Suppression of IL-2-induced SAA gene expression in mice by the administration of an IL-1 receptor antagonist

The hepatic acute phase response induced by the administration of interleukin (IL)-2 is most likely mediated by secondary cytokines. In this investigation, we examined the role of endogenous IL-1 in the synthesis of the hepatic acute phase protein serum amyloid A (SAA) during IL-2 treatment. The injection of IL-2 induced SAA gene expression in the liver. The concurrent administration of an IL-1 receptor antagonist (IL-1RA) markedly reduced hepatic SAA mRNA levels and, to a lesser extent, SAA protein levels in the serum. Although IL-1 is an inducer of IL-6 production, the administration of the IL-1RA had no effect on circulating IL-6 levels in IL-2-treated mice. These findings suggest that the production of IL-1 is an important factor in the induction of SAA mRNA in mice undergoing immunotherapy with IL-2.     

应用椭偏光学显微成像对IL-6与其受体的相互应用的初步观察

白细胞介素 6 (ＩＬ 6 )是一种具有复杂生物功能的细胞因子 ,可由多种淋巴类和非淋巴类细胞产生。它对机体多种组织及细胞均有不同程度的作用[1～ 3 ] 。近年来发现 ,临床上免疫异常性疾病 ,如发热、淋巴结肿大、血沉增快、急性期蛋白增高、高γ球蛋白血症、自身抗体阳性等症状都与ＩＬ 6的异常表达密切相关。ＩＬ 6的生物活性是通过细胞膜表面特异性受体介导的[4] 。研究ＩＬ 6与其受体的相互作用对于揭示某些疾病的发病机制 ,监测疾病进程以及指导临床治疗等均具有重要意义。用于研究ＩＬ 6与其受体相互作用的方法主要有ＩＬ 6依赖株细胞…     

Human interleukin 24 (MDA-7/IL-24) protein kills breast cancer cells via the IL-20 receptor and is antagonized by IL-10

The melanoma differentiation-associated gene-7 (mda-7/IL-24) is a unique member of the interleukin 10 (IL-10) family of cytokines, with ubiquitous tumor cell pro-apoptotic activity. Recent data have shown that IL-24 is secreted as a glycosylated protein and functions as a pro-Th1 cytokine and as a potent anti-angiogenic molecule. In this study, we analyzed the activity of Ad-mda7 and its protein product, secreted IL-24, against human breast cancer cells. We show that Ad-mda7 transduction of human breast cancer cells results in G₂/M phase cell cycle arrest and apoptotic cell death, which correlates with secretion of IL-24 protein. Neutralizing antibody against IL-24 significantly inhibited Ad-mda7 cytotoxicity. IL-24 and IL-10 both engage their cognate receptors on breast cancer cells resulting in phosphorylation and activation of STAT3, however, IL-10 receptor binding failed to induce cell killing, indicating that tumor cell killing by IL-24 is independent of STAT3 phosphorylation. Treatment with exogenous IL-24 induced apoptosis in breast cancer cells and this effect was abolished by addition of anti-IL-24 antibody or anti-IL-20R1, indicating that bystander cell killing is mediated via IL-24 binding to the IL-20R1/IL-20R2 heterodimeric receptor complex. Co-administration of the related cytokine IL-10 inhibited killing mediated by IL-24 and concomitantly inhibited IL-24 mediated up-regulation of the tumor suppressor proteins, p53 and p27^Kip1. In summary, we have defined a tumor-selective cytotoxic bystander role for secreted IL-24 protein and identified a novel receptor-mediated death pathway in breast cancer cells, wherein the related cytokines IL-24 and IL-10 exhibit antagonistic activity.     

