Killing effect of interleukin-13 receptor alpha 2 (IL-13Rα2) sensitized DC-CTL cells on human glioblastoma U251 cells

Recent studies show that IL-13Rα2, a brain tumor-associated antigen for IL-13, may play a role in immunotherapy for glioblastoma. Thus, we stimulated the lymphocyte by monocyte-derived dendritic cells (DCs). The DCs were pulsed with IL-13Rα2 in vitro and then co-cultured with lymphocytes. After inducing cytotoxic T cells (CTLs) and co-culturing with U₂₅₁ cells for 24 h in 96 wells, Cell Count Kit-8 (CCK-8) was added to every well equally. The optical density (OD) value was detected and recorded after 2 h. The DCs efficiently presented the antigen to the CTLs, resulting in CTLs activation and proliferation. The induced CTLs showed specific cytotoxic against U₂₅₁ cells (P < 0.01). The results demonstrated that IL-13Rα2 induced CTLs could kill glioma U₂₅₁ in vitro, which suggests that IL-13 Rα2 might have such an impact in vivo and thus recombinant IL-13Ra2 protein might be used as an anti-tumor vaccine, providing a promising new strategy for the treatment of brain malignant gliomas.     

人白介素2受体γ链胞外区在巴斯德毕赤酵母中的表达

目的:在毕赤酵母GS115中表达重组人白细胞介素2受体γ链(rhsIL-2Rγ)胞外区。方法:用RT-PCR法从正常人淋巴细胞中获得IL-2Rγ胞外区基因;构建重组质粒pPIC9K-hsIL-2Rγ,用聚乙二醇法转入感受态GS115菌株,MD平板筛选His+转化子,用BMMY培养基诱导表达rhsIL-2Rγ;对重组蛋白进行免疫酶染色、SDS-PAGE及Western印迹鉴定。结果:克隆到目的片段,构建了重组质粒pPIC9K-hsIL-2Rγ;免疫酶染色、Western印迹等结果显示,重组质粒已成功转化GS115,并获得诱导表达的rhsIL-2Rγ。结论:在毕赤酵母GS115中表达了rhsIL-2Rγ,其蛋白条带有上移现象,分子较大,可能其糖基化过度或存在二聚体。     

A comparative study of the inhibitory effects of interleukin-1 receptor antagonist following administration as a recombinant protein or by gene transfer

Anakinra, the recombinant form of IL-1 receptor antagonist (IL-1Ra), has been approved for clinical use in the treatment of rheumatoid arthritis as the drug Kineret™, but it must be administered daily by subcutaneous injection. Gene transfer may offer a more effective means of delivery. In this study, using prostaglandin E₂ production as a measure of stimulation, we quantitatively compared the ability of anakinra, as well as that of IL-1Ra delivered by gene transfer, to inhibit the biologic actions of IL-1β. Human synovial fibroblast cultures were incubated with a range of doses of anakinra or HIG-82 cells genetically modified to constitutively express IL-1Ra. The cultures were then challenged with recombinant human IL-1β either simultaneously with addition of the source of IL-1Ra or 24 hours later. In a similar manner, the potencies of the two sources of IL-1Ra were compared when human synovial fibroblasts were challenged with IL-1β produced constitutively by genetically modified cells. No significant difference in inhibitory activity was observed between recombinant protein and IL-1Ra provided by the genetically modified cells, under static culture conditions, even following incubation for 4 days. However, under culture conditions that provided progressive dilution of the culture media, striking differences between these methods of protein delivery became readily apparent. Constitutive synthesis of IL-1Ra by the genetically modified cells provided sustained or increased protection from IL-1 stimulation over time, whereas the recombinant protein became progressively less effective. This was particularly evident under conditions of continuous IL-1β synthesis.     

Interleukin-13 sensitivity and receptor phenotypes of human glial cell lines: non-neoplastic glia and low-grade astrocytoma differ from malignant glioma

