Rat interleukin 6: expression in recombinant Escherichia coli, purification and development of a novel ELISA

Interleukin 6 (IL-6) is a cytokine involved in many aspects of the acute phase and immune responses. Cloning of rat IL-6 cDNA into the pET-21d expression plasmid under control of a bacteriophage T7 RNA polymerase promoter system allowed isopropylthio-galactopyranoside (IPTG)-inducible production of recombinant rat IL-6 in Escherichia coli. The cloning, expression and purification of rat IL-6 is described. In this expression system, rat IL-6 was produced in insoluble inclusion bodies. The protein was solubilized in 6 M guanidine hydrochloride and refolded in a glutathione redox system. Refolded rat IL-6 was purified to homogeneity using anion-exchange chromatography on SP-Trisacryl. The purified recombinant rat IL-6 had a molecular mass of 21 756.38+/-0.25 Da, which is within 0.01% of the predicted value, taking into account cleavage of the N-terminal methionine residue and the formation of two disulfide bridges. Recombinant rat IL-6 was 2-3-fold more bioactive than the human standard preparation in the B9 hybridoma bioassay. Purified rat IL-6 was used to raise polyclonal antibodies in sheep and these reagents were used to develop a novel rat IL-6 enzyme-linked immunosorbent assay (ELISA). The ELISA is sensitive to 10 pg/ml and has been shown to detect IL-6 in plasma from rats injected with lipopolysaccharide (LPS).     

Enhancement of in vivo cell-mediated immune responses by three distinct cytokines

The ability of recombinant/purified cytokines to augment delayed-type hypersensitivity (DTH) responses was investigated. Suboptimal doses of haptenized SC were treated in vitro with purified or recombinant derived cytokines and tested for their ability to enhance DTH in vivo. With the use of this protocol, it was shown that both human and mouse rIL-6, as well as mouse rTNF-alpha, potentiated DTH in a dose-dependent manner. In accordance with these data, IL-6/TNF-alpha-containing supernatant from long term nonlymphoid cell lines also possessed the ability to augment DTH. By using the same protocol, we have also identified T cell hybridomas that produce DTH-augmenting activity constitutively. The hybridoma-derived factor, termed the T cell enhancing factor (TCEF), was functionally distinguishable from the defined cytokines IL-1 through IL-6, IFN-gamma, and TNF by bioassay. Furthermore, RNA derived from the hybridoma failed to hybridize with cDNA probes specific for IL-1 to IL-6, IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF. Further characterization of the serum-free conditioned media derived from the hybridoma indicated that the TCEF was a soluble acid labile glycoprotein (Mr greater than 30,000). Finally, we investigated the cellular requirements for DTH augmentation by IL-6, TNF-alpha, and TCEF; all are dependent upon the presence of T cells in the immunizing inoculum. We propose that these cytokines play a critical role in the development of DTH responses in vivo.     

Tumor necrosis factors alpha and beta differ in their capacities to generate interleukin 1 release from human endothelial cells

The capacity of the tumor necrosis factors, TNF-alpha and TNF-beta, products of activated macrophages and lymphocytes, respectively, to stimulate interleukin 1 (IL-1) release from endothelial cells derived from human umbilical veins was examined in vitro. Recombinant TNF-alpha caused IL-1 release by 4 hr with maximal levels of 17 U/ml by 24 hr; half-maximal stimulation occurred at approximately 80 pM. In contrast, recombinant TNF-beta was a relatively poor stimulus for IL-1 release. Even at concentrations as high as 600 pM, only 3 U of IL-1/ml were recovered; maximal IL-1 release (10 to 12 U/ml) required up to 5 nM TNF-beta. Natural, glycosated human TNF-beta was comparable in activity to recombinant TNF-beta. TNF-beta did not directly inhibit the IL-1 comitogenesis assay, nor was there evidence that TNF-beta induced the release of an IL-1 inhibitor, in that supernatants generated in the presence of TNF-beta did not inhibit thymocyte proliferation to a recombinant IL-1 standard. Binding of the recombinant TNF to endothelial monolayers was assessed by using [125I]TNF-alpha in competition studies with cold TNF-alpha and TNF-beta. Binding of TNF-alpha was half-maximal at 80 pM with an average of 664 receptors/cell and Kd = 0.043 nM. Although TNF-beta was capable of fully competing for [125I]TNF-alpha binding, half-maximal binding occurred at 800 pM TNF-beta. These data suggest that the TNF receptors on human endothelial cells may reflect the structural differences between these two homologous cytokines.     