WSB-1, a novel IL-21 receptor binding molecule, enhances the maturation of IL-21 receptor

Interleukin-21 (IL-21) is a pleiotropic cytokine that regulates T-cell, B-cell, NK-cell, and myeloid-cell functions. IL-21 binds with its cognate receptor complex, which consists of the IL-21 receptor (IL-21R) and the common gamma chain. We identified a novel IL-21R-binding molecule, WSB-1, which contains WD-40 repeats and a SOCS-box domain. WSB-1 associates with the middle part of intracytoplasmic region of IL-21R and enhances the maturation of IL-21R from N-linked glycosylated form to fully glycosylated mature form. Furthermore, WSB-1 moderates IL-21R degradation. Taken together, our present study suggests that WSB-1 has a role in the tuning of the maturation and degradation of IL-21R.     

Activation of IL-4 and IL-6 secreting cells by antigen

The number, antigenic specificity and phenotype of cells secreting IL-4 and IL-6 in mice immunized with ovalbumin or keyhole limpet haemocyanin (KLH) in Freund's adjuvant (FA) was studied. The frequency of cells producing either of these cytokines began to rise 6 days post immunization, peaked at 11–14 days post-immunization, and fell to background by 21 days. The number of spleen cells secreting IL-6 was higher than the number producing IL-4 at all time points. Boosting elicited an anamnestic response characterized by a significant increase in the number of cytokine secreting cells within 4 days. Cytokine production was induced in multiple strains of normal mice, and was critically dependent on the use of Complete FA in addition to antigen. Immunization induced IL-4 and IL-6 production in vivo while ‘priming’ additional cells to release these cytokines when reexposed to soluble antigen in vitro. The latter response was antigen specific and was dominated by non-B/non-T cells. Those cells may serve to boost the immune response in cases of persistent or repeated antigenic challenge.     

IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: regulation by SOCS3

The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21cip1 protein expression in primary mouse hepatocytes. Disruption of the p21cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3+/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3+/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21cip1-dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration.     

SOCS-1 inhibits expression of the antiviral proteins 2',5'-OAS and MxA induced by the novel interferon-lambdas IL-28A and IL-29

Recently, we have shown that SOCS-1/3 overexpression in hepatic cells abrogates signaling of type I interferons (IFN) which may contribute to the frequently observed IFN resistance of hepatitis C virus (HCV). IFN-lambdas (IL-28A/B and IL-29), a novel group of IFNs, also efficiently inhibit HCV replication in vitro with potentially less hematopoietic side effects than IFN-alpha because of limited receptor expression in hematopoietic cells. To further evaluate the potential of IFN-lambdas in chronic viral hepatitis, we examined the influence of SOCS protein expression on IFN-lambda signaling. First, we show that hepatic cell lines express the IFN-lambda receptor complex consisting of IFN-lambdaR1 (IL-28R1) and IL-10R2. Whereas in mock-transfected HepG2 cells, IL-28A and IL-29 induced STAT1 and STAT3 phosphorylation, overexpression of SOCS-1 completely abrogated IL-28A and IL-29-induced STAT1/3 phosphorylation. Similarly, IL-28A and IL-29 induced mRNA expression of the antiviral proteins 2',5'-OAS and MxA was abolished by overexpression of SOCS-1. In conclusion, we assume that despite antiviral properties of IFN-lambdas, their efficacy as antiviral agents may have similar limitations as IFN-alpha due to inhibition by SOCS proteins.     

SR141716A, a potent and selective antagonist of the brain cannabinoid receptor

SR141716A is the first selective and orally active antagonist of the brain cannabinoid receptor. This compound displays nanomolar affinity for the central cannabinoid receptor but is not active on the peripheral cannabinoid receptor. In vitro, SR141716A antagonises the inhibitory effects of cannabinoid receptor agonists on both mouse vas deferens contractions and adenylyl cyclase activity in rat brain membranes. After intraperitoneal or oral administration SR141716A antagonises classical pharmacological and behavioural effects of cannabinoid receptor agonists. This compound should prove to be a powerful tool for investigating the in vivo functions of the anandamide/cannabinoid system.     