Many of the actions and receptor components of interleukin-13 (IL-13), a pleiotrophic cytokine with immunotherapeutic potential, are shared with IL-4. Because human low-grade astrocytoma cells express IL-4 receptors and their growth is arrested by IL-4, we speculated that IL-13 sensitivity and receptor expression might also be present. The purpose of the current study was to investigate IL-13 receptor components and sensitivity in a series of glial cell lines derived from adult human non-neoplastic cerebral cortex, low-grade astrocytoma, anaplastic astrocytoma, and glioblastoma multiforme. Unlike peripheral blood lymphocytes (PBL), glial cells did not express IL-2 receptor γ chain. IL-13 receptor α-1 (IL-13Rα1), however, was present in 11/13 glial lines and PBL. Deficient cell lines were all glioblastoma-derived. All anaplastic astrocytoma and glioblastoma but not other glial lines or PBL expressed IL-13 receptor α-2 (IL-13Rα2). In non-neoplastic glia, low-grade, and anaplastic astrocytoma, IL-13 decreased DNA synthesis, an effect reversible with antibody to IL-4Rα. Results indicate that low-grade astrocytoma cells resemble non-neoplastic glia in terms of IL-13 sensitivity and IL-4Rα/IL-13Rα1 receptor profile but alterations occur with malignant progression. Glioblastoma cells were uniformly insensitive to IL-13 and, unlike other glia, failed to phosphorylate STAT6 after IL-13 challenge. Data suggest that IL-13 and analysis of IL-13 receptors may have clinical application in glial tumors. Received: 23 December 1999 / Accepted: 24 February 2000     

The balance between IL-1 and IL-1Ra in disease

IL-1 is an important mediator of inflammation and tissue damage in multiple organs, both in experimental animal models of disease and in human diseases. The IL-1 family consists of two agonists, IL-1alpha and IL-1beta, two receptors, biologically active IL-1RI and inert IL-1RII, and a specific receptor antagonist, IL-1Ra. The balance between IL-1 and IL-1Ra in local tissues plays an important role in the susceptibility to and severity of many diseases. An allelic polymorphism in the IL-1Ra gene has been associated with a variety of human diseases primarily of epithelial and endothelial cell origin. This association may be secondary to an imbalance in the IL-1 system with enhanced production of IL-1beta and reduced production of the major intracellular isoform of IL-1Ra. Treatment of RA with daily subcutaneous injections of recombinant IL-1Ra protein has been shown to be efficacious. Gene therapy approaches with IL-1Ra are being evaluated for the treatment of RA and other human diseases.     

The cloning and nucleotide sequence of human ST2L cDNA

The ST2 gene is a member of the IL-1 receptor family and is hypothesized to be involved in helper T cell function, but its functional ligand and physiological role remain unknown. We have cloned the human ST2L cDNA that encodes a distinct type of membrane-bound ST2 protein. The predicted 556-amino-acid sequence showed 67% identity to the mouse ST2L protein. The human ST2 gene (IL1RL1) contains 13 exons and spans 40 kb in length. Its exon-intron organization was elucidated from a registered human genomic sequence derived from chromosome 2q, which contains three other genes belonging to the IL-1 receptor family in an approximately 202-kb genomic region. The tissue distribution of ST2 expression was examined by RT-PCR, and the soluble form (ST2, IL1RL1-a) and ST2L (IL1RL1-b) appear to be expressed differentially. We also established stable transfectants of a human glioblastoma cell line, T98G, that express human ST2L constitutively, and we confirmed cell-surface expression of human ST2L protein on the transfectants.     

Phosphorylation of the human interleukin-2 receptor and a synthetic peptide identical to its C-terminal, cytoplasmic domain

The recombinant human interleukin-2 (IL-2) receptor was expressed in mouse mammary epithelial cells following the transfection of these cells with an expression vector containing the human IL-2 receptor cDNA. The recombinant IL-2 receptor in these cells was rapidly phosphorylated in response to phorbol myristate acetate (PMA), but its phosphorylation could not be detected in the absence of PMA or upon addition of human IL-2. The C-terminal, cytoplasmic peptide domain of the IL-2 receptor, Gln-Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-Ile, was synthesized and used as a substrate for protein kinase C. The Km for phosphorylation of the peptide by protein kinase C was 23 microM. The stoichiometry of phosphorylation was 1 mol of phosphate/mol of peptide and serine was the predominant amino acid phosphorylated. Because this peptide was a good substrate for protein kinase C in vitro, it was possible that the same serine (serine 247) was also phosphorylated in the receptor in the cell. The IL-2 receptor gene in the expression vector was therefore altered by site-directed mutagenesis to code for an IL-2 receptor containing an alanine in the place of serine 247. The IL-2 receptor expressed by these cells was not phosphorylated in the presence of PMA. These data suggest that protein kinase C, in response to PMA, phosphorylates the C-terminal serine residue (serine 247) in the human IL-2 receptor.     

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.     