Enhancement of in vivo immune response by tumor necrosis factor

Interleukin 1 (IL-1) has been shown to regulate several immunologic functions. Since tumor necrosis factor (TNF) shares many biologic properties with IL-1, we have investigated here the role of TNF in the modulation of the immune response. We have thus tested low doses of human recombinant TNF-alpha (hu rTNF-alpha) for its capacity to enhance the in vivo antibody responses evaluated at the cellular level in the hemolytic plaque assay. It was found that hu rTNF-alpha, like human IL-1 beta, is able to enhance the immune response to a T cell-dependent antigen (sheep red blood cells). Interestingly, at variance with human recombinant IL-1 beta, hu rTNF-alpha was not able to enhance the in vivo antibody response to a T cell-independent antigen (type III pneumococcal polysaccharide). These results suggest that low levels of TNF may have a role in the modulation of the immune response in vivo and shed new light on the biologic significance of this mediator.     

Characterisation of a monoclonal antibody to carp IL-1beta and the development of a sensitive capture ELISA

A carp IL-1beta gene was identified from a subtraction hybridisation technology based cDNA library from activated carp leucocytes. This gene was cloned into pQE vector carrying 6xHis tag and the protein was expressed. Recombinant IL-1beta was used to produce hybridomas specific for carp IL-1beta. Monoclonal antibodies were purified by affinity column and a sandwich ELISA for IL-1beta was developed with a detection limit of 10 ng of the recombinant protein. Using the capture ELISA, the presence of native IL-1beta in culture supernatant of PHA-stimulated leucocytes from carp was identified, which was confirmed by SDS-PAGE and Western blot. Since IL-1beta is known to stimulate proliferation of T & B cells and macrophages, its ability to stimulate proliferation of carp leucocytes was studied using tritiated thymidine. The recombinant protein was found to significantly stimulate proliferation of head kidney and spleen cells from carp.     

Human immunodeficiency virus does not induce interleukin-1, interleukin-6, or tumor necrosis factor in mononuclear cells.

The production of interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) by fresh peripheral blood mononuclear cells was evaluated after exposure to human immunodeficiency virus (HIV) or purified recombinant HIV-1 envelope glycoprotein (rgp120). To exclude the role of contaminating endotoxin in this study, all media were subjected to ultrafiltration and reagents contained less than 25 pg of endotoxin per ml by Limulus assay. Under endotoxin-free conditions, no increases in IL-1 beta, IL-6, or TNF-alpha mRNA or protein were detectable in cell cultures exposed to HIV-1, HIV-2, or rgp120 (0.1 to 10 micrograms/ml), as compared with cytokine levels in mock-exposed cultures. However, concentrations of endotoxin (lipopolysaccharide) as low as 0.5 ng/ml induced significant production of mRNA and protein for these three cytokines. Preincubation of mononuclear cells with "shake" HIV-1 preparations and also mock-infected shake preparations prior to lipopolysaccharide stimulation resulted in a two- to threefold increase in IL-1 beta and TNF-alpha production. This priming effect was not observed with rgp120 (0.1 to 10 micrograms/ml) or standard HIV-1 or mock-infected supernatants, suggesting the presence of biologically active material independent of virus in the shake preparations. Our studies indicate that, in the absence of endotoxin, HIV-1, HIV-2, and HIV gp120 do not induce production of IL-1 beta, IL-6, or TNF-alpha by peripheral blood mononuclear cells.     