Structure-based virtual screening and identification of a novel androgen receptor antagonist

Hormonal therapies, mainly combinations of anti-androgens and androgen deprivation, have been the mainstay treatment for advanced prostate cancer because the androgen-androgen receptor (AR) system plays a pivotal role in the development and progression of prostate cancers. However, the emergence of androgen resistance, largely due to inefficient anti-hormone action, limits the therapeutic usefulness of these therapies. Here, we report that 6-(3,4-dihydro-1H-isoquinolin-2-yl)-N-(6-methylpyridin-2-yl)nicotinamide (DIMN) acts as a novel anti-androgenic compound that may be effective in the treatment of both androgen-dependent and androgen-independent prostate cancers. Through AR structure-based virtual screening using the FlexX docking model, fifty-four compounds were selected and further screened for AR antagonism via cell-based tests. One compound, DIMN, showed an antagonistic effect specific to AR with comparable potency to that of the classical AR antagonists, hydroxyflutamide and bicalutamide. Consistent with their anti-androgenic activity, DIMN inhibited the growth of androgen-dependent LNCaP prostate cancer cells. Interestingly, the compound also suppressed the growth of androgen-independent C4-2 and CWR22rv prostate cancer cells, which express a functional AR, but did not suppress the growth of the AR-negative prostate cancer cells PPC-1, DU145, and R3327-AT3.1. Taken together, the results suggest that the synthetic compound DIMN is a novel anti-androgen and strong candidate for useful therapeutic agent against early stage to advanced prostate cancer.     

Identification of ghrelin receptor blocker, D-[Lys3] GHRP-6 as a CXCR4 receptor antagonist

[D-Lys3]-Growth Hormone Releasing Peptide-6 (DLS) is widely utilized in vivo and in vitro as a selective ghrelin receptor (GHS-R) antagonist. Unexpectedly, we identified that DLS also has the ability to block CXCL12 binding and activity through CXCR4 on T cells and peripheral blood mononuclear cells (PBMCs). Moreover, as CXCR4 has been shown to act as a major co-receptor for HIV-1 entry into CD4 positive host cells, we have also found that DLS partially blocks CXCR4-mediated HIV-1 entry and propagation in activated human PBMCs. These data demonstrate that DLS is not the specific and selective antagonist as thought for GHS-R1a and appears to have additional effects on the CXCR4 chemokine receptor. Our findings also suggest that structural analogues that mimic DLS binding properties may also have properties of blocking HIV infectivity, CXCR4 dependent cancer cell migration and attenuating chemokine-mediated immune cell trafficking in inflammatory disorders.     

Adenosine receptor A2b confers ovarian cancer survival and PARP inhibitor resistance through IL-6-STAT3 signalling

Ovarian cancer is the deadliest gynecologic cancer worldwide, and the therapeutic options are limited. PARP inhibitor (PARPi) represents an effective therapeutic strategy and has been approved for maintenance therapy. However, the intrinsic or acquired resistance to PARPi becomes a big challenge. To investigate the mechanisms for PARPi resistance, we analysed public databases and established Olaparib-resistant ovarian cancer cells for exploration. Our results showed that the inflammatory pathway and adenosine receptor A2b (Adora2b/A_2B) expression were significantly increased in Olaparib-resistant cells. A_2B was highly expressed in recurrent ovarian tumours and negatively correlated with the clinical outcomes in cancer patients. Olaparib treatment enhanced A_2B expression through NF-κB activation. The elevated A_2B contributed to Olaparib resistance by sensing adenosine signal and promoting tumour cell survival, growth and migration via IL-6-STAT3 signalling. Therefore, inhibition of A_2B-IL-6-STAT3 axis could overcome Olaparib resistance and synergize with Olaparib to reduce cancer cell growth and lead to cell death. Our findings reveal a critical role of A_2B signalling in mediating PARPi resistance independent of DNA damage repair, providing insights into developing novel therapies in ovarian cancers.     

