Induction of interleukin 6 by human and murine recombinant interleukin 1 in mice

Interleukin (IL) 6 is a pleistropic cytokine with activities, among others, on immune cells, hematopoietic precursor cells and hepatocytes. We have investigated the kinetics and amplitude of its in vivo induction in mice after injection of four different IL 1 species as well as murine (m) and human (h) tumor necrosis factor (TNF) and bacterial lipopolysaccharide (LPS) using a sensitive bioassay on 7TD1 cells to measure the IL 6 concentrations. Recombinant mIL 1 beta, administered as a single i.v. injection in mice, induced the appearance of IL 6 in the plasma with peak levels observed after 2 h. A dose-response correlation was found between serum IL 6 levels and injected IL 1 alpha concentrations at 3 and 8 h after the injection. We then compared the ability of h/mIL 1 alpha, h/mIL 1 beta, h/mTNF and LPS to induce IL 6 in mice. We found: (a) LPS is the most potent inducer of IL 6; (b) 3 h after injection, the four IL 1 preparations had induced IL 6 levels comparable with the IL 6 levels observed after TNF injection; (c) high doses of mIL 1, alpha or beta, but not hIL 1, resulted in a high IL 6 level persisting for over 8 h. We conclude that IL 1 is a potent inducer of IL 6 in vivo and that no major differences are observed between the four IL 1 preparations, as evaluated at 3 h after the injection. However, mIL 1 alpha and mIL 1 beta, in contrast to hIL 1 alpha and hIL 1 beta, induced a sustained IL 6 level over a longer time period. This pattern of prolonged IL 6 induction is even much more pronounced after mTNF injection, but not after hTNF injection.     

Action of recombinant human interleukin 6, interleukin 1 beta and tumor necrosis factor alpha on the mRNA induction of acute-phase proteins

The rat hepatoma cell line Fao was used to study the role of three inflammatory mediators on the mRNA regulation of several acute-phase proteins. In the presence of 10(-6) M dexamethasone beta-fibrinogen mRNA levels increased 6-fold after addition of recombinant human IL 6 (rhIL 6). rhIL 1 beta or recombinant human tumor necrosis factor alpha (rhTNF alpha) had essentially no effect on beta-fibrinogen mRNA induction but led to a 20-fold increase in alpha 1-acid glycoprotein mRNA in the presence of dexamethasone. On the other hand, rhIL 6 was a much weaker stimulator of alpha 1-acid glycoprotein mRNA synthesis. All three mediators reduced albumin mRNA concentrations to about 30% of controls. Whereas the induction of beta-fibrinogen mRNA was potentiated by dexamethasone, the synthetic glucocorticoid analog was an absolute requirement for the stimulation of alpha 1-acid glycoprotein mRNA. The mRNA levels of the negative acute-phase protein albumin were induced 5-fold by dexamethasone alone. The beta-fibrinogen mRNA induction started immediately after addition of rhIL 6 and reached a maximum between 12 and 18 h. In contrast, the time-course for alpha 1-acid glycoprotein mRNA synthesis showed a lag phase of 8 h followed by an increase up to 20 h after rhIL 1 beta. rhTNF alpha led to an even more delayed increase in alpha 1-acid glycoprotein mRNA. Whereas in the case of beta-fibrinogen mRNA induction no synergistic effect was observed between various concentrations of the three mediators, the combination of rhIL 6/rhIL 1 beta as well as rhIL 6/rhTNF alpha or rhIL 1 beta/rhTNF alpha regulated synergistically alpha 1-acid glycoprotein and albumin mRNA. It is concluded that discrete acute-phase proteins are regulated differently by the inflammatory mediators IL 6, IL 1 beta and TNF alpha, indicating that the acute-phase response is more complex than previously assumed. The Fao cell line used in this study turned out to be an ideal model for acute-phase protein regulation, suitable for the discrimination between the inflammatory mediators IL 6 and IL 1/TNF alpha.     

