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.     

Native low-density lipoprotein-dependent interleukin-8 production through pertussis toxin-sensitive G-protein coupled receptors and hydrogen peroxide generation contributes to migration of human aortic smooth muscle cells

Purpose

Stimulation of human aortic smooth muscle cells (hAoSMCs) with native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. However, the process of signal transduction of nLDL was currently uncharacterized. Therefore, the aim of this study was to investigate the signal transduction pathway of nLDL-dependent IL-8 production and the effect of IL-8 on hAoSMCs migration.

Materials and Methods

nLDL was prepared by ultracentrifugation with density-adjusted human serum of normocholesterolemia. In hAoSMCs, IL-8 secreted to medium was measured using ELISA assay, and Western blot analysis was performed to detect p38 MAPK activation as a key regulator of IL-8 production. nLDL-dependent H₂O₂ generation was determined by microscopic analysis using 2'',7''-dichlorofluoroscein diacetate (DCF-DA). IL-8-induced migration of hAoSMCs was evaluated by counting the cell numbers moved to lower chamber using Transwell plates.

Results

nLDL-induced IL-8 production was completely blocked by preincubation of hAoSMCs with pertussis toxin (PTX), which inhibited nLDL-dependent p38 MAPK phosphorylation. PTX-sensitive G-protein coupled receptor was responsible for nLDL-dependent H₂O₂ generation that was abrogated with preincubation of the cells with of polyethylene glycol-conjugated catalase (PEG-Cat). Pretreatment of PEG-Cat prevented nLDL-induced p38 MAPK phosphorylation and IL-8 production, which was partly mimicked by treatment with exogenous H₂O₂. Finally, IL-8 increased hAoSMCs migration that was completely blocked by incubation with IL-8 neutralizing antibody.

Conclusion

PTX-sensitive G-protein coupled receptor-dependent H₂O₂ generation by nLDL plays a critical role in IL-8 production in hAoSMC, and IL-8 may contribute to atherogenesis through increased migration of hAoSMCs.     

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.     

Influence of fluoride, hydrogen peroxide and lactic acid on the corrosion resistance of commercially pure titanium

Titanium is widely used in dental implantology and orthopaedics due to its excellent corrosion resistance and mechanical properties. However, it has been reported that Ti is sensitive to F(-), H(2)O(2) and lactic acid. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to investigate the corrosion resistance of CP-Ti disks after 9 days immersion in different test solutions, based on artificial saliva containing F(-) (0.5% and 2.5%), H(2)O(2) (0.1% and 10%) and/or lactic acid. Because activated macrophages and bacteria can also release locally some of these oxidative compounds, we investigated the role of these cells when plated onto titanium disks. The surface roughness (R(a)) was highly increased when titanium disks were immersed in artificial saliva containing F(-), H(2)O(2) and lactic acid. After 21 days of cell culture, R(a) was significantly increased on disks incubated with activated-J774.2 cells or Streptococcus mitis. AFM appeared to be more sensitive than SEM in evaluating the corrosion of the titanium. Chemical species, either environmental or those released by macrophages and bacteria, can provoke a marked attack of the titanium surface.     

Influence of the oral administration of different lactic acid bacteria on intestinal microflora and iga‐secreting cells in mice treated with ampicillin

It is well known that ampicillin induces an important change in the ecological balance of normal intestinal microflora. The present paper studies the effect of Lactobacillus casei, L. acidophilus, L. delbrueckii subsp bulgaricus and Streptococcus salivarius subsp ther‐mophilus administered orally to mice treated with ampicillin, on the intestinal microflora, on the number of immune cells associated with the gut mucosa and on the IgA‐secreting cells of the small intestine. The animals received 75 mg kg^‐1 of ampicillin orally per day over 3 consecutive days. A quantity of 1.2 X 10⁹ bacteria/day/mouse of the different lactic acid bacteria (LAB) was administered over 2 consecutive days; one group received the LAB on days 0 and 1 and the other on days 2 and 3 following the start of antibiotic treatment. Viable enterobacteria and strict and facultative anaerobic bacteria were determined in faeces and liver, in all the groups, at various intervals. The IgA‐secreting cells in histological sections of small intestine were counted by immunofluorescence test. It was observed that ampicillin therapy produced a decrease in the number of strictly anaerobic bacteria with both a remarkable overgrowth of enterobacteria and translocation to the liver. The study demonstrated that the oral administration of L. casei, L. delbrueckii subsp bulgaricus and L. acidophilus improved the intestinal microflora avoiding the bacterial translocation and increasing the number of IgA‐secreting cells which reach values similar to those in control mice (ampicillin untreated). This effect was more evident when the LAB were administered together with ampicillin (on days 0 and 1). S. thermophilus was not effective in any case. These results suggest that the oral administration of some LAB species could produce beneficial effects in preventing ampicillin‐associated gastrointestinal side‐effects, especially in the immuno‐compromised host.     

