The expression of interleukin-6 (IL-6), IL-6 receptor, and gp130-kilodalton glycoprotein in the rat decidua and a decidual cell line: regulation by 17beta-estradiol and prolactin.

The cytokine interleukin 6 (IL-6), a major mediator of immune and acute phase responses of the liver, has been implicated in the termination of pregnancy once expressed in the uterus. This study was undertaken to investigate the expression and regulation of genes encoding IL-6 and IL-6 receptor (IL-6R) in rat decidual tissue. Total RNA obtained from rat decidual tissue on different days of pseudopregnancy was analyzed by RT-PCR using specific primers for IL-6, IL-6R, and 130-kDa glycoprotein (gp130). Ribosomal L19 primers served as an internal control. IL-6R and gp130 were found to be expressed in the decidua throughout development, while no messenger RNA (mRNA) for IL-6 was detected. Interestingly, within several hours of culture, decidual explants acquired the ability to express IL-6. The apparent ability of decidual cells to express IL-6 and its lack of expression in vivo led us to examine whether the IL-6 gene is actively inhibited. Primary decidual cells were cultured in the presence of estradiol, progesterone, or PRL. Progesterone showed no effect, whereas estradiol and PRL reduced the level of IL-6 mRNA expression. To examine the mechanism by which these hormones inhibit IL-6 expression, we used a simian virus 40-transformed decidual cell line (GG-AD), which expresses only estrogen receptor-beta (ERbeta). Like primary decidual cells in culture, GG-AD cells express IL-6, IL-6R, and gp130 mRNA. When cultured in the presence of estradiol (0-100 ng/ml), mRNA for IL-6 and its receptor components were down-regulated in a dose-dependent manner. Estradiol also caused a dose-dependent decrease in IL-6 protein secretion into the culture medium. The inhibitory effect of estradiol on IL-6 mRNA expression was reversed by the antiestrogen ICI-164,384. Similar inhibition of IL-6 and gp130 mRNA expression was observed with PRL treatment. However, PRL had no effect on IL-6R mRNA levels. PRL inhibition of IL-6 expression was totally reversed by tyrphostin AG490, a JAK2 inhibitor. In summary, the results of this investigation indicate that IL-6 expression, which is detrimental to the maintenance of pregnancy, is inhibited in the rat decidual tissue. This inhibition is induced by PRL and estradiol, which down-regulate not only IL-6 expression, but also the expression of IL-6 receptor and signaling proteins. The results also suggest that PRL signaling to the IL-6 gene is mediated through the long form of PRL receptor and involves JAK2 activation, whereas that of estradiol can be transduced by estrogen receptor-beta.     

Regulation of FSH receptor, PKIbeta, IL-6 and calcium mobilization: Possible mediators of differential action of FSH

Sertoli cells support the development of germ cells by providing a microenvironment in the seminiferous tubules. FSH stimulates Sertoli cell proliferation only during neonatal period till day 18 in the immature rat whereas FSH regulates only functional parameters in the adult rat Sertoli cells. This suggests that FSH exerts differential action in immature and adult Sertoli cells. In an attempt to elucidate the mechanism by which FSH exerts the differential effects, we have carried out both in vivo and in vitro studies using Sertoli cells isolated from immature (7-10 days old) and adult (90 days old) rats. The differential role of FSH was studied at the receptor as well as at the signaling level. Monitoring the level of expression of FSH receptor by RTPCR and northern blot analysis revealed that the expression was more in immature Sertoli cells. Furthermore, it was found that FSH up (1.8-fold) regulates its receptor level only in the immature Sertoli cells and not in the adult. Results also revealed that PKIbeta and calcium, which are the downstream signaling molecules, are involved in FSH regulated Sertoli cells proliferation. It was also observed that FSH up (1.4-fold) regulates the levels of expression of IL-6 mRNA only in the immature rat Sertoli cells suggesting the possibility of its involvement in FSH regulated Sertoli cell proliferation.     

