Lipopolysaccharide in concentrations above 40 ng/ml stimulates proliferation of the IL-6-dependent B9 cell line

The B9 assay is known to be a specific and sensitive assay for the estimation of interleukin-6 activity. This assay was found to be compromised by lipopolysaccharide in concentrations ≥ 40 ng lipopolysaccharide per ml. The lipopolysaccharide stimulates proliferation of the B9 cell line in a dose-dependent manner both when measuring the proliferation by thymidine incorporation and when using the MTT assay. However the LPS dose-response curve is different compared to the dose-response curve for IL-6. A sample containing 100 ng LPS/ml but no IL-6 would be estimated erroneously to contain 12 pg IL-6. The interference of lipopolysaccharide is totally abolished by the addition of polymyxin B to the samples but the addition has no effect on the IL-6 induced proliferation.     

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.     

Heparanase accelerates the proliferation of both hepatocytes and endothelial cells early after partial hepatectomy

Background and aims

Heparanase (HPSE) is an endo-β-D-glucuronidase, which cleaves heparan sulfate in the extracellular matrix (ECM) and has pro-angiogenic and pro-proliferative properties. The aim of this investigation was to study the effect of HPSE on hepatocytes and endothelial cells (EC) during liver regeneration.

Methods

Following 70% hepatectomy (PHP), rats were injected daily with 1–50 μg HPSE/rat. Liver samples were stained with H&;E and anti-bromodeoxyuridine (BrdU) antibody. mRNAs of hepatocyte growth factor (HGF), stem cell factor, tumor necrosis factor (TNF)-α, interleukin(IL)-6, and cyclinD1 were tested by real-time qPCR. Matrix metalloproteinases (MMPs) were tested by gel zymography.

Results

Compared to the saline control, HPSE increased hepatocyte proliferation 24 h, 48 h and 72 h after PHP, with the maximal effect found at 24 h with 50 μg HPSE (40.9 ± 2.5% vs. 8.6 ± 4.3%, p < 0.01 for BrdU staining; 5.5 ± 0.9% vs. 0.8 ± 0.5%, p < 0.05 for mitosis). Proliferation of the sinusoidal and the portal vein radical ECs was also increased (p < 0.05). HPSE caused a twofold increase in cyclinD1 mRNA (p < 0.05) and in pro-MMP-9 levels (p < 0.05). HPSE at all doses also caused significant reductions of TNF-α mRNA (p < 0.05) and IL-6 mRNA, and no change in HGF mRNA.

Conclusions

HPSE enhances liver regeneration by inducing proliferation of hepatocytes and both sinusoidal and vascular ECs. Since the effect of HPSE on hepatocytes occurred earlier than that observed in ECs, this effect is not related to HPSE's effect on ECs. The mechanism of HPSE action is probably indirect and is mediated by HPSE-dependent ECM cleavage and the release of pre-existing enzymes.     

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。     

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。     

IL-6在诱导M1白血病细胞分化后凋亡中的作用

目的探讨IL-6能否诱导M1小鼠急性髓样白血病细胞凋亡及其分子机制。方法透射电镜观察细胞形态判断有无凋亡发生;RT-PCR及狭缝杂交分析bcl-2家族成员表达水平的改变。结果IL-6诱导M1细胞向巨噬细胞方向分化后,M1细胞发生凋亡;bcl-2、bcl-XL在IL-6诱导下表达下调,bax表达上调。结论IL-6诱导Bcl-2/Bax比例改变,使M1细胞分化后凋亡。     

睾丸局部感染时Sertoli细胞IL-1、IL-6 mRNA的表达

目的；研究大鼠Sertoli细胞在抗感染中的免疫调节作用。方法：以溶脲脲原体（ureaplasma urealyicum,UU)和致病性大肠杆务直接注入大肠膀胱模拟上行性感染的途径，分别在1，2和3周处死大鼠，从大鼠睾丸组织分离获得高纯度的Sertoli细胞，然后抽提总RNA，用RT－PCR方法比较正常组与UU感染组，致病性大肠杆菌组之间IL－1，IL－6 mRNA表达的差异。结果：与正常组相比，UU感染后，其IL－1 mRNA在1，2周时升高，3周时下降，IL－6在1，2周时下降，3周时升高；而致病性大肠杆菌感染后，其IL－1 mRNA在1，2周时均升高，3周下降；IL－6在1周时升高，2周时下降，3周又升高，结论：大鼠Sertoli细胞在抗感染免疫中，可能IL－1，IL－6的表达来发挥免疫调节作用。     

Involvement of serum and lipopolysaccharide in the production of interleukin-1- and interleukin-6-like molecules by human sperm cells