Anti-inflammatory properties of heat shock protein 70 and butyrate on Salmonella-induced interleukin-8 secretion in enterocyte-like Caco-2 cells

Intestinal epithelial cells secrete the chemokine interleukin (IL)-8 in the course of inflammation. Because heat shock proteins (Hsps) and butyrate confer protection to enterocytes, we investigated whether they modulate Salmonella enterica serovar Enteritidis (S. serovar Enteritidis)-induced secretion of IL-8 in enterocyte-like Caco-2 cells. Caco-2 cells incubated with or without butyrate (0-20 m M, 48 h) were infected with S. serovar Enteritidis after (1 h at 42 degrees C, 6 h at 37 degrees C) or without prior heat shock (37 degrees C). Levels of Hsp70 production and IL-8 secretion were analysed using immunostaining of Western blots and enzyme-linked immunosorbent assay (ELISA), respectively. The cells secreted IL-8 in response to S. serovar Enteritidis and produced Hsp70 after heat shock or incubation with butyrate. The IL-8 secretion was inhibited by heat shock and butyrate concentrations as low as 0.2 m M for crypt-like and 1 m M for villous-like cells. In a dose-dependent manner, higher butyrate concentrations enhanced IL-8 secretion to maximal levels followed by a gradual but stable decline. This decline was associated with increasing production of Hsp70 and was more vivid in crypt-like cells. In addition, the higher concentrations abolished the heat shock inhibitory effect. Instead, they promoted the IL-8 production in heat-shocked cells even in the absence of S. serovar Enteritidis. We conclude that heat shock and low concentrations of butyrate inhibit IL-8 production by Caco-2 cells exposed to S. serovar Enteritidis. Higher butyrate concentrations stimulate the chemokine production and override the inhibitory effect of the heat shock. The IL-8 down-regulation could in part be mediated via production of Hsp70.     

Effect of tumor necrosis factor-α on thein vitro synthesis of interleukin-8 by human monocytes and lymphocytes

The addition of recombinant tumor necrosis factor or monoclonal antibodies to this factor into human monocyte culture does not change the synthesis of interleukin-8. By contrast, tumor necrosis factor induces the synthesis of interleukin-8 in human lymphocytes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 8, pp. 200–202, August, 1996     

重构型人caspase-8基因的表达及其对HeLa细胞生长的影响

目的 克隆人caspase-8催化结构域基因片段，并将其改建成2种大、小亚基基因次序颠倒的重构型人caspase-8基因，转染HeLa细胞，观察重构型人caspase-8基因的表达及其对HeLa细胞生长的影响。方法 用RT－PCR法，克隆人caspase-8催化结构域基因片段，经重组PCR改造，构建大、小亚基基因次序颠倒的重构型人caspase-8基因。将其克隆入绿色荧光蛋白（GFP）真核表达载体pIRES2-EGFP，转染HeLa细胞，用荧光显微镜和倒置显微镜镜观察细胞的形态和结构。结果 用RT－PCR法，成功地克隆了人caspase-8催化结构域基因片段，构建了3种重构型人caspase-8基因及其真核表达载体。转染HeLa细胞后，重构型人caspase-8基因的表达可导致HeLa细胞死亡。结论 重构型人caspase-8基因在HeLa细胞中的表达可以有效地引起HeLa细胞死亡。     

Effect of phorbol myristate acetate on T cell colony formation, interleukin-2 (IL-2) receptor expression and IL-2 production by cells from patients at all stages of HIV infection.