The extra domain A from fibronectin targets antigens to TLR4-expressing cells and induces cytotoxic T cell responses in vivo

Vaccination strategies based on the in vivo targeting of Ags to dendritic cells (DCs) are needed to improve the induction of specific T cell immunity against tumors and infectious agents. In this study, we have used a recombinant protein encompassing the extra domain A from fibronectin (EDA), an endogenous ligand for TLR4, to deliver Ags to TLR4-expressing DC. The purified EDA protein was shown to bind to TLR4-expressing HEK293 cells and to activate the TLR4 signaling pathway. EDA also stimulated the production by DC of proinflammatory cytokines such as IL-12 or TNF-alpha and induced their maturation in vitro and in vivo. A fusion protein between EDA and a cytotoxic T cell epitope from OVA efficiently presented this epitope to specific T cells and induced the in vivo activation of a strong and specific CTL response. Moreover, a fusion protein containing EDA and the full OVA also improved OVA presentation by DC and induced CTL responses in vivo. These EDA recombinant proteins protected mice from a challenge with tumor cells expressing OVA. These results strongly suggest that the fibronectin extra domain A may serve as a suitable Ag carrier for the development of antiviral or antitumoral vaccines.     

Role of cytokines in the monocyte-mediated augmentation of human natural killer cell activity

Previous work has shown that normal human monocytes can augment natural killer (NK) cell activity both when mixed with enriched null cells in the assay and when precultured with enriched null cells and removed prior to testing. The data presented here show that a 4-hr preculture period is superior to slightly longer periods (10-12 hr) for demonstrating the augmentation. The role of cytokines in the monocyte effect was then investigated using a variety of antibody and recombinant reagents. Both monoclonal and rabbit polyclonal antibodies to IL-1 and IL-2 inhibited the monocyte effect, whereas antibodies against IFN-alpha and IFN-gamma from both sources had no effect. Of these cytokines, only IL-1 could be demonstrated (using a sensitive IL-1-dependent-IL-2 synthesis assay) in the supernatants of 4-hr cultures of monocytes plus null cells or null cells only. The ability to detect IL-1 was specifically inhibited by rabbit antibody to human IL-1 at 1:20 and 1:200 dilutions, but only the greater concentration inhibited the monocyte effect on NK activity. In contrast, the detection of soluble IL-1 was not inhibited by including monoclonal anti-IL-1 (1:20 dilution) in the 4-hr culture, although the same reagent abrogated the monocyte effect under these conditions. Recombinant IL-1 (up to 100 units/ml) did not augment NK activity either when added to the assay or when precultured for 4 hr with enriched null cells, whereas either recombinant IL-2 or monocytes were effective under these conditions. These results provide the first evidence for a cellular, and potentially physiologic, basis for the regulation of NK activity by IL-1 and IL-2, which had been previously known to act at pharmacologic levels in vitro.     

The presence of autoantibodies specific for NZB serum factors in adult NZB mice and the establishment of monoclonal autoantibodies against these humoral factors