Recombinant soluble Fc epsilon receptor II (Fc epsilon RII/CD23) has IgE binding activity but no B cell growth promoting activity

We have undertaken the production of recombinant soluble Fc epsilon receptor II (Fc epsilon RII) as a secretory protein, but not as a cleavage product of membrane-bound receptor. Several plasmid constructs containing soluble receptor sequence were prepared. Only a chimeric gene containing the sequences encoding IL-6 signal peptide and the soluble moiety of Fc epsilon RII could be expressed in Xenopus laevis oocytes and CHO cells, resulting in the secretion of soluble Fc epsilon RII. The recombinant soluble Fc epsilon RII was also produced in the yeast expression system. The NH2-terminal sequence analysis of the chimeric gene product generated by oocytes demonstrated the correct cleavage of IL-6 leader sequence by a signal peptidase. Moreover, most of CHO cell and all of the yeast-derived recombinant molecules were products identical with the native B cell-derived soluble Fc epsilon RII. These recombinant products as well as the natural soluble receptor derived from a human B cell line could bind both human IgE and two different anti-Fc epsilon RII mAb and could competitively inhibit the binding of IgE to Fc epsilon RII-expressing cells. However, the recombinant soluble Fc epsilon RII and highly purified native molecules did not display any B cell growth-promoting activity.     

Cloning and characterization of IL-1HY2, a novel interleukin-1 family member

The interleukin-1 (IL-1) family members play an important role in the process of inflammation and host defense. We describe here the identification and characterization of a novel member of the IL-1 family, IL-1HY2. The human IL-1HY2 protein shares significant amino acid sequence similarity (37%) with the IL-1 receptor antagonist and has a predicted three-dimensional structure similar to that of the IL-1 receptor antagonist. The IL-1HY2 gene is located in close proximity to other IL-1 family genes on human chromosome 2, and the genomic organization of the IL-1HY2 gene is highly conserved with other IL-1 family members. IL-1HY2 protein is secreted from mammalian cells, and the purified recombinant IL-1HY2 protein binds soluble IL-1 receptor type I. IL-1HY2 is expressed in human skin, spleen, and tonsil. Immunohistochemical analysis showed that the IL-1HY2 protein is expressed in the basal epithelia of skin and in proliferating B cells of the tonsil. These data suggest that IL-1HY2 is a novel IL-1 family member and that it may participate in a network of IL-1 family members to regulate adapted and innate immune responses.     

Interleukin-6-Mediated Autocrine Growth Promotion in Human Glioblastoma Multiforme Cell Line U87MG

Abstract: Human glioblastoma multiforme cell lines, brain tumor biopsy tissue, and normal human fetal brain synthesize interleukin (IL)-6 and IL-6 receptor (IL-6R). Neither of these is expressed in human neurons or neuroblastoma cell lines in culture. Astrocytes from fetal brain grown in culture retain the ability to synthesize IL-6 but do not express IL-6R as inferred from RT-PCR and Southern blot studies. Coexpression of IL-6 and IL-6R in the glioblastoma cell line U87MG is confirmed by immunofluorescence staining. Both specific monoclonal antibodies against IL-6 and IL-6R and antisense oligonucleotide to IL-6 mRNA inhibit the growth of U87MG cells in culture, suggesting the existence of a functional autocrine growth loop. Anti-IL-6 antibodies also inhibit the growth of glioblastoma cell lines U373 and U118. The expression of IL-6 by human fetal astrocytes in culture is highly suggestive of its role as an oncofetal protein responsible for rapid proliferation of fetal and tumor cells but not cells of adult brain.     

Human herpes virus 8 interleukin-6 homologue triggers gp130 on neuronal and hematopoietic cells.

Human herpes virus-8 (HHV8) encodes a cytokine named viral interleukin-6 (vIL-6) that shares 25% amino-acid identity with its human homologue. Human IL-6 is known to be a growth and differentiation factor of lymphatic cells and plays a potential role in the pathophysiology of various lymphoproliferative diseases. vIL-6 is expressed in HHV8-associated-diseases including Kaposi's sarcoma, Body-cavity-based-lymphoma and Castleman's disease, suggesting a pathogenetic involvement in the malignant growth of B-cell associated diseases and other malignant tumours. We expressed vIL-6 in Escherichia coli as a fusion protein with recombinant periplasmic maltose binding protein. After cleavage from the maltose binding protein moiety and purification, vIL-6 was shown to be correctly folded using circular dichroism spectroscopy. A rabbit antiserum was raised against the recombinant vIL-6 protein. vIL-6 turned out to be active on cells that expressed gp130 but no IL-6 receptor (IL-6-R) suggesting that, in contrast to human IL-6, vIL-6 stimulated gp130 directly. Accordingly, vIL-6 activity could be inhibited by a soluble gp130 Fc Fusion protein. vIL-6 was shown to induce neuronal differentiation of rat pheochromocytoma cells and to stimulate colony formation of human hematopoietic progenitor cells. Thus, vIL-6 exhibits biologic activity that has only been observed for the IL-6/soluble IL-6-R complex but not for IL-6 alone. These properties are important for the evaluation of the pathophysiological potential of vIL-6.     