TNF-alpha、IL-6及IL-8在不同程度溃疡性结肠炎患者血清中的表达及意义

目的：探讨不同严重程度溃疡性结肠炎患者血清TNF-alpha、IL-6 及IL-8 的表达及意义。方法：选择2011 年1 月~2014 年7 月 我院收治的溃疡性结肠炎患者47 例,根据严重程度将患者分为轻度组、中度组和重度组。另选取同期在我院接受健康体检的志 愿者80 例作为对照组。采用酶联免疫吸附试验（ELISA）检测各组患者血清TNF-alpha、IL-6 及IL-8 的水平。结果：溃疡性结肠炎患者 血清TNF-琢、IL-6 及IL-8 水平高于对照组,差异有统计学意义（P<0.05）;不同严重程度溃疡性结肠炎患者的TNF-alpha、IL-6 及IL-8 水平呈显著差异（P<0.05）。结论：检测溃疡性结肠炎患者血清TNF-alpha、IL-6 及IL-8的水平变化有利于预测病情进展。     

Novel Insights into Interleukin 6 (IL-6) Cis- and Trans-signaling Pathways by Differentially Manipulating the Assembly of the IL-6 Signaling Complex

The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.     

Test of a novel transparent floating carrier by recombinant cho cells expressing sIL6 receptor protein

We describe a carrier with some unusual properties.“Cellrafts” increase yields from adherent cells in conventional static T150 flasks, by floating multilayer strips of transparent film at the oxygen-rich surface of unstirred medium. This new technique allowed microscopic inspection of cell growth inside the carriers during bulk culture. Individual carriers could be picked out for subculture of selected colonies. A novel surface treatment by hypochlorite/uv allowed recycling of used carriers. Cellrafts' open-deck structure facilitated trypsinization with90% release as viable single cells from bulk carriers. Macro size (10mm by 1 mm) enables retention in flask by a coarse sieve insert in its neck, facilitating separation of product cells or media. Residual cells in carriers regenerated repeated harvests without need for reseeding. Carriers were tested with shear-sensitive CHO clones expressing soluble human IL6 receptor (sIL6R). Control was monolayer bulk culture on trays. Floating multilayer cultures remained viable longer than monolayers, had higher cellular activity of protein expression, and were less serum dependent (resembling cells on porous carriers). Purity and anti-sIL6R binding were identical to control product. Cellrafts were also tested in a small spinner vessel, but for litre batches this proved less convenient than in T-flasks. Though yields are low compared to well established porous carrier technology (spinner or packed bed) static transparent carriers might provide transitional scaleup from normal cytogenetics laboratory culture. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ligand-dependent corepressor acts as a novel androgen receptor corepressor, inhibits prostate cancer growth, and is functionally inactivated by the Src protein kinase

The activated androgen receptor (AR) promotes prostate cancer (PCa) growth. AR antagonists repress the AR by recruitment of corepressors. Not much is known about the inactivation of AR by corepressors in the presence of agonists (androgens). Here we show that the corepressor LCoR acts as an androgen-dependent corepressor that represses human PCa growth in vivo. In line with this, progressive decrease of ligand-dependent corepressor expression was observed in the PCa TRAMP mouse model with increasing age. LCoR interacts with AR and is recruited to chromatin in an androgen-induced manner. Unexpectedly, the LXXLL motif of LCoR is dispensable for interaction with the AR. Rather, the data indicate that LCoR interacts with the AR DNA binding domain on DNA. Interestingly, the interaction of LCoR with AR is inhibited by signaling pathways that are associated with androgen-independent PCa. Here we also show that the Src kinase inactivates the corepressive function of LCoR. Interfering with endogenous Src function by a dominant negative Src mutant, the growth inhibitory activity of LCoR is enhanced in vivo in a xenograft mouse model system. Thus, our studies indicate a role of LCoR as an AR corepressor and a tumor suppressor. Further, the decreased expression or inactivation of LCoR is as an important step toward PCa carcinogenesis in vivo.     

Discovery of AAT-008, a novel,potent, and selective prostaglandin EP4 receptor antagonist

Starting from acylsufonamide HTS hit 2, a novel series of para-N-acylaminomethylbenzoic acids was identified and developed as selective prostaglandin EP4 receptor antagonists. Structural modifications on lead compound 4a were explored with the aim of improving potency, physicochemical properties, and animal PK predictive of QD (once a day) dosing regimen in human. These efforts led to the discovery of the clinical candidate AAT-008 (4j), which exhibited significantly improved pharmacological profiles over grapiprant (1).     