Humoral factors in serum of young NZB mice enhance maturation of B-lymphocyte precursors in vitro. A blot ELISA assay identified autoantibodies against the serum factors. NZB-SFs (designated NZB-SF alpha, pI 3.5-4.0, and NZB-SF beta, pI 7.8) were purified by sequential steps. Both had a molecular weight (MW) of approximately 23,000 in SDS-PAGE. NZB mice develop autoantibodies against NZB-SFs by 2 months of age; titers increased progressively with age. Non-autoimmune-prone mice did not produce autoantibodies against NZB-SFs. We then developed two hybridoma clones, IIC1C1 and IIC1M4, which produce monoclonal autoantibodies against NZB-SF alpha and NZB-SF beta, respectively. Both IgM autoantibodies could be affinity purified with a column of CNBr-Sepharose 4B gel conjugated with anti-mouse IgM antibody. Neither IIC1C1 nor IIC1M4 abolished bioactivity of recombinant mouse IL-1 alpha, human IL-1, mouse, rat, or human IL-2, mouse IL-3, or colony-stimulating factor. Neither antibody reacted to recombinant mouse IL-1 alpha, IL-4, TNF alpha, or IFN gamma in blot ELISA assays. Monoclonal autoantibodies IIC1C1 and IIC1M4 were used to purify NZB-SFs. SDS-PAGE of the affinity-purified NZB-SFs revealed bands of 23 and 60 kDa, and proteins extracted from the bands were reactive to our monoclonal autoantibodies.     

Anti-human cytomegalovirus activity of cytokines produced by CD4+ T-cell clones specifically activated by IE1 peptides in vitro.

The control of latent cytomegalovirus (CMV) infections by the immune system is poorly understood. We have previously shown that CD4+ T cells specific for the human CMV major regulatory protein IE1 are frequent in latently infected healthy blood donors. In order to learn about the possible role of these cells, we have developed IE1-specific CD4+ T-cell clones and, in this study, analyzed their epitope specificity and function in vitro. We measured their cytokine production when stimulated with specific IE1 peptides or whole recombinant IE1 protein. Their cytokine profiles, as deduced from gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-4 (IL-4) and IL-6 production, were of the Th0- and Th1-like phenotypes. Supernatants from IE1-specific clones producing IFN-gamma and TNF-alpha were shown to inhibit CMV replication in U373 MG cells. This effect was due, as found by using cytokine-specific neutralizing antibodies, mostly to IFN-gamma, which was secreted at higher levels than TNF-alpha. To better assess the anti-CMV activity of cytokines, recombinant IFN-gamma and TNF-alpha were used and shown to have a synergistic effect on the inhibition of CMV replication and protein expression. Thus, IE1-specific CD4+ T cells display in vitro anti-CMV activity through cytokine secretion and may play a role in the control of in vivo latent infections.     

Expression, purification, and characterization of Tara, a novel telomere repeat-binding factor 1 (TRF1)-binding protein

Tara was originally identified as a binding protein of guanine nucleotide exchange factor Trio. Although Tara may be involved in many fundamental cellular processes, ranging from actin remodeling, directed cell movement, to cell cycle regulation, aging, and cancer, the exact molecular mechanisms are poorly understood. We expressed recombinant Tara in Escherichia coli and purified the protein to approximately 99% purity using affinity chromatography and gel-filtration chromatography. The identity of the purified protein was confirmed by mass spectrometry. Non-denaturing polyacrylamide gel electrophoresis and gel-filtration chromatography showed that Tara forms multimer in vitro. The purified Tara was used to generate polyclonal antibody, which could specifically recognize both the recombinant and endogenous Tara. Using the pull-down assay, we showed that the purified Tara interacted with TRF1, suggesting that the purified protein is functional and biologically active. The availability of purified Tara and anti-Tara antibody provides critical reagents for elucidating Tara's cellular function and its molecular mechanism.     

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins.

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.     

马传贫驴白细胞弱毒疫苗株酸性蛋白p9基因的克隆与表达

从感染驴白细胞的马传贫驴白细胞弱毒疫苗株前病毒DNA中克隆了编码p9蛋白的基因,并在大肠杆菌中进行了表达。所表达的蛋白是一种可溶性的融合蛋白,其氨基端带有6个组氨酸的标签,因此可以用固定化金属离子亲和层析法在非变性条件下进行纯化。在间接酶联免疫吸附试验(ELISA)和免疫印迹试验中,重组的酸性蛋白p9可与马传贫阳性血清样品发生反应,而与健康马血清无任何反应。这表明该重组蛋白具有良好的抗原性和特异性,可用于马传贫弱毒疫苗株在体内外复制及在接种马体内免疫应答的研究 。     