Interleukin-1 is a potent growth factor for immature rat sertoli cells

Testes from rats of different maturational ages were explored for presence of paracrine sertoli cell growth factors. Pubertal and adult testes contained a 17 kDa protein, with potent stimulatory effect on immature Sertoli cell multiplication in vitro. The bioactivity of this protein was mimicked by rat interleukin-1 (IL-1) and neutralized by IL-1 receptor antagonist. A receptor-mediated action was further supported by the demonstration of IL-1 receptor type I mRNA and protein expression in the cultured sertoli cells and in intact immature rat testes. IL-1alpha showed higher efficacy in stimulating proliferation than IL-1beta and follicle-stimulating hormone (FSH), and displayed synergistic action in combination with FSH. As IL-1alpha is constitutively produced by the rat testis and IL-1beta readily inducible by proinflammatory stimuli, our results suggest that IL-1 may serve as a growth factor for Sertoli cells under physiological and pathophysiological conditions.     

Signaling through interleukin-6 receptor supports blast cell proliferation in acute myeloblastic leukemia

Abstract: The role of the interleukin-6/interleukin-6 receptor (IL-6/IL-6R) system in regulating blast cell growth in 8 acute myeloblastic leukemia (AML) patient-derived cell lines was investigated. As they all expressed IL- 6R and as none of them responded to exogenous IL-6 under conventional serum-supplemented culture conditions, we investigated whether signaling through IL-6R plays any role in maintaining their spontaneous colony growth. This was done by treating the cells with monoclonal antibodies made against the ligand-specific IL-6R α-chain or the signal transducer gp130. In serum-supplemented cultures inhibition of gp 130 function did not affect the cell line growth, whereas anti-IL-6R α-chain antibody reduced colony growth. While some of the cell lines also showed similar growth characteristics in a serum-free environment, some others changed their growth pattern and stopped responding to anti-IL-6R α-chain treatment. At the same time, these cell lines also began to respond to exogenously added IL-6 and, interestingly, were stimulated by anti-gp130 antibody. Hence, in some of the blast cells, clonogenic cell growth seemed to be also negatively controlled by an endogenously produced growth-depressing cytokine or cytokines that utilize gp130. All the cell lines, whether cultured in the presence or absence of serum expressed IL-6 both at mRNA and protein level. The current results indicate that AML cells can use IL-6 as a growth stimulating factor, supplied either paracrinely or autocrinely. This could implicate the use of anti-IL-6R α-chain antagonists in AML treatment, not IL-6.     

Synergistic effects of glycoprotein 130 binding cytokines in combination with interleukin-1 on cartilage collagen breakdown

OBJECTIVE: To determine whether other glycoprotein 130 (gp130) binding cytokines can mimic the effects of oncostatin M (OSM) in acting synergistically with interleukin-1alpha (IL-1alpha) to induce cartilage collagen breakdown and collagenase expression, and to determine which receptors mediate these effects. METHODS: The release of collagen and proteoglycan was assessed in bovine and human cartilage explant cultures. Messenger RNA (mRNA) and protein production from immortalized human chondrocytes (T/C28a4) was analyzed by Northern blotting and specific enzyme-linked immunosorbent assays. Collagenase activity was measured by bioassay. Cell surface receptors were detected by flow cytometry. RESULTS: OSM in combination with IL-1alpha caused a rapid synergistic induction of matrix metalloproteinase 1 mRNA, which was sustained over a 72-hour period. Flow cytometric analyses detected both the OSM-specific receptor and the gp130 receptor at the chondrocyte cell surface, but failed to detect the leukemia inhibitory factor receptor (LIFR). Cartilage degradation assays revealed that, of the gp130 binding cytokines, only OSM and IL-6, in the presence of its soluble receptor (sIL-6R), were able to act synergistically with IL-1alpha to promote collagen release. CONCLUSION: This study demonstrates that IL-6 can mimic OSM in synergizing with IL-1alpha to induce chondrocyte-mediated cartilage collagen breakdown and collagenase production. In order to have this effect, IL-6 requires the presence of its soluble receptor. The apparent absence of LIFR explains why other gp130 binding cytokines do not act in synergy with IL-1alpha. Since OSM, IL-6, and sIL-6R levels have all been shown to be elevated in the rheumatoid joint, our findings suggest that these cytokines may be key mediators of cartilage collagen catabolism in the inflammatory arthritides.     

