Treponema pallidum flagellin FlaA2 induces IL-6 secretion in THP-1 cells via the Toll-like receptor 2 signaling pathway

Treponema pallidum subsp. pallidum membrane proteins are considered as potent inducers in the initiation and development of inflammation. In the present study, the mechanism that leads to the production of interleukin 6 (IL-6), one of the key proinflammatory cytokines, by human monocytic THP-1 cells when these cells are treated with T. pallidum flagellin FlaA2 was investigated. Stimulation with flagellin FlaA2 can induce IL-6 expression in human monocytes and augment the phosphorylation of ERK, p38, and NF-κB, but has no effect on the phosphorylation of JNK. Likewise, FlaA2-induced IL-6 production was found to be attenuated by inhibitors for ERK, p38, and NF-κB, but not by JNK inhibitor. Immunofluorescence analysis showed that flagellin FlaA2 could stimulate the translocation of IκBα from the cytosol to the nucleus, and this phenomenon could be inhibited by the specific inhibitor BAY11-7082. FlaA2–induced IL-6 expression was also proved to be abrogated by transfection with dominant negative (DN) plasmid of MyD88. We further demonstrated that transfection with DN-TLR2 was sufficient to attenuate IL-6 expression and the phosphorylation of ERK, p38, and IκBα. These results suggest that flagellin FlaA2 induces IL-6 production via signaling pathways involving TLR2, MyD88, ERK, p38, and NF-κB in monocytes, which could contribute to the pathogenesis of T. pallidum.     

白细胞介素-6诱导相关新基因LX3的细胞定位研究

目的 研究IL-6诱导相关新基因KX3在真核细胞内的表达定位。方法构建真核融合表达载体pEGFP-N1／LX3,运用Lipofectin转染293T和NIH3T3细胞。共聚焦荧光显微镜检测GFP荧光,以确定IL-6诱导相关新基因KX3在细胞内的表达定位情况。结果获得真核表达载体pEGFP-N1／LX3,并成功转染293T和NIH3T3细胞,共聚焦荧光显微镜观察了KX3-GFP在细胞内的荧光分布。结论IL-6诱导相关新基因在细胞内为全细胞分布。     

Autostimulatory effects of IL-6 on excessive B cell differentiation in patients with systemic lupus erythematosus: analysis of IL-6 production and IL-6R expression.

Introducing avidin-biotin complex ELISA for anti-DNA antibody, the mechanism of in vitro production of anti-ssDNA antibody as well as of polyclonal immunoglobulin mediated by an IL-6-IL-6R loop was studied in patients with systemic lupus erythematosus (SLE). Regardless of the presence or absence of T cells, B cells from SLE patients could produce IgG anti-ssDNA antibody as well as total IgG without any stimulation. Low density B cells obtained by Percoll gradient density centrifugation responded to rIL-6 to produce IgG and IgG anti-ssDNA antibody. rIL-2 and rIL-4 had lesser effects on the differentiation of low density B cells. In fact, IL-6R was preferentially expressed on low density B cells from active SLE patients, as detected by anti-IL-6R MoAb, MT18, which did not inhibit IL-6 binding. SLE B cells, especially high density B cells, produced greater amounts of IL-6 in culture supernatants than did T cells, regardless of whether disease was active or inactive. Normal T cells and B cells did not produce significant amounts of IL-6. Thus, endogenous IL-6 produced by high density B cells bound to the IL-6R preferentially expressed on the low density B cells, and drove them into terminal differentiation, especially in active SLE patients. Further, addition of polyclonal anti-IL-6 or anti-IL-6R MoAb (PM1), which inhibited IL-6 binding, both inhibited IgG anti-ssDNA antibody as well as total IgG production by SLE B cells in a dose-dependent manner. These results suggest that interruption of the autocrine IL-6 loop would be of therapeutic value in SLE.     

Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells

A number of cell types situated along interfaces of various tissues and organs such as the peritoneum and the intestine have been shown to secrete inflammatory cytokines in a polarized fashion. Retinal pigment epithelial (RPE) cells are positioned at the interface between the vascularized choroid and the avascular retina, forming part of the blood–retina barrier. These cells are potent producers of inflammatory cytokines and are therefore considered to play an important role in the pathogenesis of ocular inflammation. Whether cytokine secretion by these cells also follows a vectorial pattern is not yet known, and was therefore the subject of this study. Monolayers of human RPE cells (primary cultures and the ARPE-19 cell line) cultured on transwell filters were stimulated to produce IL-6 and IL-8 by adding IL-1β (100 U/ml) to either the upper or the lower compartment. After stimulation, the human RPE cell lines showed polarized secretion of IL-6 and IL-8 towards the basal side, irrespective of the side of stimulation. The ARPE-19 cell line also secreted IL-6 and IL-8 in a polarized fashion towards the basal side after basal stimulation; polarized secretion was, however, not apparent after apical stimulation. The observation that human RPE cells secrete IL-6 and IL-8 in a polarized fashion towards the choroid may represent a mechanism to prevent damage to the adjacent fragile retinal tissue.     

Interferon-gamma stimulates the secretion of IL-1, but not of IL-6, by glomerular mesangial cells.

IL-1 activity in culture supernatant and cell lysate from rat mesangial cells stimulated with interferon-gamma (IFN-gamma) was measured by a thymocyte proliferation assay. While IFN-gamma alone had no effect on the secretion or the intracellular pool of IL-1, the enhancement by IFN-gamma of IL-1 secretion in response to lipopolysaccharide (LPS) was observed. The stimulatory effect of culture supernatant on thymocyte proliferation was abrogated by preincubation with the anti-IL-1 antibody. At least 4-h incubation with IFN-gamma and LPS was required to detect enhancing effect of IFN-gamma. The addition of as little as 1 U/ml IFN-gamma significantly increased IL-1 secretion in the presence of 10 micrograms/ml LPS. The IL-6 activity in culture supernatants was determined by measurement of thymidine uptake in mouse IL-6-dependent cell line (MH60.BSF2). Mesangial cells secreted IL-6 in culture supernatant without additional stimuli and LPS distinctly increased it as described previously. However, in contrast to IL-1 production, no effect of IFN-gamma on IL-6 secretion was observed in the presence or absence of LPS. Moreover, we determined whether enhanced IL-1 release is associated with Ia expression on mesangial cells. IFN-gamma alone and the combination with LPS induced marked expression of Ia antigen, whereas LPS alone did not. We conclude that IFN-gamma stimulates the production of IL-1, but not IL-6, by mesangial cells and suggest an important role of IFN-gamma in the pathogenesis of glomerulonephritis by regulating the mesangial production of IL-1 and the accessory cell function of mesangial cells.     

卵巢癌细胞IL-6、IL-8及其受体表达的研究

目的分析比较五种常见的上皮性卵巢癌细胞系IL-6、IL-8及其受体表达的差异。方法IL-6、IL-8的表达分别采用RT-PCR和ELISA法进行检测,IL-6受体（IL-6Rα和gp130）及IL-8受体（IL-8RA和IL-8RB）的表达采用免疫印迹技术进行测定。结果①五种上皮性卵巢癌细胞均组成性表达IL-6和IL-8。IL-6和IL-8在CAOV-3细胞中的表达水平均最高,而在HO-8910PM细胞中的表达水平均最低,IL-6在SKOV-3、HO-8910、OVCAR-3细胞中的表达水平依次降低,IL-8在OVCAR-3、SKOV-3、HO-8910细胞中的表达水平依次降低。②五种上皮性卵巢癌细胞均表达IL-6Rα、gp130及IL-8RA;除CAOV-3细胞外,其它细胞均表达IL-8RB。结论本研究旨在筛选表达IL-6和IL-8及其相应受体的细胞株,为研究IL-6、IL-8与卵巢癌发生、发展关系奠定基础,同时也为今后卵巢癌的免疫治疗提供一个新的思路。     