PROBLEM: To examine the capacity of sperm cells from fertile and infertile men to secrete interleukin (IL)-6, and the involvement of serum factors and lipopolysaccharide (LPS) in the regulation of IL-6 and IL-1 production by sperm cells. METHODS: Swim-up sperm cells from fertile (donors) and oligoteratoasthenospermic (OTA)-infertile men were incubated with or without 5% fetal calf serum (FCS) and LPS (10 microg/mL) for 2-24 hr. After incubation, IL-6 and IL-1 bioactivities were measured in supernatants and lysates by specific bioassays (B9 cell proliferation assay and 1A-5 system, respectively). RESULTS: IL-6- and IL-1-like activities were observed to be produced by swim-up sperm cells from both study groups. Stimulation of swim-up sperm cells with either LPS or FCS or both together did not affect their capacity to produce IL-1. However, LPS, but not serum increased the secretion levels of IL-6 by swim-up sperm cells. CONCLUSIONS: Swim-up sperm cells from both study groups constitutively produce IL-6 and IL-1, and serum components did not affect this capacity. However, LPS was shown to increase the capacity of swim-up sperm cells of both study groups to secrete IL-6, but not IL-1. Cytokines may be involved in the physiology and pathophysiology of sperm functions and, thus, may affect male fertility.     

IFN-{gamma} expression in CD8+ T cells regulated by IL-6 signal is involved in superantigen-mediated CD4+ T cell death

Infection with pathogens containing superantigens (Sags) canresult in massive excessive CD4⁺ T cell activation and deathin such conditions as toxic shock, food poisoning and autoimmunediseases. We here showed how enhancement of IL-6 signaling suppressesSag-mediated activated CD4⁺ T cell death. Sag-induced CD4⁺ Tcell death increased in IL-6 knockout (KO) mice, whereas itdecreased in mice characterized by enhanced IL-6–gp130–STAT3signaling. The serum concentration of IFN- was inversely correlatedwith the magnitude of IL-6 signaling, and IFN- deficiency inhibitedSag-induced activated CD4⁺ T cell death, suggesting that IL-6suppresses CD4⁺ T cell death via IFN- expression. Interestingly,depletion of activated CD8⁺ T cells inhibited Sag-mediated increasesin IFN- expression in IL-6 KO mice as well as the augmentedCD4⁺ T cell death. The results demonstrate that IL-6–gp130–STAT3signaling in activated CD8⁺ T cells contributes to Sag-inducedCD4⁺ T cell death via IFN- expression, highlighting this signalingaxis in CD8⁺ T cells as a potential therapeutic target for Sag-relatedsyndromes.     

Characteristics of IL-6 and TNF-α production by respiratory syncytial virus-infected macrophages in the neonate

The production of IL-6 and TNF-α and the expression of their mRNA were studied with neonatal (cord blood) and adult blood monocyte-derived macrophages (MDM) after in vitro infection with respiratory syncytial virus (RSV). Cord blood MDM exhibited production of high levels of IL-6 within 24 hr after infection. Little or no IL-6 production was detected after 24–48 hr and after in vitro stimulation with inactivated (nonreplicating) virus. Adult blood MDM also produced high levels of IL-6 within 24 hr of RSV infection. Unlike cord blood MDM, adult MDM demonstrated significant activity of IL-6 after 24 hr of infection with live RSV and after exposure to the inactivated virus. The pattern of TNF-α production by cord and adult blood MDM after live RSV infection resembled closely the pattern of IL-6 production. Both cell types produced TNF-α in the first 24 hr after infection. However, little or no production was observed after 24 hr of infection and after exposure to the inactivated virus. The profile of mRNA expression was similar to the production of IL-6 or TNF-α. mRNA expression occurred over a shorter period in cord blood MDM. These observations suggest that inflammatory and immunoregulatory cytokines, such as IL-6 and TNF-α, are produced by neonatal as well as previously primed adult macrophages. However, neonatal cells may be less efficient in inducing IL-6 production. © 1996 Wiley-Liss, Inc.     

Knockdown of HOXC6 inhibits glioma cell proliferation and induces cell cycle arrest by targeting WIF-1 in vitro and vivo

Background

Homeobox C6 (HOXC6) is one of several HOXC genes and is frequently overexpressed in multiple cancers. However, the function and mechanism of HOXC6 in glioma remain unclear.