Inhibitory effect of all-trans retinoic acid on the growth of freshly isolated myeloma cells via interference with interleukin-6 signal transduction [see comments]

We showed the dose-dependent growth inhibition by alltrans retinoic acid (ATRA) of myeloma cells freshly isolated from patients. ATRA downregulated the cell surface expression of interleukin-6 receptor (IL- 6R) and/or glycoprotein (gp) 130. The growth-inhibitory activity of ATRA was well correlated with that of anti-gp 130 antibody in every sample. Furthermore, ATRA inhibited the production of IL-6 from both myeloma cells and marrow stromal cells, and recombinant IL-6 (rIL-6) could partially recover the myeloma cell growth that had been inhibited by ATRA. These data suggest that ATRA may inhibit the proliferation of myeloma cells both by the downregulation of IL-6R and gp130 expression on myeloma cells and by the inhibition of IL-6 production from myeloma and stromal cells. Prednisolone (PSL) and interferon-gamma (IFN-gamma) also inhibited the myeloma growth, while their effects were different from those of ATRA on IL-6 R and gp130 expression, IL-6 production, and morphological change. The inhibitory effect of ATRA on myeloma cell proliferation was observed in 10 of 14 samples obtained from eight patients, which suggests that ATRA may be a potent new therapeutic agent for some myeloma patients.     

Effect of recombinant human interleukin-11 on expressions of interleukin-11 receptor α-chain and glycoprotein 130 in intestinal epithelium cell line-6 after neutron irradiation

AIM: To explore the effect of recombinant human interleukin-11 (rhIL-11) on the expressions of interleukin-11 receptor α-chain (IL-11Rα) and an additional signal transducer glycoprotein 130 (gp130) in intestinal epithelium cell line-6 (IEC-6) after neutron irradiation.METHODS: Cultured IEC-6 cells were exposed to 4.0Gy neutron and treated with 100 ng/mL rhIL-11 12 h prior to or immediately after irradiation. The apoptosis and necrosis rates and expressions of IL-11Rα and gp130 were observed by flow cytometry, immunohistochemistry, Western blot and image analysis.RESULTS: The apoptosis rate of IEC-6 cells was increased by irradiation at 6 h (P ＜ 0.01), IL-11 stimulation resulted in a decreased apoptosis rate in irradiated IEC-6 cells (P ＜ 0.05). In normal control IEC-6 cells, intense immunoreactivity of IL-11Rα was located within the cell membrane and cytoplasm. The level of IL-11Rα expression significantly decreased at 6 h after irradiation (P ＜ 0.01) and restored at 24 h after irradiation. In IEC-6 cells treated with both radiation and rhIL-11, the level of IL-11Rα expression was higher than that of irradiated cells (P ＜ 0.05). When it came to gp130 protein, it was located in the cytoplasm of IEC-6 cells. After irradiation, we found a progressive decrease in the expression of gp130 protein (P ＜ 0.05) in 48 hours post-radiation, while in rhIL-11-stimulated cells, it came back to normal level at 24 h after irradiation and decreased at 48 h, but was still higher than that of only irradiated cells (P ＜ 0.05).CONCLUSION: rhIL-11 can protect IEC-6 cells from neutron irradiation. The protective effect of rhIL-11 might be connected with its ability to up-regulate the expressions of specific ligand-binding subunit IL-11Rα and signal-transducing subunit gp130.     

Interleukin-1 receptor antagonist is produced by sertoli cells in vitro

The interleukin-1 (IL-1) system has been suggested to be involved in the cell-to-cell cross-talk within the testis. To identify a testicular cell source of IL-1 receptor antagonist (IL-1ra), mouse Sertoli cells were isolated, purified, cultured, and examined for IL-1ra. Our investigation revealed that Sertoli cells produce large amounts of immunoreactive IL-1ra under basal culture conditions, as examined by enzyme-linked immunosorbent assays. Its expression can be induced, showing maximum concentrations after 8 h of stimulation. Lipopolysaccharide, as well as IL-1alpha and -beta, were found to stimulate IL-1ra production in Sertoli cells. FSH is capable to induce IL-1ra production in Sertoli cells in a dose-dependent manner. Immunocytochemical staining confirmed the presence of IL-1ra in the cytoplasma of Sertoli cells. The presence of IL-1ra messenger RNA was demonstrated by RT-PCR analysis. Our results may help to better evaluate the IL-1 activity in the testis and may indicate the involvement of IL-1ra in the autocrine and paracrine regulation of testicular cell function.     