Development of a p38 Kinase Binding Assay for High Throughput Screening

p38 is a member of the mitogen-activated protein kinase (MAPK) family of serine/threonine kinases, which is activated by cellular stressors and has been shown to be a critical enzyme in the synthesis and action of proinflammatory cytokines, tumor necrosis factor-a (TNF-alpha) and interleukin-1beta (IL-1beta). A group of pyridinyl imidazole compounds such as SB202190 have been identified as selective inhibitors of p38 that bind directly to the ATP pocket of the enzyme. These compounds inhibit the p38 kinase activity, block TNF-alpha and IL-1beta secretion both in vivo and in vitro and are found to be effective in animal models of arthritis, bone resorption, and endotoxin shock. We postulated that other classes of compounds capable of competing the binding of pyridinyl imidazole with p38 enzyme could have efficacy in the treatment of inflammatory diseases. Therefore, a simple and robust assay was developed to measure the ability of small molecules to inhibit the binding of tritium-labeled pyridinyl imidazole, SB202190, to recombinant p38 kinase. For assay development, the human p38 gene was cloned in baculovirus and then expressed in insect cells. Tritiated SB202190 was synthesized and used as the p38 ligand for a competitive filter binding assay. This assay has been used successfully to screen both synthetic and combinatorial chemical libraries for other classes of p38 kinase inhibitors.     

降钙素原的原核表达及单克隆抗体制备

目的:高效表达与纯化可溶性重组人PCT蛋白,制备高灵敏度和高特异性的抗人PCT医用诊断单克隆抗体。方法:大肠杆菌表达重组人PCT蛋白后,利用饱和硫酸铵沉淀和亲和层析方法纯化PCT蛋白后,经质谱、Western blot和间接ELISA法进行性质鉴定和分析重组蛋白的表达与免疫反应性;重组蛋白免疫小鼠,经细胞融合及筛选制备抗PCT单克隆抗体(m Ab)。结果:在大肠杆菌中高效表达了人PCT蛋白;重组人PCT蛋白具有良好的免疫反应性与免疫原性;经筛选获得7株抗PCT单克隆抗体细胞株,经ELISA鉴定,筛选抗体可与PCT抗原有良好的特异性反应。结论:利用重组人PCT蛋白免疫制备了抗人PCT单克隆抗体,为进一步研发PCT快速诊断试剂提供了原料。     

GM-CSF production from human airway smooth muscle cells is potentiated by human serum

Recent evidence suggests that airway smooth muscle cells (ASMC) actively participate in the airway inflammatory process in asthma. Interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) induce ASMC to release inflammatory mediators in vitro. ASMC mediator release in vivo, however, may be influenced by features of the allergic asthmatic phenotype. We determined whether; (1) allergic asthmatic serum (AAS) modulates ASMC mediator release in response to IL-1beta and TNF-alpha, and (2) IL-1beta/TNF-alpha prime ASMC to release mediators in response to AAS. IL-5 and GM-CSF were quantified by ELISA in culture supernatants of; (1) ASMC pre-incubated with either AAS, nonallergic non-asthmatic serum (NAS) or Monomed (a serum substitute) and subsequently stimulated with IL-1beta and TNF-alpha and (2) ASMC stimulated with IL-1beta/TNF-alpha and subsequently exposed to either AAS, NAS or Monomed. IL-1beta and TNF-alpha induced GM-CSF release in ASMC pre-incubated with AAS was not greater than that in ASMC pre-incubated with NAS or Monomed. IL-1beta and TNF-alpha, however, primed ASMC to release GM-CSF in response to human serum. GM-CSF production following IL-1beta/TNF-alpha and serum exposure (AAS or NAS) was significantly greater than that following IL-1beta/TNF-alpha and Monomed exposure or IL-1beta/TNF-alpha exposure only. Whilst the potentiating effects of human serum were not specific to allergic asthma, these findings suggest that the secretory capacity of ASMC may be up-regulated during exacerbations of asthma, where there is evidence of vascular leakage.     

