鼠Ⅰ型可溶性白细胞介素1受体基因在昆虫细胞的克隆和表达

白细胞介素１（ＩＬ－１）是一种重要的细胞因子,具有广泛的生物学活性。它通过与细胞表面的白细胞介素 １受体（ＩＬ－１Ｒ）结合而起作用。以杆状病毒为载体在昆虫细胞中克隆表达了小鼠Ｉ型可溶性白细胞介素１受体（ｓＩＬ－１　ＲＩ）基因。以ＮＩＨ／３Ｔ３细胞ＲＮＡ为模板,采用ＲＴ－ＰＣＲ方法扩增得到小鼠ｓＩＬ－ＩＲＩ的ｃＤＮＡ,克隆至杆状病毒转移载体ｐＡｃＧＰ６７Ｂ,将转移重组质粒与野生病毒ＡＣＮＰＶ ＤＮＡ共转染昆虫细胞Ｓｆ９,经同源重组得到重组杆状病毒ｒＡＣＮＰＶ。应用经纯化的ｒＡｃＮＰＶ感染昆虫细胞Ｓｆ９,表达获得重组的ｓＩＬ－１ＲＩ。经对亲和层析样品的ＳＤＳ－ＰＡＧＥ分析和对ＩＬ－１β生物活性阻断作用实验证实,表达产物能够与其配基结合,并且能够分泌至细胞培养上清中。     

Effects of interleukin-33 on cardiac fibroblast gene expression and activity

Interleukin-33 (IL-33) is a recently described member of the interleukin-1 (IL-1) family. It is produced by diverse cell types in response to a variety of stresses including hemorrhage and increased mechanical load. Though only relatively recently discovered, IL-33 has been shown to participate in several pathological processes including promoting type 2 T helper cell-associated autoimmune diseases. In contrast, IL-33 has been also found to have protective effects in cardiovascular diseases. Recent studies have illustrated that IL-33 attenuates cardiac fibrosis induced by increased cardiovascular load in mice (transaortic constriction). Since cardiac fibrosis is largely dependent on increased production of extracellular matrix by cardiac fibroblasts, we hypothesized that IL-33 directly inhibits pro-fibrotic activities of these cells. Experiments have been carried out with isolated rat cardiac fibroblasts to evaluate the effects of IL-33 on the modulation of cardiac fibroblast gene expression and function to test this hypothesis. The expression of the IL-33 receptor, interleukin-1 receptor-like 1 (ST2), was detected at the mRNA and protein levels in isolated adult rat cardiac fibroblasts. Subsequently, the effects of IL-33 treatment (0-100 ng/ml) on the expression of extracellular matrix proteins and pro-inflammatory cytokines/chemokines were examined as well as the effects on rat cardiac fibroblast activities including proliferation, collagen gel contraction and migration. While IL-33 did not directly inhibit collagen I and collagen III production, it yielded a dose-dependent increase in the expression of interleukin-6 and monocyte chemotactic protein-1. Treatment of rat cardiac fibroblasts with IL-33 also impaired the migratory activity of these cells. Further experiments illustrated that IL-33 rapidly activated multiple signaling pathways including extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, c-Jun N-terminal kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) in a dose-dependent manner. Experiments were carried out with pharmacological inhibitors to determine the role of specific signaling pathways in the response of fibroblasts to IL-33. These experiments illustrated that the activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinases are critical to the increased production of interleukin-6 and monocyte chemotactic protein-1 in response to IL-33. These studies suggest that IL-33 has an important role in the modulation of fibroblast function and gene expression. Surprisingly, IL-33 had no effect on the expression of genes encoding extracellular matrix components or on proliferation, markers typical of fibrosis. The major effects of IL-33 detected in these studies included inhibition of cell migration and activation of cytokine/chemokine expression. The previously reported inhibition of cardiac fibrosis may include more complicated mechanisms that involve other cardiac cell types. Future studies aimed at determining the effects of IL-33 on other cardiac cell types are warranted.     

Induction of interleukins by mycoplasma in culture of human mononuclear leukocytes

Mycoplasma shows a variety of effects on immune system, including the activation of macrophage, the increase in T cell cytotoxicity, and the enhancement of the proliferation and maturation of B cells, etc. As it is well known that many cytokines regulate the immune system, it is interesting to examine whether or not human peripheral blood mononuclear cells (PBMC) produce interleukin (IL) in response to mycoplasmas. In the present study, human PMBC were incubated with 7 species of mycoplasmas for 48 hours, and IL-1 beta, IL-2 and IL-6 activities in the supernatants were determined by ELISA. All the species of mycoplasmas were able to induce IL-1 beta and IL-6, although IL-2 was induced only by M. pneumoniae. These results suggest that the influence of mycoplasma infection on immune system may be partly due to the interleukins induced by mycoplasmas.     