Epigallocatechin-3-gallate inhibits IL-6 synthesis and suppresses transsignaling by enhancing soluble gp130 production

Regulation of IL-6 transsignaling by the administration of soluble gp130 (sgp130) receptor to capture the IL-6/soluble IL-6R complex has shown promise for the treatment of rheumatoid arthritis (RA). However, enhancing endogenous sgp130 via alternative splicing of the gp130 gene has not yet been tested. We found that epigallocatechin-3-gallate (EGCG), an anti-inflammatory compound found in green tea, inhibits IL-1β–induced IL-6 production and transsignaling in RA synovial fibroblasts by inducing alternative splicing of gp130 mRNA, resulting in enhanced sgp130 production. Results from in vivo studies using a rat adjuvant-induced arthritis model showed specific inhibition of IL-6 levels in the serum and joints of EGCG-treated rats by 28% and 40%, respectively, with concomitant amelioration of rat adjuvant-induced arthritis. We also observed a marked decrease in membrane-bound gp130 protein expression in the joint homogenates of the EGCG-treated group. In contrast, quantitative RT-PCR showed that the gp130/IL-6Rα mRNA ratio increased by ~2-fold, suggesting a possible mechanism of sgp130 activation by EGCG. Gelatin zymography results showed EGCG inhibits IL-6/soluble IL-6R–induced matrix metalloproteinase-2 activity in RA synovial fibroblasts and in joint homogenates, possibly via up-regulation of sgp130 synthesis. The results of these studies provide previously undescribed evidence of IL-6 synthesis and transsignaling inhibition by EGCG with a unique mechanism of sgp130 up-regulation, and thus hold promise as a potential therapeutic agent for RA.     

Glycoprotein 130 ligand oncostatin-M induces expression of vascular endothelial growth factor in human adult cardiac myocytes

OBJECTIVE: In murine and rat cardiac myocytes the gp130 system transduces survival as well as hypertrophic signals and via induction of the expression of the potent angiogenic factor VEGF in these cells also indirectly contributes to cardiac repair processes through the development of new blood vessels. There are, however, species differences in receptor specificity and receptor crossreactivity in the gp130-gp130 ligand system. We asked whether gp130 signaling is also involved in the regulation of VEGF in human cardiac myocytes and if so which gp130 ligands are critical for such an effect. METHODS: Human adult cardiac myocytes (HACMs) were isolated from myocardial tissue and characterised by positive staining for myocardial actin, troponin-I and cardiotin. HACMs were treated with the gp130 ligands CT-1, IL-6, LIF or OSM and VEGF-1 was determined by a specific ELISA in the conditioned media of these cells. RT-PCR and Western blot analysis was used in order to detect gp130, IL-6-receptor, LIF-receptor or OSM-receptor specific protein and mRNA in human adult cardiac myocytes and for detection of VEGF-1 specific mRNA in cardiac myocytes after incubation with OSM. Pieces of myocardial tissue were incubated ex vivo in the presence and absence of OSM and VEGF was determined in supernatants of these cultures and immunohistochemistry was performed on the tissue using specific antibodies for VEGF-1. Immunohistochemistry was also employed to detect VEGF in sections from a healthy human heart and in a heart from a patient suffering from acute myocarditis. RESULTS: OSM, but not CT-1, IL-6 or LIF increased VEGF-1 production in human adult cardiac myocytes dose-dependently derived from five different donors. This selective stimulation of VEGF by gp130 ligands was also reflected by a specific receptor expression on these cells. We detected high levels of mRNA for gp130 and the OSM receptor in freshly isolated human cardiac myocytes but only low amounts of mRNA for the IL-6 receptor whereas mRNA for the LIF receptor was hardly detectable by RT-PCR. OSM receptor and IL-6 receptor were also detectable by Western blotting whereas LIF receptor was only present as a faint band. OSM also increased the expression of VEGF-1 mRNA in cardiac myocytes. When pieces of human myocardial tissue were incubated with the gp130 ligands in an ex vivo model only OSM resulted in an increase in VEGF-1 in the supernatants of these cultures. Furthermore, VEGF increased in tissue samples treated with OSM in cardiac myocytes as evidenced by immunohistochemistry. In addition, we found increased VEGF-1 expression in myocardial tissue from a patient suffering from acute myocarditis. CONCLUSION: The gp130-gp130 ligand system is also involved in VEGF regulation in human cardiac myocytes and OSM is the gp130 ligand responsible for this effect in the human system whereas LIF and CT-1 which had been shown to regulate VEGF expression in mouse and rat cardiac myocytes had no effect. Thus we have added OSM, which is produced by activated T lymphocytes and monocytes, to the list of regulatory molecules of VEGF production in the human heart. Our results lend further support to the notion that besides hypoxia, inflammation via induction of VEGF through autocrine or paracrine pathways plays a key role in (re)vascularisation of the myocardium.     