Hyper-IL-6基因诱导的体内抗肝癌免疫功能

目的:建立稳定表达Hyper-IL-6基因的小鼠肝癌细胞株,探讨Hyper-IL-6用于诱导抗肝癌主动免疫治疗的可行性。方法:用脂质体法将Hyper-IL-6基因转染小鼠肝癌细胞系MM45T.Li,通过G418筛选获得抗性细胞株,命名为MM45T-HIL-6,RT-PCR和ELISA法检测目的基因的表达。同时筛选空载体pEGFP-C1转染的阳性克隆命名为MM45T-mock作为对照。将BALB/c小鼠随机分为3组,于各组小鼠右侧腋部皮下分别接种细胞MM45T.Li、MM45T-mock和MM45T-HIL-6 5×105个/只,体内实验观察MM45T.Li、MM45T-mock及MM45T-HIL-6的成瘤性,流式细胞仪检测荷瘤小鼠外周血中CD4+,CD8+T淋巴细胞亚群水平。结果:RT-PCR和ELISA检测结果表明,筛选到的小鼠肝癌细胞株MM45T-HIL-6中存在Hyper-IL-6的表达,而MM45T.Li和MM45T-mock不表达。体内成瘤实验结果显示,与MM45T.Li、MM45T-mock相比,MM45T-HIL-6的成瘤性下降;流式细胞分析结果显示,与接种MM45T.Li和MM45T-mock的小鼠相比,接种MM45T-HIL-6稳定表达HYPER-IL-6基因的小鼠肝癌细胞株的小鼠外周血中CD4+,CD8+T淋巴细胞水平明显升高,差异有统计学意义(P<0.05)。结论:转染Hyper-IL-6基因的小鼠肝癌细胞株能够诱导小鼠抗肝癌主动免疫反应。     

Relation between acute-phase proteins and enhanced bleomycin-induced pulmonary fibrosis in the rat

Intratracheal application of Bleomycin (Bleo) in rats induces interstitial pneumonitis followed by progressive fibrosis. As the presence of high levels of acute-phase proteins (= reactants = APR), especially alpha 2-macroglobulin of the rat (alpha 2M), enhances liver fibrosis, we investigated whether this phenomenon also occurs in rats with Bleo-induced lung fibrosis. The experiments showed that this is the case; lung fibrosis assessed by measuring hydroxyproline, hexosamine, and prolyl-4-hydroxylase was enhanced when just before Bleo application an acute-phase reaction was induced. This effect can be explained by the inhibitory effect of alpha 2M on collagenase. The experiments showed a significant positive correlation between alpha 2M and parameters of fibrosis. This is especially the case in the third week after Bleo application. Bleo itself does not induce a strong acute-phase reaction, notwithstanding the pneumonitis during the first weeks. The increased fibrosis is accompanied by progressive ventilatory disturbances demonstrated by high arterial pCO2 and low pO2. In patients undergoing Bleo treatment, varying levels of APR can be expected, and this could explain the rapid development of fibrosis in individual cases.     

顺铂诱导大鼠C6胶质瘤细胞凋亡

为深入进行胶质瘤细胞凋亡的分了生物学研究及提高胶质瘤辅助化疗疗效打下基础。通过光镜,电镜,荧光显微镜分析,DNA断裂分析及流式细胞仪分析,进行顺铂诱导C6胶质瘤细胞调亡研究。本研究证实在3μg／mLCDDP作用72h,C6细胞出观细胞凋亡;光镜和电镜可见细胞形态学上出现细胞皱缩,染色质浓集贴达;流式细胞仅结果提示有凋亡峰出现,凋亡细胞占细胞总数17.3％±1.2％;荧光显微镜观察出现染色质浓集,染色质断裂：DNA电泳未表现出DNA呈梯状带型断裂。上述结果提示用顺铂成功地诱导大鼠C6胶质瘤细胞发生凋亡。     

Endotoxin, tumor necrosis factor-alpha and interleukin 1 induce interleukin 6 production in vivo