New Agents for Targeting of IL-13RA2 Expressed in Primary Human and Canine Brain Tumors

Interleukin 13 receptor alpha 2 (IL-13RA2) is over-expressed in a vast majority of human patients with high-grade astrocytomas like glioblastoma. Spontaneous astrocytomas in dogs resemble human disease and have been proposed as translational model system for investigation of novel therapeutic strategies for brain tumors. We have generated reagents for both detection and therapeutic targeting of IL-13RA2 in human and canine brain tumors. Peptides from three different regions of IL-13RA2 with 100% sequence identity between human and canine receptors were used as immunogens for generation of monoclonal antibodies. Recombinant canine mutant IL-13 (canIL-13.E13K) and canIL-13.E13K based cytotoxin were also produced. The antibodies were examined for their immunoreactivities in western blots, immunohistochemistry, immunofluorescence and cell binding assays using human and canine tumor specimen sections, tissue lysates and established cell lines; the cytotoxin was tested for specific cell killing. Several isolated MAbs were immunoreactive to IL-13RA2 in western blots of cell and tissue lysates from glioblastomas from both human and canine patients. Human and canine astrocytomas and oligodendrogliomas were also positive for IL-13RA2 to various degrees. Interestingly, both human and canine meningiomas also exhibited strong reactivity. Normal human and canine brain samples were virtually negative for IL-13RA2 using the newly generated MAbs. MAb 1E10B9 uniquely worked on tissue specimens and western blots, bound live cells and was internalized in GBM cells over-expressing IL-13RA2. The canIL-13.E13K cytotoxin was very potent and specific in killing canine GBM cell lines. Thus, we have obtained several monoclonal antibodies against IL-13RA2 cross-reacting with human and canine receptors. In addition to GBM, other brain tumors, such as high grade oligodendrogliomas, meningiomas and canine choroid plexus papillomas, appear to express the receptor at high levels and thus may be appropriate candidates for IL-13RA2-targeted imaging/therapies. Canine spontaneous primary brain tumors represent an excellent translational model for human counterparts.     

Identification and gene organization of three novel members of the IL-1 family on human chromosome 2

Members of the IL-1 family of cytokines are important in mediating inflammatory responses. The genes encoding IL-1alpha, IL-beta, and the IL-1 receptor antagonist (IL-1Ra) are clustered within 450 kb on human chromosome 2q. By searching the EST databases and sequencing this region of chromosome 2, we have identified three novel genes that show homology to the IL-1 family, which we have named IL-1-related protein 1, 2, and 3 (IL-1RP1, IL-1RP2, and IL-1RP3). All three genes contain a signature motif common to the IL-1 family and appear to be more closely related to IL-1Ra. Similar to the intracellular form of IL-1Ra, these genes lack conventional hydrophobic signal sequences. The expression of these genes appears to be highly restricted to various epithelial cell populations. Our results demonstrate the existence of additional IL-1 gene family members within the previously defined IL-1 cluster and point to this region of chromosome 2 as an evolutionary hotspot for IL-1 gene duplication. These genes may prove to have an important role in inflammatory responses.     

Human BCAS3 expression in embryonic stem cells and vascular precursors suggests a role in human embryogenesis and tumor angiogenesis

Cancer is often associated with multiple and progressive genetic alterations in genes that are important for normal development. BCAS3 (Breast Cancer Amplified Sequence 3) is a gene of unknown function on human chromosome 17q23, a region associated with breakpoints of several neoplasms. The normal expression pattern of BCAS3 has not been studied, though it is implicated in breast cancer progression. Rudhira, a murine WD40 domain protein that is 98% identical to BCAS3 is expressed in embryonic stem (ES) cells, erythropoiesis and angiogenesis. This suggests that BCAS3 expression also may not be restricted to mammary tissue and may have important roles in other normal as well as malignant tissues. We show that BCAS3 is also expressed in human ES cells and during their differentiation into blood vascular precursors. We find that BCAS3 is aberrantly expressed in malignant human brain lesions. In glioblastoma, hemangiopericytoma and brain abscess we note high levels of BCAS3 expression in tumor cells and some blood vessels. BCAS3 may be associated with multiple cancerous and rapidly proliferating cells and hence the expression, function and regulation of this gene merits further investigation. We suggest that BCAS3 is mis-expressed in brain tumors and could serve as a human ES cell and tumor marker.     