The gp130 cytokines interleukin-11 and ciliary neurotropic factor regulate through specific receptors the function and growth of lactosomatotropic and folliculostellate pituitary cell lines

Two of the most potent cytokines regulating anterior pituitary cell function are leukemia inhibitory factor and interleukin-6 (IL-6), which belong to the cytokine receptor family using the common gp130 signal transducer. We studied the actions of two other members of this family, IL-11 and ciliary neurotropic factor (CNTF), on folliculostellate (FS) cells (TtT/GF cell line) and lactosomatotropic cells (GH3 cell line). The messenger RNA (mRNA) for the alpha-chain specific for the IL-11 receptor (1.7 kb) and CNTF receptor (2 kb) are expressed on both cell types. In addition, we detected CNTF receptor mRNA in normal rat anterior pituitary cells. IL-11 (1.25-5 nM) dose dependently stimulated the proliferation of FS cells. CNTF, at doses from 0.4-2 nM, also significantly stimulated the growth of these cells. In addition, both cytokines significantly stimulated proliferation of lactosomatotropic GH3 cells, and CNTF stimulated hormone production (GH and PRL) at 24 h by these cells. At 16-72 h, IL-11 stimulates the secretion of the angiogenic factor vascular endothelial growth factor by FS cells. In addition, both GH3 and FS cells express CNTF mRNA. These data suggest that IL-11 and CNTF may act as growth and regulatory factors in anterior pituitary cells.     

Reciprocal regulation of activin A and inhibin B by interleukin-1 (IL-1) and follicle-stimulating hormone (FSH) in rat Sertoli cells in vitro

In several biological systems, the inhibin beta(A) homodimer activin A is stimulated by, and in turn, inhibits the action of interleukin (IL)-1 (both IL-1alpha and IL-1beta) and IL-6. The possibility that a similar regulatory relationship operates within the testis was investigated. Sertoli cells from immature (20-day-old) rats were cultured with human IL-1alpha or IL-1beta, human IL-6 and/or ovine FSH or dibutyryl cAMP. Activin A and the inhibin dimers, inhibin A and inhibin B, were measured by specific ELISA. Immunoreactive inhibin (ir-inhibin) was measured by RIA. Activin/inhibin subunit mRNA expression was measured by quantitative real-time PCR. Both IL-1 isoforms, but not IL-6, stimulated activin A secretion through increased synthesis of beta(A)-subunit mRNA. IL-1 also stimulated activin A secretion by testicular peritubular cells. In contrast to the effect on activin A, IL-1 suppressed inhibin beta(B)-subunit and, to a lesser extent, alpha-subunit mRNA expression, thereby reducing basal and FSH-stimulated inhibin B secretion by the Sertoli cells. Conversely, FSH inhibited basal activin A secretion and antagonised the stimulatory effects of IL-1. Dibutyryl cAMP partially inhibited the action of IL-1 on activin A secretion, but had no significant effect on basal activin A secretion. Secretion of inhibin A was low in all treatment groups. These data demonstrate that IL-1 and FSH/cAMP exert a reciprocal regulation of activin A and inhibin B synthesis and release by the Sertoli cell, and suggest a role for activin A as a potential feedback regulator of IL-1 and IL-6 activity in the testis during normal spermatogenesis and in inflammation.     