The ability of Escherichia coli-derived lipopolysaccharide (LPS), recombinant (r) interleukin 1-beta (rIL-1 beta), and r murine tumor necrosis factor-alpha (rMuTNF-alpha) to induce interleukin 6 (IL-6) production in vivo was investigated. Peak serum IL-6 concentration was attained after 2 hr of LPS injection into mice. The coinjection of antiserum against rMuTNF-alpha with LPS resulted in a reduction of the induced serum IL-6 level, indicating the involvement of endogenous TNF-alpha in LPS induction of IL-6. Recombinant IL-1 beta and rMuTNF-alpha injected directly caused the production of substantial amounts of IL-6 within 30 min. The injection of a combination of rIL-1 beta and rTNF-alpha induced a significantly greater level of IL-6 than either agent alone. The greater level of serum IL-6 was associated with hypothermia and an increased lethality among mice injected with both cytokines. These data demonstrate the abilities of IL-1 beta and TNF-alpha to induce IL-6 production in vivo and indicate that LPS induction of IL-6 may be mediated, at least partially, through TNF-alpha action. The data describe a new in vivo biologic activity shared between IL-1 beta and TNF-alpha and suggest that IL-6 may be an important effector in the manifestation of TNF-alpha and IL-1 beta actions in vivo.     

The relation among stress, adrenalin, interleukin 6 and acute phase proteins in the rat.

Stress reactions exist in many conditions in which plasma interleukin 6 (IL-6) is elevated. Examples are burns and sepsis. In these situations fever is often present. These stress situations are always accompanied with high levels of adrenalin and corticosteroids. These hormones, especially when given together, elicit a definite response of acute phase proteins in normal rats. In two stress models, (i) laparotomy and (ii) fever induced by administration of PGE2 in the lateral intracerebral ventricle, we observed a rise of adrenalin and corticosteron followed by an elevated level of plasma IL-6. Therefore, we studied the effect of adrenalin and corticosteron on the plasma level of IL-6. Adrenalin evokes high levels of IL-6, and this effect can be blocked by propranolol. When IL-6 release is blocked in this way, the response of alpha 2 macroglobulin and the cysteine protease inhibitor, both fast-reacting acute phase proteins in rat, is strongly depressed. Isoprenalin, an adreno beta 2 agonist, also causes very high levels of IL-6, indicating that the release of IL-6 can be mediated by an adreno beta 2 receptor whose presence has been demonstrated in monocytic cells. The results suggest a relation between stress situations and IL-6 and may be another factor besides the presence of endotoxins, virus, etc. explaining the high levels of IL-6 observed in many serious clinical situations.     

Quantitative changes in interleukin proteins following focal stroke in the rat

Changes in the phosphoinositide (PPI) signal transduction system induced by E-5842, a new sigma₁ (σ₁) receptor ligand and potential atypical antipsychotic, were studied in the rat frontal cortex, hippocampus and striatum. Acute treatment with E-5842 increased phospholipase C (PLC) activity in the striatum and the hippocampus. Chronic treatment with E-5842 induced an increase in the activity of PLC in the frontal cortex and the striatum. Similar up-regulation of the activity of the enzyme was also observed in rat frontal cortex membranes in presence of GTPγS. After chronic treatment with E-5842, it was also observed a significant increase of the immunoreactivity levels of G_q/11 in the frontal cortex. Our results suggest that part of the antipsychotic effects of E-5842 could be related to the regulation of the PPI signal transduction pathway, especially after a prolonged treatment.     

Induction of endogenous retroviral gene product (SU) as an acute-phase protein by IL-6 in murine hepatocytes.

The effect of Lps locus and IL-6 on the production of SU (previously termed gp70), a mouse endogenous retroviral gene product, was studied. Back-cross studies using the progeny between (NZB x C3H/HeJ)F1 and C3H/HeJ mice indicate that the basal level of SU is not associated with the Lps locus on chromosome 4. Lipopolysaccharide (LPS) mitogen response-negative mice did not show the enhancement of serum SU production after LPS injection. Spleen cells from LPS-mitogen response-positive but not from negative mice showed increase of IL-6 synthesis in the presence of LPS. Since IL-6 may be involved in the production of serum SU, we tested the effect of IL-6 in a primary hepatocyte culture system. SU production was clearly enhanced in the presence of recombinant IL-6, indicating that IL-6 induced by LPS can enhance the expression of retroviral genome.     