Retinoic acid modulates the in vivo and in vitro growth of IL-6 autocrine human myeloma cell lines via induction of apoptosis

We previously showed that IL-6 is an autocrine growth factor for two human myeloma cell lines, RPMI 8226 and U266. We investigated here the in vitro and in vivo effects of all-trans retinoic acid (RA) on the growth and survival of these two cell lines. RA induced a dramatic dose- and time-dependent inhibition of the proliferation of both cell lines. This inhibition was correlated with a down-modulation of the cell surface expression of the IL-6 binding chain (gp80) and the transducing chain (gp130) of the IL-6 receptor (IL-6R). Long-term culture experiments showed that down-modulation of gp80 expression was complete at days 15 and 30 in the presence of 10^–5 and 10^–7 mol/l of RA, respectively. Gp130 expression was greatly decreased, albeit still detectable, in similar culture conditions. RA-mediated interruption of the IL-6 autocrine loop was associated with a decrease of bcl-2 oncoprotein expression and apoptosis of the myeloma cells which was RA concentration- and time-dependent. The in vivo relevance of the effects of RA was studied on tumours which developed in nude mice inoculated with a subclone of RPMI 8226. Whereas tumours grew in all control mice, 40% of tumours regressed within 20 days in RA-treated mice. Cells from regressing tumours featured characteristics of apoptosis and exhibited low gp80 and gp 130 expression. Our study indicate that long-term RA treatment interferes in vivo and in vitro with IL-6 autocrine growth of myeloma cell lines, leading to apoptosis.     

卵巢癌细胞IL-6、IL-8分泌与其化疗敏感性及耐药相关基因和凋亡抑制基因表达关系的初步探讨

目的 分析比较4种常见的人E皮性卵巢癌细胞系IL-6、IL-8分泌与其化疗敏感性及部分耐药相关基因和凋亡抑制基因表达的关系,为今后研究IL-6、IL-8诱导卵巢癌细胞对化疗药物产生耐药的机制奠定基础.方法 IL-6、IL-8及其受体的表达采用RT-PCR、ELISA及免疫印迹技术进行检测,卵巢癌细胞对顺铂和紫杉醇的敏感性采用M1Tr试验进行分析,耐药相关基因和凋亡抑制基因的表达则采用RT-PCR进行测定.结果 1)4种卵巢癌细胞除A2780细胞外均组成性分泌IL-6和IL-8,二者转录水平与其蛋白水平基本一致.2)4种卵巢癌细胞均表达IL-6受体和IL-8受体.3)4种卵巢癌细胞对顺铂和紫杉醇的敏感性不同,其中A2780细胞最敏感,ES-2细胞次之,CAOV-3和SKOV-3细胞则有不同程度的耐药性.4)部分耐药相关基因和凋亡抑制基因在A2780和ES-2细胞中的表达水平均较低,而在CAOV-3和SKOV-3细胞中的表达水平均较高.结论 卵巢癌细胞IL-6、IL-8的自分泌水平(尤其是IL-6的水平)与其对顺铂和紫杉醇的敏感性基本呈负相关趋势,与其部分耐药相关基因和凋亡抑制基因的表达水平相一致.     

The cannabinoid R(+)methanandamide induces IL-6 secretion by prostate cancer PC3 cells