The vanilloid capsaicin induces IL-6 secretion in prostate PC-3 cancer cells

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a constituent of green and red peppers, has been linked with suppression of tumorigenesis through a mechanism that is not well understood. In the present study, we examined the effects of capsaicin on the production of the cytokine interleukin (IL)-6 by PC-3 cells at both protein and mRNA levels which were evaluated by ELISA and real-time PCR, respectively. Capsaicin-treated PC-3 cells increased the synthesis and secretion of IL-6 which was abrogated by the transient receptor potential vanilloid receptor subtype 1 (TRPV1) antagonist capsazepine, as well as by inhibitors of PKC-α, phosphoinositol-3 phosphate kinase (PI-3K), Akt and extracellular signal-regulated protein kinase (ERK). We analyzed the role of capsaicin in the tumor necrosis factor (TNF)-α secretion by PC-3 cells which was increased at shorter times than IL-6 production. Furthermore, incubation of PC-3 cells with an anti-TNF-α antibody blocked the capsaicin-induced IL-6 secretion. These results raise the possibility that capsaicin-mediated IL-6 increase in prostate cancer PC-3 cells is regulated at least in part by TNF-α secretion and signaling pathway involving Akt, ERK and PKC-α activation.     

猪白细胞介素4,6融合基因和CpG基序对猪蓝耳病疫苗免疫的强化效应

为探索新型高效安全的猪蓝耳病疫苗免疫佐剂,本实验用离子交联法制备壳聚糖纳米颗粒包裹猪融合基因PIL-46和CpG基序的真核表达质粒(CNP-(VRIL-46 VR1C)),将CNP-(VRIL-46 VR1C)肌注接种过猪蓝耳病灭活苗的长白川白杂交猪,接种后1、2、4、6和8周采取实验猪静脉血,用Sandwich ELISA检测血清中白细胞介素2(IL-2)、IL-4、IL-6和IgG、IgA、IgM及特异抗体的含量,同时检测外周血液免疫细胞数量的变化。结果发现实验猪接种CNP-(VRIL-46 VR1C)后1～8周内,其血清中IL-2、IL-4、IL-6和IgG、IgA、IgM及特异抗体的含量较对照组均显著增多(P<0.05),淋巴细胞和单核细胞数量也明显升高(P<0.05)。这些表明接种CNP-(VRIL-46 VR1C)的实验猪的特异体液和细胞免疫水平均显著多于对照组,提示PIL-46基因和CpG基序组合能显著增强猪的特异体液和细胞免疫机能,明显提高其免疫防御力,具有研制为高效安全的分子免疫增强剂的应用前景。     

Neutralization of interleukin (IL)-10 released by monocytes/macrophages enhances the up-regulatory effect of monocyte/macrophage-derived IL-6 on expressions of IL-6 and MUC1, and migration in HT-29 colon cancer cells

The interactions between monocyte-derived IL-6 and IL-10 in colon cancer are unknown. We continued previous work that showed monocyte/macrophage-derived IL-6 induces IL-6 and MUC1 expression in HT-29 cancer cells, and evaluated if IL-10 present in monocyte/macrophage is involved in this IL-6-mediated effect. We treated HT-29 cells with monocyte/macrophage supernatant following neutralization of monocyte/macrophage-released IL-10. Neutralization markedly enhanced monocyte/macrophage-derived IL-6 effects on HT-29 cells including IL-6 and MUC1 production and cell migration. Double blocking of IL-6 and IL-10 in monocyte/macrophage supernatants abolished this enhancement. Western blot analysis of STAT3 phosphorylation showed that this augmented response in HT-29 cells following IL-10 neutralization is probably mediated through enhanced IL-6-induced phosphorylation (Tyr⁷⁰⁵) of STAT3 proteins. Therefore, monocytes/macrophages have the capacity to release the functionally associated cytokines IL-6 and IL-10 whose interactions can account for the pathogenesis and progression of colon cancer.