Induction of unusual tumors in vivo by rat fibroblasts treated with diepoxybutane.

Diploid rat embryo fibroblasts exposed to diepoxybutane, a carcinogenic alkylating agent, exhibited chromosome aberrations and a high proportion of tetraploid cells immediately after treatment. Following a prolonged period of carcinogen-free growth, a diepoxybutane-treated cuture showed morphologic transformation and produced transplantable malignant tumors upon inoculation into neonatal rats. Morphologic and histochemical characteristics of the tumors derived from cells exposed to diepoxybutane in culture were also found in tumors of animals exposed directly to this carcinogen. The tumors from diepoxybutane-treated cell cultures were composed of highly anaplastic giant cells with centrally located cytoplasmic inclusions. Clusters of identical cells were found in some of the tumors obtained by direct in vivo treatment of rats with diepoxybutane. The morphologic similarities of these tumors suggest that similar processes of cellular evolution to malignancy occur in cell culture systems and in vivo.     

Enhanced expression of interleukin 6 in rat and murine arthritis models

Interleukin 6 (IL-6) is a multifunctional cytokine and plays an important role in host defense mechanisms. Enhanced production of IL-6 has been reported in polyclonal B-cell abnormalities and autoimmune diseases such as rheumatoid arthritis (RA). To investigate the role of IL-6 in inflammatory joint diseases, serum IL-6 levels of three animal models of RA, namely type II collagen (CII)-induced murine, rat arthritis and adjuvant-induced rat arthritis, were monitored. In these models, serum IL-6 increased with the development of arthritis. Serum IL-6 was not elevated by immunization with a non-arthritogenic immunogen such as bovine type I collagen (CI) and bovine serum albumin (BSA) to DBA/1J mice. The serum IL-6 level was correlated well with the severity of adjuvant-induced arthritis. The elevated IL-6 in sera may be associated with the overproduction of IL-6 at the arthritic paws, because higher IL-6 activity was detected in the homogenates of arthritic paws as compared with the control paws. Synovial fibroblasts were isolated from the arthritic knee joints of DBA/1J mice. These cells expressed type I interleukin 1 (IL-1) receptor constitutively and produced large amounts of IL-6 in response to IL-1 in vitro. Enhanced production of IL-1 was also detected at the arthritic paws. These results suggest that the elevated IL-6 in sera may be associated with the overproduced IL-6 in response to the increased IL-1 at the arthritic joints. Serum IL-6 may be a useful parameter for monitoring disease activity.     

Changes of gene expression of iron regulatory proteins during turpentine oil-induced acute-phase response in the rat

In the present study, turpentine oil was injected in the hind limb muscle of the rat to stimulate an acute-phase response (APR). The changes in the gene expression of cytokines and proteins known to be involved in the iron regulatory pathway were then studied in the liver and in extra-hepatic tissue. In addition to the strong upregulation of interleukin-6 (IL-6) and IL-1 beta observed in the inflamed muscle, an upregulation of the genes for IL1-beta and tumor necrosis factor-alpha, but not IL-6, were detectable in the liver. Hepatic Hepc gene expression increased to a maximum at 6 h after the onset of APR. An upregulation of transferrin, transferrin receptor 1 (TfR1), TfR2, ferritin-H, iron responsive element binding protein-1 (IRP1), IRP2 and divalent metal transporter gene expression was also found. Hemojuvelin (Hjv)-, ferroportin 1-, Dcytb-, hemochromatosis-gene- and hephaestin gene expression was downregulated. Hepcidin (Hepc) gene expression was not only detectable in extra-hepatic tissues such as heart, small intestine, colon, spleen and kidney but it was also upregulated under acute-phase conditions, with the Hjv gene being regulated antagonistically. Fpn-1 gene expression was downregulated significantly in heart, colon and spleen. Most of the genes of the known proteins involved in iron metabolism are expressed not only in the liver but also in extra-hepatic tissues. Under acute-phase conditions, acute-phase cytokines (eg IL-6) may modulate the gene expression of such proteins not only in the liver but also in other organs.     