Rabbit IL-1. Cloning, expression, biologic properties, and transcription during endotoxemia

The cloning, sequencing, expression, and biologic activities of rabbit IL-1 alpha and beta are described. A cDNA library was constructed in lambda gt10 by using polyadenylated RNA extracted from rabbit adherent splenic macrophages 4 h after stimulation with endotoxin. By using the cDNA for human IL-1 beta and IL-1 alpha as hybridization probes, cDNA for both forms of rabbit IL-1 were isolated. The cDNA for rabbit IL-1 beta encodes a precursor polypeptide of 268 amino acids with an overall homology to human IL-1 beta of 74% (81% in the mature region coding for a 17.5 kDa carboxyl-terminal protein). The similarity between the two rabbit IL-1 forms is 31% for the entire molecule and 34% for the mature protein. The mature polypeptides of both forms were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity and tested in a variety of biologic assays. Both forms produced typical endogenous pyrogen fevers in rabbits and augmented murine thymocyte and Th cell proliferation. Rabbit IL-1 alpha and beta were more pyrogenic in rabbits than human rIL-1 beta, whereas human rIL-1 alpha and beta were slightly more potent lymphocyte-activating factors. The recombinant rabbit proteins induced PGE and IL-1 production from human PBMC in vitro. A RIA for human IL-1 alpha did not recognize rabbit IL-1 alpha or beta, but rabbit IL-1 beta cross-reacted (as much as 30%) in a RIA for human IL-1 beta. Rabbits were injected with endotoxin and mRNA for both forms of IL-1 were observed primarily in the spleen and liver. The mRNA reached maximal levels after 60 min, then declined rapidly over the next 3 h, but were still present after 24 h. Liver tissue removed 4 h after endotoxin infusion produced lymphocyte-activating factors which were neutralized by more than 90% with a combination of goat anti-rabbit IL-1 alpha and anti-IL-1 beta.     

Interleukin-15 increases Paracoccidioides brasiliensis killing by human neutrophils

Interleukin-15 is a cytokine produced by a wide range of different cell types, including macrophages, in response to lipopolysaccharide or microbial infection. This cytokine may play a crucial role in the activation of phagocytic cells against pathogens, especially during innate immune response. The effects of IL-15 on human polymorphonuclear leukocyte fungicidal activity against a highly virulent Paracoccidioides brasiliensis strain were investigated. Pretreatment of human neutrophils from healthy individuals with IL-15 for 18 hours increased cell fungicidal activity in a dose-dependent manner. In addition, the exposure to IL-15 induced an increase in neutrophil oxidative burst as evaluated by superoxide anion and H(2)O(2) release. Catalase inhibited fungicidal activity supporting a role for H(2)O(2) in fungus killing. In contrast, IL-8 and TNF-alpha levels were not affected by IL-15 suggesting that its effects were not mediated by these cytokines. Together, these results show that IL-15 is a potent stimulant of antifungal activities in human neutrophils, at least in part by a mechanism dependent on oxidative metabolism.     

Natural Killer (NK) Cells in Antibacterial Innate Immunity: Angels or Devils?

Natural killer (NK) cells were first described as immune leukocytes that could kill tumor cells and soon after were reported to kill virus-infected cells. In the mid-1980s, 10 years after their discovery, NK cells were also demonstrated to contribute to the fight against bacterial infection, particularly because of crosstalk with other leukocytes. A wide variety of immune cells are now recognized to interact with NK cells through the production of cytokines such as interleukin (IL)-2, IL-12, IL-15 and IL-18, which boost NK cell activities. The recent demonstration that NK cells express pattern recognition receptors, namely Toll-like and nucleotide oligomerization domain (NOD)-like receptors, led to the understanding that these cells are not only under the control of accessory cells, but can be directly involved in the antibacterial response thanks to their capacity to recognize pathogen-associated molecular patterns. Interferon (IFN)-γ is the predominant cytokine produced by activated NK cells. IFN-γ is a key contributor to antibacterial immune defense. However, in synergy with other inflammatory cytokines, IFN-γ can also lead to deleterious effects similar to those observed during sepsis. Accordingly, as the main source of IFN-γ in the early phase of infection, NK cells display both beneficial and deleterious effects, depending on the circumstances.     