汉逊酵母重组表达的人胃蛋白酶原Ⅱ校准品研制

目的：探索一种高效制备人胃蛋白酶原Ⅱ（pepsiongenⅡ,PGⅡ）体外诊断试剂用校准品的方法。方法：以汉逊酵母ATCC26012作为宿主菌,以携带Zeocin及G418双重筛选标记的pRMHP2.1质粒为载体,以遗传密码子优化设计后的PGⅡ序列为目的基因,通过电转化及后续多轮次的传代与稳定,筛选获得高水平分泌表达PGII蛋白的重组菌株26012/PGⅡ。采用Ni柱亲和层析的方法从200L发酵罐大量制备的培养液中纯化出高纯度的重组PGⅡ蛋白,将该蛋白质进行校准定值后配制成6种不同浓度的成套PGⅡ校准品,并对该系列校准品的实时稳定性、加速破坏性及开瓶稳定性展开评价。结果：筛选获得的重组汉逊酵母菌株26012/PGⅡ外源基因整合拷贝数高于40个且整合于染色体的rDNA位置,该菌株以分泌形式表达重组PGⅡ蛋白,表达量超过50mg/L,发酵培养液经Ni柱亲和层析纯化后,重组PGⅡ蛋白纯度达93.8%。通过免疫比浊试剂盒定量检测,该PGⅡ蛋白的活性定值与实际蛋白质量之间的比值达0.85,配制后的PGⅡ校准品在4℃实时保存1年、37℃加速破坏2周及4℃开盖保存2周后,校准品定值的平均下降幅度都不超过10%,其稳定性能不逊色于商品化的校准品。结论：汉逊酵母重组表达的PGⅡ蛋白可用作体外诊断试剂的校准品。     

Preparation of recombinant rat interleukin-5 by baculovirus expression system and analysis of its biological activities.

Rat interleukin-5 (IL-5) cDNA was subcloned from peritoneal cells collected 4 h after intraperitoneal injection of Ascaris suum antigen solution into the immunized rats. Cysteine proteinase-deleted (CPd) rat IL-5 recombinant virus was constructed by inserting rat IL-5 cDNA into CPd virus having a deletion in the cysteine proteinase gene of the silkworm Bombyx mori nuclear polyhedrosis virus. On infection with the CPd rat IL-5 recombinant virus, the silkworm B. mori larvae produced rat IL-5 as a dimeric form in hemolymph. Recombinant rat IL-5 was purified more than 95.5% by anion-exchange chromatography and hydrophobic chromatography. The purified recombinant rat IL-5 promoted the proliferation of T88-M cells in a concentration-dependent manner, and its effect was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. When bone marrow cells from normal rats were incubated with recombinant rat IL-5 in medium containing methylcellulose, the colony formation by eosinophilic cells was induced. Furthermore, when rat peritoneal eosinophils were incubated with recombinant rat IL-5, the spontaneous decrease in the eosinophil viability was inhibited in time- and concentration-dependent manners. In addition, the recombinant rat IL-5-induced eosinophil survival was inhibited by an anti-murine IL-5 neutralizing polyclonal antibody. These findings suggest that rat IL-5 acts as B-cell growth factor II (BCGF-II), eosinophil differentiation factor (EDF), and eosinophil survival-enhancing factor.     