In the present study, we have investigated the effect of the cannabinoid R(+)methanandamide (MET) in the androgen-resistant prostate cancer PC3 cells. MET induced a dose-dependent decrease in PC3 cell viability as well as a dose-dependent increase in the secretion of the cytokine IL-6. Looking deeper into the mechanisms involved, we found that MET-induced de novo synthesis of the lipid mediator ceramide that was blocked by the ceramide synthase inhibitor Fumonisin B1. Pre-incubation of cells with the cannabinoid receptor CB2 antagonist SR 144528 (SR2), but not the CB1 antagonist Rimonabant or the TRPV1 antagonist capsazepine, partially prevented the anti-proliferative effect, the ceramide accumulation, and the IL-6-induced secretion, suggesting a CB2 receptor-dependent mechanism. Fumonisin B1 did not have any effect in the IL-6 secretion increase induced by MET. However, even an incomplete down-regulation of (i.e., not a total silencing of) ceramide kinase expression by specific siRNA prevented the MET-induced IL-6 secretion. These results suggest that MET regulates ceramide metabolism in prostate PC3 cells which is involved in cell death as well as in IL-6 secretion. Our findings also suggest that CB2 agonists may offer a novel approach in the treatment of prostate cancer by decreasing cancer epithelial cell proliferation. However, the interaction of prostate cancer cells with their surrounding, and in particular with the immune system in vivo, needs to be further explored.     

miR-24-3p靶向KLF6基因调控IL-6/STAT3信号通路影响食管癌细胞的活力和凋亡

目的:探讨微小RNA-24-3p(miR-24-3p)对食管癌细胞活力和凋亡的影响及机制。方法:以人正常食管上皮细胞HEEC为对照,采用RT-qPCR检测食管癌细胞TE11、Eca109和EC9706中miR-24-3p和KLF6 mRNA的表达,Western blot检测KLF6蛋白的表达。用anti-miR-24-3p和KLF6 siRNA转染EC9706细胞,MTT检测细胞活力,流式细胞术检测细胞凋亡率,Western blot检测检测细胞中与增殖、凋亡相关的蛋白以及IL-6/STAT3信号通路相关蛋白的表达,ELISA法检测IL-6的表达。双萤光素酶报告基因实验验证miR-24-3p与KLF6靶向调控的关系。结果:食管癌癌细胞TE11、Eca109和EC9706中miR-24-3p表达上调(P<0.05),KLF6的mRNA和蛋白表达下调(P<0.05)。敲减EC9706细胞miR-24-3p表达可抑制其细胞活力,诱导其凋亡,并抑制细胞CDK4、cyclin D1、CDC25A、p-STAT3、IL-6及Bcl-2的表达,促进caspase-3和Bax的表达。结论...     

Cytokine-specific ELISPOT assay single cell analysis of IL-2, IL-4 and IL-6 producing cells

In order to assess cytokine-producing cells at the single cell level, the cytokine-specific ELISPOT assay has proven to be an important and sensitive method. The purpose of this study was to adapt this method to elucidate individual cells producing murine IL-2, IL-4 or IL-6. In order to establish these cytokine-specific ELISPOT assays, IL-2-, IL-4- and IL-6-specific cDNA transfected myeloma cell lines, e.g., X63-Ag8-653 X2, X63-Ag8-653 X4 and X63-Ag8-653 X6, respectively, were used as specific cytokine-producing cells. In the IL-2 ELISPOT assay, the coating reagent, monoclonal antibody (mAb) rat IgG2a anti-mouse IL-2 (CR #40014) was used while rabbit IgG polyclonal anti-mouse IL-2 was employed for detection of IL-2 spot forming cells (SFC). The mAbs anti-mouse IL-4, BVD4-1D11 and BVD6-24G2 were selected as capture and detection antibodies for enumeration of IL-4 SFC. For the IL-6 ELISPOT assay, anti-mouse IL-6 (MP5-20F3) mAb was used for coating and MP5-32C11 mAb was used for detection of IL-6 SFC. When IL-2 producing X63-Ag8-653 X2 cells were subjected to these three different ELISPOT assays, IL-2-specific SFC were only noted with the IL-2 ELISPOT system. In the case of IL-4 SFC, only X63-Ag8-653 X4 cells formed specific spots using the tandem of BVD4-1D11 and BVD6-24G2 mAbs. IL-6-specific spots developed in MP5-20F3 mAb pre-coated wells containing X63-Ag8-653 X6 cells, when developed with mAb anti-IL-6 (MP5-32C11). Addition of cycloheximide (50 μg/ml) inhibited formation of IL-2, IL-4 and IL-6 SFC by approximately 90%. When an unrelated mAb was used as detection antibody in these three different cytokine-specific ELISPOT assays, IL-2-, IL-4-and IL-6-specific SFC were not detected. Further, when concanavalin A stimulated T cells from Peyer's patch of normal mice were subjected to the respective cytokine-specific ELISPOT assay, IL-2, IL-4 and IL-6 SFC were enumerated. These results have shown that cytokine-specific IL-2, IL-4 and IL-6 ELISPOT assays have now been established and will allow analysis of the frequency of cytokine-secreting cells at the single cell level.     