Recombinant interleukin 4 promotes the growth of human T cells

Recently, we reported the isolation of a cDNA clone that encodes a polypeptide which has B cell and T cell growth factor activities. The amino acid sequence of this polypeptide deduced from the nucleotide sequence of the cDNA clone showed significant homology with mouse B cell stimulating factor-1. Because of its multiple biologic activities, it was designated interleukin 4 (IL-4). Here we describe the effects of supernatants of Cos-7 mouse cells transfected with the IL-4 coding cDNA clone in a mammalian expression vector, on human thymocyte T cells and T cell clones. The T cell growth-promoting effect of IL-4 on preactivated T cells was not inhibited by monoclonal antibodies against IL-2 or the IL-2 receptor, indicating that the IL-4 activity is independent from IL-2 or the IL-2 receptor. IL-4 induces a low proliferative response in thymocytes and peripheral blood lymphocytes, but the response was considerably enhanced by preactivation of the thymocytes or peripheral blood T cells. Both T4+ and T8+ antigen-specific proliferative and cytotoxic T cell clones and T3 natural killer clones proliferated in response to IL-4. But one of six T4+ and one of four T8+ T cell clones were consistently found to be unresponsive. The proliferative responses to IL-4 were always lower than those obtained with IL-2. Most of the T cell clones generally became unresponsive to IL-4 10 days after stimulation, but still responded well to IL-2. These results indicate that the responsiveness to IL-4 is relatively short lasting and is regulated by activation signals. Interestingly, IL-4 acted in synergy with IL-2 in promoting the growth of T cell clones. Our results establish that IL-4 can act as a T cell growth factor independently of IL-2.     

Aberrant regulation of IL-1 expression in macrophages from young autoimmune-prone mice

IL-1 is a multifunctional, immunoregulatory polypeptide produced by many cell types. Because activated macrophages are a major source of IL-1 and have also been implicated in the pathogenesis of autoimmune disease, we investigated the regulation of IL-1 expression in several autoimmune-prone strains of mice. Peritoneal macrophages derived from the autoimmune-prone strains MRL/lpr, MRL/+, NZB, and NZB/W F1, as well as NZW, displayed transient expression of IL-1 in contrast to the stable expression characteristic of control normal strains including A. Thy, A/J, B10, B10.A, B10.D2, C57BL/6, BALB/c, and C3H/HeN. The down-regulation of IL-1 by macrophages from the autoimmune-prone mice was not attributable to inherently defective signal transduction because macrophages from both the normal and autoimmune-prone strains displayed substantial initial levels of cell-associated and secreted IL-1. However, during the first 2 to 3 days in culture, macrophages from autoimmune-prone mice became progressively refractory to both induction and maintenance of IL-1, a pattern that correlated with changes in the levels of IL-1 alpha and beta mRNA. The progressive reduction in IL-1 expression by macrophages from these autoimmune-prone strains was not due to a reduction in general metabolism or viability, because expression of cell surface antigens, including MHC class I and II Ag and LFA-1, was comparable to that of control macrophages. Because IL-1 plays a critical role in the homeostasis of a variety of cell lineages, defective expression, and maintenance of IL-1 (and perhaps other cytokines) by macrophages from the autoimmune-prone strains may contribute to the immune dysregulation that develops in these mice. Alternatively, cytokine dysregulation might not contribute directly to disease, but rather reflect a more basic defect related to specific signal transducing or gene regulatory pathways.     

Evaluation of murine interleukin 4 (IL-4) receptor expression using anti-receptor monoclonal antibodies and S1 nuclease protection analyses