Graves病患者甲状腺组织内IL-6及gp130表达增强

对8例Graves病(GD)甲状腺组织内白细胞介素6(IL-6)及其受体(gp80、gp130)表达进行研究,发现GD组甲状腺组织内IL-6mRNA、gp130mRNA表达增高;GD中甲状腺滤泡细胞和淋巴细胞胞浆IL-6表达水平显著升高.提示GD患者甲状腺内IL-6/gp130信号通路的激活可能参与GD的发病.     

白细胞介素-6受体基因多态性与内分泌疾病的关系

白细胞介素-6（IL-6）是由不同种类细胞如淋巴细胞、内皮细胞、成纤维细胞、骨骼肌细胞及脂肪细胞所分泌的一种多效性细胞因子，主要参与糖代谢和脂代谢，IL-6水平与肥胖和2型糖尿病相关。IL-6发挥其生物学功能是通过结合白细胞介素-6受体（IL-6R）的双链系统（gp80、gp130）来完成的。IL6R基因的外显子和内含子中均存在单核苷酸多态性（SNPs），IL-6R基因的SNPs与2型糖尿病、肥胖、雄激素过多症和多囊卵巢综合征等代谢性疾病具有相关性。     

补肾中药对去卵巢大鼠骨髓IL-6、IL-6R基因表达及骨髓源性破骨细胞形成的影响

目的　观察补肾中药对去卵巢大鼠骨髓细胞表达白介素 6 (IL 6 )、IL 6受体 (IL 6R)、gp130基因的影响及其与骨髓干细胞诱导分化形成破骨细胞能力变化的关系。　 方法　健康 3月龄雌性SD大鼠 ,随机分为假手术对照组、去卵巢组、雌激素组、中药组。取骨髓细胞作细胞培养和提取RNA。细胞培养第 6天染色 ,以TRAP染色 (+)以及胞核≥ 3为破骨细胞。骨髓细胞用TRIZOL提取总RNA后进行RT PCR。　结果　去卵巢组术后 6周及 12周 ,骨髓破骨细胞形成数明显多于对照组 (P <0 0 5 ) ,补肾中药与雌激素均能抑制破骨细胞的形成 (P <0 0 5 )。去卵巢后 6周骨髓细胞IL 6和IL 6RmRNA表达显著增高 (P <0 0 5及 0 0 1) ,第 12周时 ,IL 6、IL 6R基因表达虽有下降 ,但仍然高于对照组。补肾中药与雌激素均显示可减少上述基因过度表达 ,以第 6周时作用最显著(P <0 0 5及 0 0 1)。以上各组全程未见 gp130基因表达水平有明显变化。　 结论　本方所用补肾药物可以抑制大鼠去卵巢后骨髓IL 6、IL 6R基因过度表达 ,这一效应与药物抑制骨髓源性破骨细胞生成的作用密切相关。     

Expression of interleukin-6 and its effects on growth of HP75 human pituitary tumor cells

The role of IL-6 in the pathogenesis of pituitary adenomas is unclear, as tumor biology is difficult to study in primary culture. We have shown here that the human pituitary cell line HP75 synthesizes IL-6 mRNA and expresses and secretes IL-6 (6167 +/- 56 pg/ml/72 h for 30,000 cells). IL-6 receptor (IL-6R) mRNA was identified by in situ hybridization and RT-PCR. Exogenous IL-6 in low dose (1 ng/ml) stimulated, whereas higher doses (100 ng/ml) inhibited, growth. This diverse effect occurs in other cell types as a result of receptor down-regulation. Cell growth was inhibited by IL-6-blocking antibody (76 +/- 6.5% inhibition; P < 0.0001). This demonstrates that IL-6 is an important growth regulator in HP75 cells, having an autocrine growth stimulatory effect under basal conditions. IL-1alpha and dibutyryl cAMP stimulated and dexamethasone inhibited IL-6 secretion; however, bacterial lipopolysaccharide, forskolin, and cholera toxin had no effect. This implies that there is a defect in the control of IL-6 secretion. Soluble IL-6R was not detected, but soluble gp130 receptor was present in the conditioned medium. Stimulation of cleavage of soluble IL-6R from the membrane-bound IL-6R could not be induced by phorbol ester or dexamethasone. Whether IL-6 has a similar effect in human pituitary adenomas requires further investigation.