We and others have shown that several T cell responses induced by the mitogen phytohaemagglutinin (PHA), including T cell colony formation, IL-2 receptor (IL-2R) expression, and IL-2 production are impaired in patients with AIDS and lymphadenopathy syndrome (LAS). We investigated whether phorbol myristate acetate (PMA) could act in synergy with PHA (as it does in healthy subjects) to enhance in vitro T cell responses of patients at all stages of infection by HIV. In AIDS patients with opportunistic infections (AIDS/OI), PHA + IL-2 + PMA led to a total disappearance of T cell colonies in 10/11 patients, among whom six already displayed very low numbers of colonies induced by PHA + IL-2 (less than 50 colonies/5 x 10(4) cells). In contrast, T cell colony formation induced by PHA + IL-2 + PMA was maintained or increased, compared with that induced by PHA + IL-2, in five out of six AIDS patients with Kaposi's sarcoma (AIDS/KS), 10/14 LAS and six out of seven HIV-seropositive asymptomatic (HIV+/AS) homosexuals. In these three groups of patients, a low percentage of colony cells induced by PHA + IL-2 + PMA expressed CD3 and CD4 molecules, but 50-89% of cells were IL-2R (Tac) positive, as in healthy controls. Studies on T cell activation and IL-2 production were performed on a selected group of 12 HIV-infected patients for whom sufficient numbers of lymphocytes could be obtained. PMA induced CD4 down-modulation in controls and in HIV-infected patients. However, CD3 down-modulation and induction of the Tac chain of IL-2R by PMA were significantly impaired in patients, compared with controls, and these two parameters were correlated. Although PHA alone induced virtually normal levels of Tac antigen on patients' cells, Tac induction by PHA + PMA was significantly decreased in patients versus controls. Cells from five out of 10 patients tested failed to produce detectable amounts of IL-2 after PHA stimulation, whereas IL-2 production increased significantly in all patients tested (n = 9) after PHA + PMA, with a level of IL-2 activity significantly higher than in controls. No correlation was found in this group of patients between the effects of PMA + PHA on T cell colony formation, Tac expression, or IL-2 production, as compared with PHA alone. Taken together, our results indicate that in vitro T cell functional studies with PMA may be useful to evaluate better the defects of T cell activation in HIV-infected patients.     

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.     

复方小柴胡汤对荷EAC鼠IL-2水平和CD4+/CD8+比值的影响

目的： 探讨复方小柴胡汤（FFXCHT）对艾氏腹水癌（EAC）荷瘤小鼠脾细胞IL-2水平和血CD4+/CD8+比值的影响。方法： 观察FFXCHT对EAC荷瘤小鼠的肿瘤重量、胸腺和脾系数的影响；用［3H］-TdR掺入法测定脾细胞IL-2的水平；用流式细胞仪测定血CD4+和CD8+T淋巴细胞含量，并计算CD4+/CD8+比值。结果： FFXCHT高、中和低浓度治疗组EAC荷瘤小鼠肿瘤重量均明显少于模型组（P<0.01）。模型组胸腺系数和脾系数高于空白对照组（P<0.05）。FFXCHT高、中和低浓度治疗组脾系数和胸腺系数均明显高于模型组（P<0.01）。与模型组相比，FFXCHT高、中和低浓度治疗均能明显提高荷瘤鼠脾细胞IL-2分泌水平（P<0.05）和血CD4+/CD8+ T淋巴细胞比值（P<0.05）,其中以中浓度治疗组最为明显。结论： FFXCHT能明显抑制EAC肿瘤的生长，提高胸腺和脾系数、脾细胞IL-2活性及血CD4+/CD8+比值，提示FFXCHT可能通过调整EAC荷瘤小鼠免疫功能而发挥其抗肿瘤作用。     