卵巢癌细胞IL-6、IL-8分泌与其他莫西芬敏感性及ER亚型及p160共激活因子表达关系的初步探讨

目的分析比较4种常见的人上皮性卵巢癌细胞系(A2780、CAOV-3、SKOV-3和ES-2)IL-6、IL-8分泌与其他莫西芬(Tamoxifen,TAM)敏感性及雌激素受体(estrogen receptor,ER)亚型及p160共激活因子表达的关系,为今后研究IL-6、IL-8诱导卵巢癌细胞对内分泌治疗产生耐药的机制奠定基础。方法 IL-6、IL-8的表达采用RT-PCR及ELISA法进行检测,卵巢癌细胞对TAM的敏感性采用MTT试验进行分析,ERα、ERβ及p160共激活因子的表达采用免疫印迹和RT-PCR技术进行检测。结果 4种卵巢癌细胞除A2780细胞外均组成性分泌IL-6和IL-8;4种细胞对TAM的敏感性不同,其中A2780细胞最敏感,CAOV-3、SKOV-3和ES-2细胞则有不同程度的耐药性(耐药倍数分别为4.98、3.75和2.66);这些细胞均不同程度地表达ERα,其中A2780细胞较低,CAOV-3和SKOV-3细胞较高,ES-2细胞居中;ERβ的表达情况则与ERα恰好相反;3种p160共激活因子mRNA的表达水平均以A2780细胞为最低,而ES-2、SKOV-3和CAOV-3细胞则依次升高。结论卵巢癌细胞IL-6、IL-8的自分泌水平与其对TAM的敏感性呈负相关,与其ERβ(而非ERα)及3种p160共激活因子的表达水平相一致。     

四种hGM-CSF/IL-6融合蛋白的基因构建、表达及活性比较

目的 构建及表达具有hGM-CSF和hIL-6双重生物学活性的hGM-CSF／IL-6融合蛋白分子。方法 应用PCR技术对hGM-CSF和hIL-6的基因分别加以改造，同时在二者之间加上连接肽序列(G-G-S-G-S)3，克隆PCR产物，并构建成克隆有4种融合蛋白基因的pBV220表达质粒，4种融合蛋白分别是GM-CSF(9～127)-IL-6(29～184)(简称GII)、GM-CSF(9～127)-IL-6(1～184)(简称GL2)、GM-CSF(1-127)-IL-6(1—184)(简称GI3)、GM-CSF(9-127)-IL-6(24～184)(简称GI4)，表达质粒分别导入E．coli BL-21中诱导表达。通过Q Sepharose HP离子交换柱和Sephacryl S-200分子筛柱二步柱纯化以获取目的蛋白。使用hGM—CSF依赖细胞株TF1和hIL-6依赖细胞株B9通过MTT法测量融合蛋白的生物学活性。结果 对4种融合蛋白基因的测序结果表明，其序列与理论设计完全一致，表达质粒在E．coli BL-21中均得到高效表达，表达的融合蛋白均占到总蛋白含量的30％以上，表达产物以包涵体的形式存在，通过Q Sepharose HP离子交换柱和Sephacryl S-200分子筛柱二步柱纯化及复性后获得4种目的蛋白，其纯度均达到95％以上。活性测定结果表明4种融合蛋白均具有较高的hGM-CSF和hIL-6的双重生物学活性。结论 获得了具有较高纯度和双重生物学活性的GM-CSF／IL-6融合蛋白。     