Interleukin-18 mRNA, but not interleukin-18 receptor mRNA, is constitutively expressed in islet beta-cells and up-regulated by interferon-gamma

Interleukin-18 (IL-18) mRNA is expressed in islets of NOD mice during the early stages of insulitis and IL-18 has therefore been implicated as a contributing factor in immune-mediated beta-cell destruction. However, a recent study failed to show any effect of human IL-18 on the function of isolated rat islets. Since species differences have been shown between human and murine IL-18, the aims of this study were to investigate 1) if species homologous IL-18 alone or following IL-12 pre-exposure affected rat islet function, 2) if IL-18 dose-dependently modulated IL-1 beta or interferon-gamma (IFN-gamma) + tumor necrosis factor-alpha (TNF-alpha) actions on islet function, and 3) if IL-18 and IL-18 receptor (IL-18R) were expressed in rat islet beta-cells. Insulin release and nitric oxide (NO) production from isolated rat islets were measured after incubation with or without cytokines. RT-PCR was used to quantitate mRNA expression of IL-18 and the IL-18R signaling chain (IL-18R beta). There were no significant effects of 0.625-10 nM recombinant murine (rm) IL-18 alone on accumulated or glucose-challenged insulin release or NO production after 24 hours. Fifteen pg/ml of recombinant human (rh) IL-1 beta as well as 200 U/ml recombinant rat (rr) IFN-gamma + 250 U/ml rhTNF-alpha significantly increased islet NO production and inhibited both accumulated and glucose-challenged islet insulin release. However, rmIL-18 failed to modulate these effects of IL-1 beta or IFN-gamma + TNF-alpha. Although IL-12 induces IL-18R expression in Th1 and B lymphocytes, 24-hours rmIL-12 preincubation neither sensitized islets to effects of 10 nM of rm or rrIL-18 alone nor primed the islets to IL-1 beta actions on insulin release and NO production. IL-18R beta mRNA, which was expressed in human peripheral blood mononuclear cells (PBMC), was not expressed in rat insulinoma (RIN) cells or in isolated rat islets, even after exposure to IL-1 beta and/or IFN-gamma + TNF-alpha or IL-12. IL-18 mRNA was constitutively expressed in RIN cells, in FACS-purified rat beta-cells and in intact rat and mouse islets, and was up-regulated by IFN-gamma in an interferon regulatory factor-1- IRF-1) and NO - independent manner. However, IL-18 protein was undetectable in lysates and supernates of RIN cells by ECL, Western blotting and immunoprecipitation. In conclusion, we show for the first time that IL-18 but not IL-18R is expressed in rodent islet beta-cells. The physiological importance and pathological role of IL-18 originating from islet beta-cells deserve further investigation.     

Immunomodulatory effects of glycine on LPS-treated monocytes: reduced TNF-alpha production and accelerated IL-10 expression.

Cytokines play a pivotal role in the pathogenesis of septic shock. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) stimulate the progression of septic shock whereas the anti-inflammatory cytokine IL-10 has counterregulative potency. The amino acid glycine (GLY) has been shown to protect against endotoxin shock in the rat by inhibiting TNF-alpha production. In the current study we investigated the role of GLY on lipopolysaccharide (LPS) -induced cell surface marker expression, phagocytosis, and cytokine production on purified monocytes from healthy donors. GLY did not modulate the expression of HLA-DR and CD64 on monocytes, whereas CD11b/CD18 expression (P<0.05) and E. coli phagocytosis (P<0.05) decreased significantly. GLY decreased LPS-induced TNF-alpha production (P<0.01) and increased IL-10 expression of purified monocytes. Similarly, in a whole blood assay, GLY reduced TNF-alpha (P<0.0001) and IL-1beta (P<0.0001) synthesis and increased IL-10 expression (P<0.05) in a dose-dependent manner. The inhibitory effects of GLY were neutralized by strychnine, and the production of IL-10 and TNF-alpha was augmented by anti-IL-10 antibodies. Furthermore, GLY decreased the amount of IL-1beta and TNF-alpha-specific mRNA. Our data indicate that GLY has a potential to be used as an additional immunomodulatory tool in the early phase of sepsis and in different pathophysiological situations related to hypoxia and reperfusion.