IRAK-1表达水平与COPD大鼠PASMCs分泌PDGF-AB和IL-6的相关性研究

目的:探讨白细胞介素1受体相关激酶1(IRAK-1)与慢性阻塞性肺疾病(COPD)大鼠肺动脉平滑肌细胞(α-平滑肌肌动蛋白(α-SMA),PASMCs)分泌血小板源性生长因子AB(PDGF-AB)和白细胞介素6(IL-6)的相关性。方法:构建COPD大鼠模型,HE染色观察肺组织病理学变化,图像分析法测定远端肺动脉管壁厚度占动脉外径的百分比(WT%)和管壁面积占血管总面积的百分比(WA%)。消化、分离和纯化COPD大鼠远端PASMCs,并采用特异性抗α-平滑肌肌动蛋白(α-SMA)抗体进行细胞免疫荧光鉴定大鼠PASMCs。将COPD大鼠PASMCs随机分为对照组(不进行干预)、脂多糖(LPS)组(终浓度为1 mg/L)、IRAK-1/4抑制剂组(终浓度为10μmol/L)和LPS+IRAK-1/4抑制剂组(IRAK-1/4抑制剂终浓度为10μmol/L,预处理30 min后加入LPS,终浓度为1 mg/L),采用Western blot检测各组PASMCs中p-IRAK-1和IRAK-1的蛋白水平;ELISA方法检测各组PASMCs上清液中PDGF-AB和IL-6的浓度。结果:COPD模型组WT%和WA%较空白对照组升高(P0.01)。光学显微镜下COPD大鼠PASMCs呈梭形,荧光镜下可见胞质α-SMA蛋白染成红色。与对照组相比,LPS组p-IRAK-1蛋白表达水平及PDGF-AB和IL-6的含量升高(P0.05);与LPS组相比,LPS+IRAK-1/4抑制剂组p-IRAK-1的蛋白水平及PDGF-AB和IL-6的含量明显降低(P0.05)。IRAK-1磷酸化水平与细胞上清液中PDGF-AB和IL-6的浓度呈正相关。结论:IRAK-1参与COPD大鼠PASMCs分泌PDGF-AB和IL-6的调控。这为COPD的早期干预提供了新依据。     

Deficiency of SHP1 leads to sustained and increased ERK activation in mast cells, thereby inhibiting IL-3-dependent proliferation and cell death

SHP-1 plays an important role for the regulation of signaling from various hematopoietic cell receptors. In this study, we examined IL-3-induced cell proliferation and IL-3 depletion-induced apoptosis in bone marrow-derived mast cells (BMMC) established from motheaten (me) that lack SHP-1 expression, viable motheaten (me^v) expressing phosphatase-deficient SHP-1, and wild-type (WT) mice. When BMMC were stimulated with IL-3, increased ERK activation was evident in resting state and sustained in me-BMMC relative to WT-BMMC. ERK is known to be involved in the regulation of cell proliferation and apoptosis in some cells. In accordance with sustained ERK activation, apoptosis was decreased in me- and me^v-BMMC compared with WT-BMMC. In contrast to the predicted role of ERK as a pro-survival molecule, IL-3-induced cell proliferation was much lower in me- and me^v-BMMC than WT-BMMC. Stimulation with lower concentration of IL-3 or addition of PD98059, a MEK inhibitor, to the culture resulted in the suppression of decreased apoptosis and cell proliferation in me- and me^v-BMMC. Collectively, these results suggest that SHP-1 positively regulates IL-3-dependent mast cell proliferation and apoptosis by inhibiting ERK activity through its phosphatase activity. Furthermore, our results indicate that ERK would act as a negative regulator for cell proliferation and induce apoptosis when its activity is highly increased.     

A T cell activation antigen,Ly6C,induced on CD4+ Th1 cells mediates an inhibitory signal for secretion of IL-2 and proliferation in peripheral immune responses

A T cell activation antigen, Ly6C, is considered to be involved in the autoimmunity of some autoimmune-prone mice; however, the function of Ly6C remains largely unknown. We prepared a rat anti-mouse Ly6C monoclonal antibody (mAb) (S14) that inhibits the proliferation of peripheral T cells stimulated with anti-CD3 mAb in vitro. S14 mAb, the specificity of which is confirmed by a cDNA transfectant, recognizes Ly6C antigen preferentially expressed on a part of CD8⁺ T cells in peripheral lymphoid organs. The immunohistochemical analysis demonstrates that Ly6C appears on CD8⁺ T cells in the conventional T cell-associated area of BALB/c but not of nonobese diabetic (NOD) mice, confirming the absence of Ly6C⁺ T cells in NOD mice. Addition of soluble S14 mAb to the culture does not influence the proliferation of T cells in vitro; however, the S14 mAb coated on the plate clearly inhibits the proliferation and IL-2 production of anti-CD3-stimulated peripheral T cells. The T cells are arrested at the transitional stage from G₀/G₁ to S+G₂/M phases, but they are not induced to undergo apoptotic changes in vitro. This inhibitory signal provided through the Ly6C molecule inhibited IL-2 secretion in a subpopulation of the activated CD4⁺ T cells. Ly6C is expressed on T cell clones of both Th1 and Th2 cells, but the cytokine secretion from Th1 clones is preferentially inhibited. These results suggest that Ly6C mediates an inhibitory signal for secretion of cytokines from Th1 CD4⁺ T cells, potentially causing the inhibition of immune response in peripheral lymphoid tissues.     