Induction of interleukin 1 secretion by adjuvant-active peptidoglycans.

The ability of differently structured, purified peptidoglycans (PG) to induce interleukin 1 (IL1) secretion was compared. PG from Bacillus megaterium and Staphylococcus aureus stimulated the production of IL1 by mouse peritoneal macrophages and human adherent mononuclear cells, whereas PG from Micrococcus lysodeikticus and Corynebacterium poinsettiae were inactive. There was a correlation between the ability of PG to induce IL1 secretion and previously demonstrated immunoenhancing activities (adjuvant effect, increase of resistance to tumor growth) of PG. PG solubilization by lysozyme decreased but did not abolish the PG effect on IL1 secretion. Active PG induced IL1 production in nude mice and in the C3H/HeJ strain (which is unresponsive to lipopolysaccharides).     

In vivo interleukin 6 gene expression in the tumoral environment in multiple myeloma

Whether interleukin 6 (IL 6) is an autocrine or paracrine myeloma cell growth factor in vivo remains unresolved. To identify which cells are producing IL 6 in vivo, we have studied the IL 6 gene expression in bone marrow mononuclear cells (BMMC) of 19 patients with multiple myeloma (MM) and in peripheral blood mononuclear cells (PBMC) of 9 patients with plasma cell leukemia (PCL). We found that the IL 6 gene was transcribed by BMMC of most patients with MM (79%). Further, IL 6 mRNA was not produced by purified myeloma cells from patients with either MM (5 patients) or PCL, but by the bone marrow environment, mainly by monocytes and myeloid cells (CD13+CD15+ cells). For 2 patients with PCL, for whom PBMC and BMMC samples were available, IL 6 mRNA could be detected in BMMC but not in PBMC. Finally, no IL 6 mRNA was detected in five freshly established IL 6-dependent myeloma cell lines. The present data give a clear-cut demonstration of the paracrine origin of IL 6 in vivo in human MM.     

Adenovirus vector expressing mouse oncostatin M induces acute-phase proteins and TIMP-1 expression in vivo in mice.

Mouse oncostatin M (MuOSM) regulates the production of acute-phase proteins by hepatocytes as well as tissue inhibitor of metalloproteinases-1 (TIMP-1) production by fibroblasts in vitro. We have generated an adenovirus (Ad) encoding MuOSM and tested the effects of administration of recombinant AdMuOSM to mice in vivo. On intramuscular injection, AdMuOSM (5 X 10(7) plaque-forming units, pfu) induced an increase in serum levels of interleukin-6 (IL-6) as well as the acute-phase proteins serum amyloid A (SAP) and alpha1-acid glycoprotein (AGP) at day 1. SAP and AGP concentrations were elevated to greater levels at day 3 and decreased to near control levels at day 7. Intratracheal treatment with AdMuOSM induced TIMP-1 mRNA levels (as assessed by Northern blots) that corresponded to the presence of transgene MuOSM mRNA levels. TIMP-1 was elevated at day 1 and day 3 and less consistently at day 7 after administration. Intraperitoneal treatment with AdMuOSM also resulted in elevation of TIMP-1 mRNA in lung tissue. These results show that AdMuOSM can induce both local and systemic effects and demonstrate in vivo effects of OSM that are consistent with in vitro studies on acute-phase protein and TIMP-1 expression.     

Elevation of serum interleukin 6 prior to acute phase proteins on the inflammation by surgical operation

Serum levels of interleukin 6 (IL-6) and acute phase proteins were measured in patients who underwent surgical operation. Elevation of IL-6 preceded that of acute phase proteins, indicating that the measurement of serum IL-6 may be helpful for the early detection of an inflammatory state.     