Anti-receptor antibodies have previously been used in two cytokine systems (IL-1 and TNF alpha) to identify the existence of different cytokine receptors on different cell types. In this study, we have similarly used two approaches to evaluate whether IL-4 receptors on different cell types are identical, or whether more than one species of IL-4 receptor exists. The first approach involved production of monoclonal antibodies specific for the IL-4 receptor expressed by the murine mast cell line, MC/9. Six anti-IL-4 receptor monoclonal antibodies were produced against the purified soluble extracellular domain of the recombinant IL-4 receptor derived from MC/9 cells. These antibodies were capable of binding to and specifically immunoprecipitating the soluble extracellular domain of the recombinant mast cell IL-4 receptor. Following biotinylation of the antibodies and addition of phycoerythrin-streptavidin, their binding to cell associated IL-4 receptors on MC/9 mast cells could be readily visualized by immunofluorescence. Using this approach, the anti-mast cell IL-4R antibodies were found to specifically bind IL-4 receptors expressed on a variety of other murine cell types, including T cells, B cells, macrophages, fibroblasts, and L cells. The antibodies did not bind to two human cell lines known to bind human but not murine IL-4. The intensity of staining was directly related to the number of IL-4 binding sites identified previously by receptor-ligand equilibrium binding analyses. As a second approach to evaluating potential receptor heterogeneity, we constructed S1 nuclease protection assay probes for two separate regions of the mast cell IL-4 receptor, one located in the extracellular domain and one in the intracellular domain. Subsequent S1 analyses showed that both regions are expressed by the following types of cells: T cells, B cells, macrophages, myeloid cells, L cells, and stromal cells. The two approaches used in this study therefore indicate that the same or highly similar IL-4 receptor species is expressed by a wide variety of hemopoietic and nonhemopoietic cells. Since the anti-IL-4 receptor antibodies produced in this study did not block binding of IL-4 to its receptor, we cannot exclude the possible existence of a second type of IL-4R coexpressed on the cells tested in this study, or expressed uniquely by other cell types that were not investigated.     

产白介素-17T细胞在肺部细菌感染中的研究进展

目前产白介素-17(IL-17)T细胞主要包括了产IL-17 CD4+T细胞即Th17细胞、产IL-17γδT细胞和产IL-17 CD8+T细胞等。随着对Th17这一系细胞功能研究的不断深入,发现它们能通过产生IL-17和IL-22等细胞因子参与炎症反应。该文将对产IL-17 T细胞在肺部细菌感染中所起作用的最近研究进展进行综述。     

Neutrophils, dendritic cells and Toxoplasma

Toxoplasma gondii rapidly elicits strong Type 1 cytokine-based immunity. The necessity for this response is well illustrated by the example of IFN-gamma and IL-12 gene knockout mice that rapidly succumb to the effects of acute infection. The parasite itself is skilled at sparking complex interactions in the innate immune system that lead to protective immunity. Neutrophils are one of the first cell types to arrive at the site of infection, and the cells release several proinflammatory cytokines and chemokines in response to Toxoplasma. Dendritic cells are an important source of IL-12 during infection with T. gondii and other microbial pathogens, and they are also specialized for high-level antigen presentation to T lymphocytes. Tachyzoites express at least two types of molecules that trigger innate immune cell cytokine production. One of these involves Toll-like receptor/MyD88 pathways common to many microbial pathogens. The second pathway is less conventional and involves molecular mimicry between a parasite cyclophilin and host CC chemokine receptor 5-binding ligands. Neutrophils, dendritic cells and Toxoplasma work together to elicit the immune response required for host survival. Cytokine and chemokine cross-talk between parasite-triggered neutrophils and dendritic cells results in recruitment, maturation and activation of the latter. Neutrophil-empowered dendritic cells possess properties expected of highly potent antigen presenting cells that drive T helper 1 generation.     

Expression of human interleukin-2 in recombinant baby hamster kidney, Ltk-, and Chinese hamster ovary cells. Structure of O-linked carbohydrate chains and their location within the polypeptide

The similarity or identity of O-glycosylation in glycoproteins from natural sources or produced in heterologous cell lines, a central problem for the development of many biotechnologically relevant production processes, was examined using interleukin-2 (IL-2) as a model. Human interleukin-2 was constitutively expressed in several mammalian cell lines in high amounts. The recombinant proteins were purified to homogeneity and their carbohydrate structures were analyzed. Only the NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GalNAc oligosaccharide structure or the NeuAc alpha 2-3Gal beta 1-3GalNAc were found in all IL-2 preparations secreted from recombinant Ltk-, Chinese hamster ovary, and baby hamster kidney cell lines. The O-linked chains were exclusively linked to Thr in position 3 of the polypeptide chain which is the carbohydrate attachment site in natural human IL-2. The proportions of O-glycosylated versus nonglycosylated forms of the protein secreted by each recombinant cell line were independent of productivity or of cell culture conditions. Our results show that O-glycosylated human IL-2 can be produced by applying recombinant DNA technology in heterologous cell lines with the same type of post-translational modification that is observed for the protein secreted from natural T lymphocytes.     

