＩＬ－６在肺巨细胞癌的转移中发挥更为重要的作用。     

β-淀粉样肽对人SH-SY5Y神经细胞膜性脂质和胆碱能受体的影响

目的研究β-淀粉样肽对人SH-SY5Y神经母细胞瘤细胞膜性脂质和胆碱能受体的影响及其意义。方法选择不同浓度B一淀粉样肽。处理SH—SY5Y细胞,用比色法测定细胞四甲基偶氮唑盐（MTT）还原率、脂质过氧化产物（丙二醛）、蛋白氧化产物（蛋白羰基）及磷脂含量,高效液相色谱法测定细胞胆固醇及辅酶Q水平,放射性配体一受体结合实验测定胆碱能受体．配体结合率,Western印迹方法测定细胞膜神经型尼古丁受体亚单位α3及α7蛋白表达水平,并对照研究维生素E的抗氧化作用。结果用低浓度（0．1μmol／L）β-淀粉样肽处理细胞后即出现MTT还原率和磷脂水平的下降,丙二醛及蛋白羰基水平升高,用较高浓度（1μmol／L）β-淀粉样肽处理细胞后胆碱能受体．配体结合密度、尼古丁受体亚单位α3,胡蛋白水平及辅酶Q含量降低,但未引起细胞胆固醇含量的改变。维生素E能明显对抗B-淀粉样肽的细胞毒性作用。结论β-淀粉样肽引起细胞膜脂质成分改变以及胆碱能受体水平降低,这些改变可能与其诱导的氧化应激增强有关。     

Hydrogen peroxide induces apoptosis of chondrocytes; involvement of calcium ion and extracellular signal-regulated protein kinase

OBJECTIVE: Recent observations demonstrated that reactive oxygen species facilitate cartilage degradation. We demonstrated that hydrogen peroxide (H2O2) caused inhibition of proteoglycan synthesis, induction of apoptosis and stimulation of extracellular signal-regulated protein kinase (ERK) of the chondrocytes (Inflamm Res 48: 399-403, 1999). To determine whether activation of ERK is involved in the induction of chondrocyte apoptosis, we examined the signal transduction pathways in this hydrogen peroxide induced apoptosis. DESIGN: Bovine articular chondrocytes were cultured. To determine the induction of apoptosis, Annexin V staining and terminal deoxynucleotidyl transferase were used. The activity of caspase-3 was measured using an apopain assay kit. Intracellular Ca2+ imaging was observed after fura2-AM loading. RESULTS: Hydrogen peroxide enhanced annexin V positive apoptotic cells and caspase-3 activity, which is an executor of apoptosis. Hydrogen peroxide also enhanced intracellular Ca2+ and preincubation with the intracellular Ca2+ chelator protected chondrocytes against hydrogen peroxide-induced cell apoptosis, indicating that an increase in the cytosolic Ca2+ plays a decisive role in this action. When ERK activity was blocked with geldanamycin and PD098059, increased apoptosis was evident. CONCLUSION: Hydrogen peroxide induces chondrocyte apoptosis via Ca2+ signaling, and ERK is involved in these signal transduction pathways.     

Generation of interleukin-8 from human monocytes in response to Trichomonas vaginalis stimulation.

Neutrophils are the predominant inflammatory cells found in the vaginal discharges of patients with Trichomonas vaginalis infection. We have investigated the possible role of interleukin-8 (IL-8) in the inflammatory response elicited by T. vaginalis infection. This study has shown that T. vaginalis induces blood monocytes to produce large amounts of bioactive IL-8, mainly by membrane components of T. vaginalis (MTV). Monocyte-derived IL-8 induced by MTV was dose and time dependent. The peak level of IL-8 was 102 +/- 11 ng/ml of conditioned media (mean +/- standard error; n = 5) obtained from MTV-stimulated monocytes (MTVCM) at 36 h of cultivation. With a multichamber chemotactic assay, we found an optimal neutrophil chemotaxis (177 +/- 14 migrated cells) induced by MTVCM collected at 16 h of cultivation when the level of IL-8 was 42 +/- 8 ng/ml. A neutralizing monoclonal antibody directed against IL-8, but not the irrelevant antibodies, significantly blocked the neutrophil chemotactic activity (decreased from 153 +/- 6 to 23 +/- 3 migrated cells; n = 3 [P < 0.001]) induced by MTVCM. Moreover, the maximum increase of the IL-8 mRNA level from MTV-treated monocytes was observed after a 5-h cultivation and decreased thereafter. Monocytes cocultured with MTV in the presence of a neutralizing monoclonal antibody directed against tumor necrosis factor alpha, but not against IL-1 beta, decreased IL-8 production by 25% (P < 0.05), indicating that the release of IL-8 in MTV-stimulated monocytes is partially dependent on tumor necrosis factor alpha. The capacity of MTV-induced monocytes to synthesize IL-8 suggests that these cells can contribute to the induction of the acute inflammatory response seen in T. vaginalis infection.     