Approach to treatment of bronchopneumonia by evaluation of selected acute-phase proteins in calf herds

The objective of this study was to evaluate the concentrations of selected acute-phase proteins serum amyloid A, haptoglobin, fibrinogen, and albumin in calves with bronchopneumonia. Bronchopneumonia is a multifactorial disease in association with various infectious agents. Negative economic impact of this disease is associated with death loss, treatment costs, reduction in live weight gain, and reduced productive life span. In this study, acute-phase proteins and alteration in hematologic values were measured for evaluation of dairy calves’ health status. Sixty Holstein calves within 2 weeks up to 6 months old were divided into treatment and control groups. Clinical findings including body temperature, pulsation, and respiratory rate were recorded after common physical examination. Blood samples were collected from the jugular vein of each calf. Hematological parameters as well as some biochemical profiles, i.e., albumin and globulin, showed no significant difference between the two groups (p?>?0.05). The results indicated a significant increase in serum amyloid A, haptoglobin, and fibrinogen between the two groups (p?<?0.05). In conclusion, this study reveals that serum amyloid A, haptoglobin, and fibrinogen are potentially useful and sensitive markers for early determination of bronchopneumonia and can be used as inflammatory indicators of health in calf herds, thereby facilitating treatment decisions.     

Induction of cytolytic cells by pure recombinant human interleukin 2

Purified recombinant human interleukin 2, produced in Escherichia coli, was sufficient to generate cytolytic cells in concanavalin A-stimulated, T helper cell-depleted (Lyt-1.1-) or accessory cell-depleted (Ia-) murine spleen cell cultures. Moreover, recombinant interleukin 2 (rIL2) was able (even at 0.5 ng/ml) to induce cytolytic cells in undepleted murine spleen cell cultures and undepleted human peripheral blood lymphocyte cultures in the absence of a mitogen. Purified recombinant human interferon-gamma, produced in Chinese hamster ovary cells, did not induce either cytolytic activity or IL2 responsiveness in human peripheral blood lymphocyte cultures. Possibly, the rIL2-induced cytolytic cells are formed as a result of nonspecific secondary cytotoxic T lymphocyte activation.     

Synergistic activation of human T cells by interleukin 1 and interleukin 6

Purified human interleukin 6 (IL 6) was found to stimulate the proliferation of human tonsillar and peripheral rosetting T cells subliminally activated with phytohemagglutinin (PHA). This response seemed independent of IL 2 but highly dependent on the presence of accessory cells. Indeed, when accessory cell-depleted tonsillar T cells were activated with PHA and exposed to IL 6, only minimal proliferations were observed. A similar result was obtained with IL 1. However, a combination of these two cytokines induced strong proliferations, indicating that IL 1 and IL 6 plays a synergistic role in the interactions between accessory cells and T lymphocytes.     

Serum levels of interleukin 4, interleukin 6 and interferon-gamma following in vivo isotype-specific activation of IgE synthesis in humans.

It is now generally accepted that interleukin 4 (IL4), interleukin 6 (IL6) and interferon-gamma (IFN gamma) play main roles in the regulation of human IgE synthesis. This concept is based mainly on in vitro data. To obtain corresponding in vivo data, we determined IL4, IL6 and IFN gamma by immunoassays in sera collected from 4 atopic patients following a clinical trial of selective IgE apheresis (plasmaimmunoadsorption). This treatment removes several milligrams of IgE from patient's blood and is suggested to induce strong and isotype-specific activation of the IgE system. Serum IgE levels restored rapidly within 3-5 days after IgE apheresis. However, very low and constant levels of IL4 (from less than 50 to 130 pg/ml) and IL6 (from less than 300 to 920 pg/ml) were detected in the sera of the treated patients. Serum IFN gamma was absent before treatment (concentrations less than 0.5 U/ml) and increased to low but detectable levels (0.90 and 8.05 U/ml) on the day following the last IgE apheresis in 2 of 4 patients. In our opinion, the data presented argue against in vivo participation of IL4 and IL6 in the activation of the human IgE system, at least in atopic patients under constant allergen exposure.