脂多糖刺激下大鼠腹膜间质细胞IL-18、IL-6的表达和氧化应激的影响

目的:研究脂多糖刺激下大鼠腹膜间皮细胞(RPMC)IL-18、IL-6和氧化应激产物丙二醛(MDA)的表达。方法:原代培养RPMC,用不同浓度脂多糖(1、10、100 mg/L)刺激RPMC 6 h;10 mg/L脂多糖刺激RPMC 3、6、12、24 h。用real time-PCR法检测IL-18mRNA的表达,ELISA法检测细胞上清液中IL-18和IL-6的蛋白水平,硫代巴比妥酸法检测细胞中MDA的含量。结果:与正常对照组比较,脂多糖刺激下RPMC IL-18、IL-6和MDA的表达明显增加(P<0.05),且呈浓度依赖性;随着刺激时间的延长,上述指标呈递增趋势,IL-18于12 h达高峰。结论:脂多糖刺激下RPMC促炎症因子IL-18、炎症因子IL-6及氧化应激产物MDA的表达均增加,引起持续放大的炎症氧化反应,损伤腹膜导致超滤失败。     

IL-2对中子照射后肠上皮细胞生长和凋亡的影响及其机制

目的:观察中子照射对体外培养的IEC-6细胞生长的影响及IL-2对其损伤后增殖和恢复的作用,并进一步探讨IL-2调节受照射肠上皮细胞生长的相关机制。方法:单独用IL-2(1×105U/L)或同时施加JAK1激酶阻断剂(A77-1726)处理受4Gy中子照射的IEC-6细胞,并于照后10、15、30min和1、3、6、12、24、48及72h,用MTT比色法和流式细胞术检测受照射后IEC-6细胞的增殖活力和死亡方式的改变。以免疫细胞化学染色和Western blot检测IEC-6细胞上IL-2Rβ的表达和JAK1活化情况。结果:4Gy中子照射后24h,IEC-6细胞的增殖活力明显降低;而IL-2处理组该细胞的增殖活力有显著提高(P<0.05)。受中子照射的IEC-6细胞经IL-2作用24h,其凋亡率明显降低(P<0.05),而坏死率变化不明显。以IL-2刺激中子照射的IEC-6细胞后,于10及15min可见JAK1发生明显磷酸化活化,24h时IL-2Rβ的表达明显增多。同时应用A77-1726和IL-2处理受中子照射的IEC-6细胞后,其增殖活力明显低于单纯IL-2处理组。结论:IL-2可促进受中子照射的IEC-6细胞增殖,具有抗辐射作用。IL-2Rβ和JAK1活化参与了IL-2对中子损伤的IEC-6细胞生长的调控。     

核受体NR6A1通过上调RIPK3基因表达诱导血管平滑肌细胞凋亡

目的:探讨核受体亚家族6A1(NR6A1)对血管平滑肌细胞凋亡的影响及可能的分子机制。方法:将腺病毒Ad-NR6A1感染大鼠血管平滑肌细胞,分别在感染后0 h、24 h和48 h时进行MTT实验,以时间为横坐标,A570为纵坐标绘制细胞生长曲线,观察NR6A1对细胞生长的影响;进行DAPI染色、TUNEL染色及caspase活性检测,观察细胞凋亡情况;进一步通过基因芯片技术,寻找NR6A1的靶基因;采用siRNA介导的基因沉默技术,观察受体相互作用丝氨酸/苏氨酸蛋白激酶3(RIPK3)基因沉默对NR6A1诱导的血管平滑肌细胞凋亡的影响。结果:腺病毒Ad-NR6A1感染细胞48 h时,NR6A1过表达组细胞数量较对照组(重组腺病毒载体Ad-Lac Z)明显减少;DAPI染色显示NR6A1过表达诱导血管平滑肌细胞出现核浓缩和核碎裂的凋亡表型,TUNEL染色显示NR6A1过表达引起细胞凋亡,caspase活性检测结果显示NR6A1过表达细胞内caspase-3、caspase-8和caspase-9活性均较对照组高;基因芯片技术检测发现,NR6A1过表达上调血管平滑肌细胞中RIPK3基因表达;RIPK3基因沉默可以显著抑制NR6A1诱导的平滑肌细胞凋亡。结论:NR6A1通过上调RIPK3基因表达诱导血管平滑肌细胞凋亡。     