慢性乙肝患者外周血单个核细胞中CXCR1、CXCR2及IL-8 mRNA表达与干扰素治疗关系

目的:了解乙型肝炎病毒(HBV)慢性感染患者外周血单个核细胞(PBMC)中CXCR1、CXCR2及IL-8 mRNA表达水平及与α干扰素(IFN-α)治疗的关系。方法:采用实时定量PCR法动态观察30例慢性乙型肝炎患者接受IFN-α治疗前、治疗3个月、6个月后其外周血单个核细胞CXCR1、CXCR2及IL-8 mRNA表达水平。结果:治疗前慢性乙肝患者CXCR1、CXCR2及IL-8 mRNA表达水平分别为(0.44740.0386)、(0.4720 0.0458)、(1.1897 0.1028),均高于正常对照组(n=36),其中CXCR1及IL-8 mRNA水平升高显著,差异有统计学意义(P<0.01)。治疗过程中CXCR1、CXCR2及IL-8表达水平均显著下降。IFN-α治疗6个月后CXCR1、CXCR2及IL-8 mRNA表达水平分别为(0.41290.0395)、(0.4461 0.0477)、(0.8660 0.1307),与治疗前相比,差异有显著性(P<0.01或P<0.05)。治疗前的CX-CR1、CXCR2及IL-8的表达水平在HBV高复制组(HBV-DNA>106,n=22)明显高于HBV低复制组(HBV-DNA<106,n=8),差异有统计学意义(P<0.05)。结论:慢性乙肝患者外周血单个核细胞中CXCR1和IL-8表达水平显著升高,在干扰素治疗后,其表达水平下调,证明其可能与慢性乙肝炎症的发生机制相关。     

DL-3-n-Butylphthalide protects rat bone marrow stem cells against hydrogen peroxide-induced cell death through antioxidation and activation of PI3K-Akt pathway

Bone marrow stem cells (BMSCs) have been one of the most important cell sources for cell replacement therapy (CRT) in cerebral infarction. However, long-lasting oxidative stress during stroke, which plays an important role in neuron damage, deteriorates the microenvironment for cell survival, differentiation and removal. Thus the outcome of CRT in ischemic diseases was poor. DL-3-n-Butylphthalide (NBP) has protective effects on ischemic brain tissue through multiple mechanisms and has been used for stroke treatment in China for several years. In this study, hydrogen peroxide (H(2)O(2)) was used to induce oxidative stress injury to rat bone marrow stem cells (rBMSCs), imitating the microenvironment surrounding transplanted cells in the ischemic brain in vitro. The protective effects of NBP on rBMSCs against apoptosis induced by oxidative stress were investigated. Our results indicated that NBP could protect rBMSCs against apoptosis due to antioxidative properties and modulation of PI3K/Akt pathway. NBP could be used in combination with BMSCs for the treatment of cerebral infarction by improving the oxidative stress microenvironments and cell survival, however, further studies remain warranted.     

Inhibition effect of arachidonic acid on hypoxia-induced [Ca(2+)](i) elevation in PC12 cells and human pulmonary artery smooth muscle cells

[Ca(2+)](i) elevation is a key event when O(2) sensitive cells, e.g. PC12 cells and pulmonary artery smooth muscle cells, face hypoxia. Ca(2+) entry pathways in mediating hypoxia-induced [Ca(2+)](i) elevation include: voltage-gated Ca(2+) channels (VGCCs), transient receptor potential (TRP) channel and Na(+)-Ca(2+) ex-changer (NCX). In the pulmonary artery, accumulated evidence strongly suggests that prostaglandins (PGs) are involved in pulmonary inflammation and cause vasoconstriction during hypoxia. In this study, we investigated the effect of arachidonic acid (AA), the upstream substrate for PGs, on hypoxia response in O(2) sensitive cells. Exogenous application of AA significantly inhibited hypoxia-induced [Ca(2+)](i) elevation. This effect was due to AA itself rather than its degenerative products. The pharmacological modulation of endogenous AA showed that the prevention of AA generation by blockage of cPLA2, diacylglycerol (DAG) lipase and fatty acid hydrolysis (FAAH), augments hypoxia-induced [Ca(2+)](i) elevation, whereas prevention of AA degeneration attenuates hypoxia-induced [Ca(2+)](i) elevation. Over-expression of COX2 enhances hypoxia-induced [Ca(2+)](i) elevation and this enhancement is reversed by exogenous AA. Our results suggest that AA inhibits hypoxia response. The dynamic alterations in cellular lipids might determine cell response to hypoxia.