The comparative study of reactive oxygen species generated by polymorphonuclear leukocytes as alpha 1-proteinase inhibitor inactivators-possible application for antioxidant prevention of emphysema

The oxidative inactivation of alpha 1-proteinase (alpha 1AP) inhibitor is a one of mechanisms that may lead to the pulmonary emphysema. This process is caused by oxidants derived from atmosphere and released from lung phagocytes. These cells produce various oxidants hydrogen peroxide (H2O2), hypochlorous acid (HClO), hydroxyl (OH.) and superoxide (O2-) radicals after inflammatory stimulation. In this study I have investigated the effects of H2O2 (1.5 x 10(-5) to 1.5 x 10(-2) M) alone or with addition of FeCl2 (50 microM) in order to generate OH., chloramine-T (1.5 x 10(-5) to 1.5 x 10(-3) M) which generates HClO, glucose 10 mg/ml-glucose oxidase (12.5 to 80 mU/ml)-H2O2 generating system, xanthine 0.2 mM-xanthine oxidase (12.5 to 80 mU/ml)-O2-2 generating system on the elastase inhibitory activity of alpha 1AP in vitro. H2O2 was weak in alpha 1AP inactivation--only concentration of H2O2 1.5 x 10(-2) caused severe loss of its activity to 23 +/- 8% inhibition of elastase. Addition of FeCl2 to H2O2 and following OH. generation did not enhance its alpha 1AP inactivation. O2-2 generating system inhibited moderately alpha 1AP. The % inhibition of elastase at concentration of xanthine oxidase 80 mU/ml was 65 +/- 7. HClO was most effective as an alpha 1AP inactivator. All used chloramine-T concentrations completely suppressed alpha 1AP activity. The obtained results and in vivo consumption of H2O2 by polymorphonuclear leukocyte myeloperoxidase for HClO production suggest that scavenging of these reactive oxygen species may be useful in prevention of emphysema.     

Response of the rabbit VH subgroups a and y following antigenic stimulation.

Phorbol myristate acetate (PMA)-activated neutrophils were found to destroy B lymphoblast tumour cells (Raji) as determined by the 51Cr release assay. The target cell lysis was prevented by azide, suggesting the involvement of the myeloperoxidase enzyme. Catalase and cytochrome c caused a marked impairment of the neutrophil-mediated cytolysis, whereas superoxide dismutase significantly enhanced the target cell destruction. These data indicate that hydrogen peroxide plays a key role in the target cell injury; superoxide anion appears to be devoid of direct cytotoxic activity, despite its requirement as a precursor of hydrogen peroxide. The target cell destruction required the presence of the iodide ion as oxidizable co-factor for the myeloperoxidase-hydrogen peroxide system. The chloride ion alone was uneffective. Inhibition of target cell metabolic pathways, involved in the cellular defences against oxidative injury, by the anti-neoplastic agent 1,3-bis-(2-chloroethyl)-1-nitrosurea (BCNU) resulted in an increased neutrophil-mediated cytolysis. Under the experimental conditions employed, PMA-activated neutrophils incubated with BCNU-treated Raji cells became cytotoxic also in the presence of the chloride ion alone as myeloperoxidase co-factor. Our results suggest that Raji target cell destruction by PMA-activated neutrophils depends on the myeloperoxidase-hydrogen peroxide-halide system. The cytolytic event is influenced by target cells themselves, which should be regarded as an active component of the cytotoxic system, capable of interfering with the lytic mediators of the effector cells.     

Overexpression of Cu-Zn superoxide dismutase protects neuroblastoma cells against dopamine cytotoxicity accompanied by increase in their glutathione level

Dopamine (DA) was shown to exert toxic effects on cultured neurons through autoxidation or oxidative deamination, followed by formation of highly reactive quinone compounds and superoxide radicals. In the present study, therefore, any involvement of Cu-Zn superoxide dismutase (SOD) in DA toxicity was evaluated by transfection of Cu-Zn SOD cDNA. The transient transfection of Cu-Zn SOD cDNA inhibited the DA-induced decrease of dopaminergic neuroblastoma cells. Moreover, Cu-Zn SOD cDNA-transfection significantly increased the glutathione (GSH) level when the cells were exposed to DA. However, such Cu-Zn SOD-overexpression failed to show any protective effects against hydrogen peroxide. The Cu-Zn SOD-overexpressing cells also showed significantly higher levels of GSH upon DA exposure than did the empty vector-transfected cells. The increase in the GSH level in response to hydrogen peroxide remained almost identical in empty vector-transfected or Cu-Zn SOD-overexpressed cells. The level of GSH in DA-treated Cu-Zn SOD-overexpressing cells was 2.5-fold higher than that increased by hydrogen peroxide exposure. The catechol structure of DA molecule is probably involved in the mechanism of increasing GSH level. Furthermore, the Cu-Zn SOD-overexpressing cells inhibited the activation of caspase-3 upon DA exposure. Therefore, Cu-Zn SOD overexpression may temporarily inhibit or delay DA autoxidation and consequently increase the GSH level, which then prevents the activation of apoptotic pathway and subsequent cell death.