Detection of IL-1 alpha and IL-1 beta gene expression by in situ hybridization. Tissue localization of IL-1 mRNA in the normal C57BL/6 mouse

IL-1 is a cytokine with a wide variety of effects on cells involved in inflammatory and immune responses, hemopoiesis, and bone formation. Many cell types have been shown to produce IL-1 in vitro; however, very little is known about the source and role of IL-1 in vivo. By using in situ hybridization, we examined the tissue distribution of cells containing IL-1 mRNA in normal C57BL/6 mice. The results show that many organs contain IL-1 mRNA-positive cells, but the highest frequency was found in lymphoid organs. The distribution and localization of these cells suggest that many of the IL-1 mRNA-producing cells are tissue macrophages. Organs exposed to environmental Ag and microbial products (lymph nodes, liver, intestine, lung, and uterus) had high frequencies of IL-1 mRNA-producing cells, suggesting that IL-1 is produced in local inflammatory or immune responses in vivo. The production of IL-1 mRNA in the thymus and in the bone marrow suggests that IL-1 is available to play physiologic roles in T cell differentiation and in hemopoiesis.     

Interleukin‐1β: a bridge between inflammation and excitotoxicity?

Interleukin-1 (IL-1) is a proinflammatory cytokine released by many cell types that acts in both an autocrine and/or paracrine fashion. While IL-1 is best described as an important mediator of the peripheral immune response during infection and inflammation, increasing evidence implicates IL-1 signaling in the pathogenesis of several neurological disorders. The biochemical pathway(s) by which this cytokine contributes to brain injury remain(s) largely unidentified. Herein, we review the evidence that demonstrates the contribution of IL-1β to the pathogenesis of both acute and chronic neurological disorders. Further, we highlight data that leads us to propose IL-1β as the missing mechanistic link between a potential beneficial inflammatory response and detrimental glutamate excitotoxicity.     

In vivo immunomodulation following intradermal injection with DNA encoding IL-18.

IL-18, a recently identified cytokine synthesized by different cell types, including Kupffer cells, activated macrophages, and keratinocytes, induces IFN-gamma production by T cells and NK cells. The cDNA encoding IL-18 with its natural signal peptide was cloned under control of the CMV promoter and injected into the skin of mice. A single intradermal injection of this construction led to efficient in vivo expression of IL-18 in cutaneous dermal cells and induced IFN-gamma mRNA production, indicating that it was produced in a biologically active form. In addition, a massive cellular infiltrate was observed in the skin 2 days after injection. When the mice were subsequently infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG), they produced lower levels of anti-BCG Abs than control animals. However, in contrast to their lowered humoral immune response, the mice produced higher amounts of Ag-specific IFN-gamma after in vitro restimulation, as compared with the controls. Therefore, injection of DNA encoding IL-18 into the skin modulates both Ag-specific humoral and T cell responses upon mycobacterial infection. It increases the Th1 type response, which may be particularly useful for the development of new immunotherapeutic or immunoprotective approaches against infections by intracellular parasites, such as mycobacteria.     

Dual effects of pertussis toxin on lymphoid cells in culture

Pertussis toxin (Ptx), a component of Bordetella pertussis, is responsible for many of the biological activities of this bacterium, including its potent adjuvant capacity. In attempt to better understand the Ptx activity on the immune response in vivo, we have examined the effect of Ptx on certain lymphoid cell responses in vitro which could be targets for the adjuvant activity of this molecule. Ptx was found to stimulate a variety of cell responses which include (a) increased production and release of interleukin-1 (IL-1) by human monocytes and murine macrophages; (b) co-mitogenesis, in combination with IL-1, in cultures of murine thymocytes; (c) mitogenesis in cultures of various peripheral lymphocytes; (d) increased production of IL-2 in cultures of human blood lymphocytes and rodent splenocytes; and (e) elevated release of IL-3 in cultures of murine spleen cells. In addition to its stimulatory effects, however, Ptx was found to inhibit responses of both mononuclear phagocytes and lymphocytes to other stimuli. Most activities of Ptx in vitro were achieved at the optimal concentration range of 1-10 micrograms/ml, which is 100-1000 times higher than that showing adjuvant effects in vivo. Possible explanations for the dual effect of Ptx and for the discrepancy in doses optimal for the effects in vivo and in vitro are discussed.