OPG-HSP65重组蛋白的原核表达及其生物学活性研究

PCR扩增OPG-HSP65基因,构建原核重组表达载体pET-28a－OPG-HSP65,转化大肠杆菌BL21（DE3）,经IPTG诱导表达产生包涵体形式的目的蛋白。对重组蛋白进行Western blot检测表明,重组蛋白能与抗His-Tag单克隆抗体及鼠抗人OPG单克隆抗体特异性结合。对重组蛋白进行尿素洗涤纯化,进而透析、复性。经破骨细胞生长抑制实验和抑炎实验表明,重组蛋白能减少破骨细胞生成及减轻迟发型超敏反应小鼠模型炎症反应。     

Characterization of a recombinant herpes simplex virus 1 designed to enter cells via the IL13Ralpha2 receptor of malignant glioma cells

Malignant glioma tumor cells in situ exhibit on their surfaces the interleukin 13 (IL-13) receptor designated IL13Ralpha2. To target herpes simplex virus 1 to this receptor, we constructed a recombinant virus (R5111) in which the known heparan sulfate binding sites in glycoproteins B and C were deleted and IL-13 was inserted into both glycoproteins C and D. We also transduced a baby hamster kidney cell line lacking the known viral receptors (J1-1) and Vero cells with a plasmid encoding IL13Ralpha2. The J1-1 derivative (J-13R) cell line is susceptible to and replicates the R5111 recombinant virus but not the wild-type parent virus. We report the following. (i) Expression of IL13Ralpha2 was rapidly lost from the surface of transduced cells grown in culture. The loss appeared to be related to ligands present in fetal bovine serum in the medium. None of the malignant glioma cell lines cultivated in vitro and tested to date exhibited the IL13Ralpha2 receptor. (ii) Soluble IL-13 but not IL-4 or IL-2 blocked the replication of R5111 recombinant virus in J-13R cells. (iii) The endocytosis inhibitor PD98059 blocked the replication in J1-1 cells of a mutant lacking glycoprotein D (gD-/-) but not the replication of R5111 in the J-13R cells. We conclude that R5111 enters cells via its interaction with the IL13Ralpha2 receptor in a manner that cannot be differentiated from the interaction of wild-type virus with its receptors.     

Matrilin-3 Induction of IL-1 receptor antagonist Is required for up-regulating collagen II and aggrecan and down-regulating ADAMTS-5 gene expression

Introduction

Deletion or mutation of the gene encoding the cartilage extracellular matrix (ECM) protein matrilin-3 (MATN3) results in the early onset of osteoarthritis (OA), suggesting chondroprotective properties of MATN3. To understand the mechanisms underlying these properties, we determined the effects of MATN3 protein on the expression of several key anabolic and catabolic genes involved in chondrocyte homeostasis, and the dependence of such regulation on the anti-inflammatory cytokine: IL-1 receptor antagonist (IL-1Ra).

Methods

The effects of recombinant human (rh) MATN3 protein were examined in C28/I2 immortalized human chondrocytes, primary human chondrocytes (PHCs), and primary mouse chondrocytes (PMCs). Messenger RNA levels of IL-1Ra, COL2A1, ACAN, MMP-13, and ADAMTS-4 and -5 were determined using real-time RT-PCR. Knocking down IL-1Ra was achieved by siRNA gene silencing. IL-1Ra protein levels were quantified by ELISA and the Bio-Plex Suspension Array System. COL2A1 protein level was quantified using Western blot analysis. Statistic analysis was done using the two-tailed t-test or one-way ANOVA.

Results

rhMATN3 protein induced gene expression of IL-1Ra in C28/I2 cells, PHCs, and PMCs in a dose- and time-dependent manner. Treatment of C28/I2 cells and PHCs with MATN3 protein stimulated gene expression of COL2A1 and ACAN. Conversely, mRNA levels of COL2A1 and ACAN were decreased in MATN3 KO mice. MATN3 protein treatment inhibited IL-1β-induced MMP-13, ADAMTS-4 and ADAMTS-5 in C28/I2 cells and PHCs. Knocking down IL-1Ra abolished the MATN3-mediated stimulation of COL2A1 and ACAN and inhibition of ADAMTS-5, but had no effect on MATN3 inhibition of MMP-13 mRNA.

Conclusion

Our findings point to a novel regulatory role of MATN3 in cartilage homeostasis due to its capacity to induce IL-1Ra, to upregulate gene expression of the major cartilage matrix components, and to downregulate the expression of OA-associated matrix-degrading proteinases in chondrocytes. The chondroprotective properties of endogenous MATN3 depend partly on its induction of IL-1Ra. Our findings raise a possibility to use rhMATN3 protein for anti-inflammatory and chondroprotective therapy.