卵巢癌细胞IL-6、IL-8分泌与他莫西芬敏感性及MAPK、Akt活性和ER特异性位点磷酸化水平的关系

目的:分析比较4种常见的人上皮性卵巢癌细胞系(A2780、CAOV-3、SKOV-3和ES-2)IL-6、IL-8分泌与他莫西芬(TAM)敏感性及MAPK、Akt活性和雌激素受体(ER)特异性位点磷酸化水平的关系,探讨IL-6、IL-8诱导卵巢癌细胞对内分泌治疗产生耐药的机制。方法:RT-PCR及ELISA法检测IL-6、IL-8的表达,MTT法分析卵巢癌细胞对TAM的敏感性,免疫印迹法测定MAPK、Akt活性及ER特异性位点的磷酸化水平。结果:(1)4种卵巢癌细胞除A2780细胞外均组成性分泌IL-6和IL-8,二者转录水平与其蛋白水平基本一致;(2)4种卵巢癌细胞对TAM的敏感性不同,其中A2780细胞最敏感,CAOV-3、SKOV-3和ES-2细胞则有不同程度的耐药性,其耐药倍数分别为4.99、3.76和2.66;(3)4种卵巢癌细胞的MAPK、Akt活性存在差异,CAOV-3细胞的二者活性最强,SKOV-3细胞的MAPK活性弱于ES-2细胞,而其Akt活性则强于ES-2细胞,A2780细胞未检测到二者有活性;(4)ER的第167位丝氨酸(Ser167)在四种卵巢癌细胞中均存在不同程度的磷酸化,ER的第118位丝氨酸(Ser118)除A2780细胞外亦存在不同程度的磷酸化。结论:卵巢癌细胞IL-6、IL-8的自分泌水平与其对TAM的敏感性呈负相关,与其MAPK、Akt活性和ER特异性位点(Ser167、Ser118)的磷酸化水平相一致。     

Enhancement of in vitro hsp72 expression by placental IL-6

PROBLEM: To give an approach in order to elucidate the mechanism by which placental IL-6 induce modifications in the glycosylation status of immunoglobulins, in the present work, we investigate a putative relationship between a stimulus by placental IL-6 and expression of cytoplasmic "hsp70 family proteins" in an in vitro model. METHODS OF STUDY: Supernatants of cultures of placentae obtained from primiparous and multiparous AKR/J x AKR/J and AKR/J x BALB/c mouse crossbreedings were added to mouse IgGI hybridoma cultures which produced symmetric and asymmetric anti-dinitrophenol (anti-DNP) antibodies. Analyses of the expression of inducible hsp72/constitutive hsp73 in cellular lysates obtained from hybridomas cultured, in the presence of rmIL-6 or crude murine placental culture supernatants, followed by neutralization assays with anti IL-6, were performed. In addition, the level of IL-6 present in the employed placental culture supernatants was determined and compared with the placental hsp70-inducing effect. RESULTS: These experiments showed that mouse placentae were able to release IL-6 in vitro. In addition, mouse placental supernatants (PS) containing over 1,000 pg/mL of IL-6 enhanced the expression of the inducible isoform hsp72 in the employed hybridomas. This effect was abolished when the hsp70-inducing PS were previously incubated with anti-mIL6 antibody. CONCLUSIONS: These observations indicate that mouse placentae produce different titers of IL-6 and suggest that IL-6 appears to be the unique mouse placental factor able to induce in vitro hsp72 synthesis. A relationship with the increased synthesis of anti-paternal antigen asymmetric antibodies, previously observed during pregnancy, is discussed.