Angiogenic capacity and lung-colonizing potential in vivo is increased in weakly metastatic B16F1 cells and decreased in highly metastatic BL6 cells by phorbol esters

The development of tumor metastasis is a multistep process. Key aspects of this process are the interaction of tumor cells with the extracellular matrix, digestion of, and motility through the basement membrane and the induction of angiogenesis. In this study, we analysed the effects of a low dose of TPA (12-tetradecanoylphorbol-13-acetate; 0.1 microM on angiogenesis, proteolytic activity and lung colonizing potential of both weakly metastatic B16F1 cells and highly metastatic BL6 murine melanoma cells. Our results demonstrated opposite effects of TPA in the two cell lines. TPA-treated B16F1 cells showed enhanced release of basic FGF (bFGF) and vascular endothelial growth factor (VEGF) and increased angiogenic capacity and lung colony formation in vivo. In contrast, TPA-treated BL6 cells demonstrated a dramatic reduction of angiogenic and gelatinolytic activity and metastatic capacity. However, both cell lines showed an induction of VEGF as well as bFGF expression by TPA treatment suggesting that in BL6 cells antagonistic factors, inhibiting the angiogenic and metastatic capacity, are induced by this treatment.     

Angiogenic capacity and lung-colonizing potential in vivo is increased in weakly metastatic B16F1 cells and decreased in highly metastatic BL6 cells by phorbol esters

The development of tumor metastasis is a multistep process. Key aspects of this process are the interaction of tumor cells with the extracellular matrix, digestion of, and motility through the basement membrane and the induction of angiogenesis. In this study, we analysed the effects of a low dose of TPA (12-tetradecanoylphor-bol- 13-acetate; 0.1 M on angiogenesis, proteolytic activity and lung colonizing potential of both weakly metastatic B16F1 cells and highly metastatic BL6 murine melanoma cells. Our results demonstrated opposite effects of TPA in the two cell lines. TPA-treated B16F1 cells showed enhanced release of basic FGF (bFGF) and vascular endothelial growth factor (VEGF) and increased angiogenic capacity and lung colony formation in vivo. In contrast, TPA-treated BL6 cells demonstrated a dramatic reduction of angiogenic and gelatinolytic activity and metastatic capacity. However, both cell lines showed an induction of VEGF as well as bFGF expression by TPA treatment suggesting that in BL6 cells antagonistic factors, inhibiting the angiogenic and metastatic capacity, are induced by this treatment. © Rapid Science Ltd.     

Involvement of adenylate cyclase and p70S6-kinase activation in IL-10 up-regulation in human monocytes by gp41 envelope protein of human immunodeficiency virus type 1

Our previous results show that recombinant gp41 (aa565–647), the extracellular domain of HIV-1 transmembrane glycoprotein, stimulates interleukin-10 (IL-10) production in human monocytes. The signal cascade transducing this effect is not yet clear. In this study, we examined whether gp41-induced IL-10 up-regulation is mediated by the previously described synergistic activation of cAMP and NF-κB pathways. gp41 induced cAMP accumulation in monocytes in a time- and concentration-dependent manner and the adenylate cyclase inhibitor SQ 22536 suppressed gp41-induced IL-10 production in monocytes. In contrast, gp41 failed to stimulate NF-κB binding activity in as much as no NF-κB bound to the main NF-κB-binding site 2 of the IL-10 promoter after addition of gp41. We also examined the involvement of other signal transduction pathways. Specific inhibitors of p70^S6-kinase (rapamycin), and G_i protein (pertussis toxin), prevented induction of IL-10 production by gp41 in monocytes, while inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase) (wortmannin) and mitogen-activated protein kinase (MAPK) pathway (PD 98059) did not. Thus HIV-1 gp41-induced IL-10 up-regulation in monocytes may not involve NF-κB, MAPK, or PI 3-kinase activation, but rather may operate through activation of adenylate cyclase and pertussis-toxin-sensitive G_i/G_o protein to effect p70^S6-kinase activation. Received: 28 September 1998 / Received after revision: 27 October 1998 / Accepted: 3 November 1998