α7烟碱型乙酰胆碱受体激动剂抑制骨水泥微粒刺激小鼠外周血单核细胞分泌炎性因子

目的探讨α7烟碱型乙酰胆碱受体(α7n AChR)激动剂PNU282987对骨水泥微粒刺激小鼠外周血单核细胞分泌炎性反应因子的影响及其分子机制。方法分离培养小鼠外周血单核细胞,使用聚甲基丙烯酸甲酯(PMMA)微粒刺激后,ELISA检测培养上清液中TNF-α、IL-1β和IL-6的含量;RT-PCR检测细胞TNF-α、IL-1β和IL-6的mRNA表达;Western blot检测p-p65、p65、p-JAK2、JAK2、p-STAT3、STAT3及β-actin的表达;ELISA检测NF-κB DNA结合活力。结果单核细胞经PMMA微粒刺激后,上清液中TNF-α、IL-1β和IL-6的含量明显增高(P0.05);细胞TNF-α、IL-1β和IL-6 mRNA表达明显增高(P0.05);p65、JAK2和STAT3磷酸化明显增强(P0.05);NF-κB DNA结合活力明显增高(P0.05)。不同浓度PNU282987作用后,上清液中TNF-α、IL-1β和IL-6的含量呈浓度依耐性下降(P0.05);细胞TNF-α、IL-1β和IL-6 mRNA表达呈浓度依耐性下降(P0.05);总p65、JAK2和STAT3的表达不变;p-p65、p-JAK2和p-STAT3的表达呈浓度依耐性下降(P0.05);NF-κB DNA结合活力也呈浓度依耐性下降(P0.05)。结论α7n AChR激动剂PNU282987能显著抑制PMMA骨水泥微粒所诱导的小鼠血单核细胞炎性反应因子的分泌。     

Interleukin-4 but not interleukin-10 inhibits the production of leukemia inhibitory factor by rheumatoid synovium and synoviocytes

The expression of the proinflammatory cytokine leukemia inhibitory factor (LIF) has been reported in the cartilage and synovium of rheumatoid arthritis (RA) patients. Here, we show that high levels of LIF were constitutively produced by cultures of synovium pieces. Low levels of LIF were produced spontaneously by isolated synoviocytes, but interleukin (IL)-1β caused a fourfold enhancement of this secretion. The anti-inflammatory cytokine IL-4 reduced the production of LIF by synovium pieces by 75%, as observed earlier with IL-6, IL-1β and tumor necrosis factor (TNF)-α. IL-4 had a direct effect since it inhibited LIF production by unstimulated and IL-1β- or TNF-α-stimulated synoviocytes. Conversely, IL-4 enhanced the production of IL-6, which shares with LIF biological activities and receptor components. The inhibitory effect of IL-4 was dose dependent and was reversed using a blocking anti-IL-4 receptor antibody. Similar inhibitory action of IL-4 on LIF production was observed on synovium pieces from patients with osteoarthritis and on normal synoviocytes. IL-10, another anti-inflammatory cytokine acting on monocytes, had no effect on LIF production by either synovium pieces or isolated synoviocytes. Thus, the production of LIF by synovium tissue was inhibited by IL-4 through both a direct effect on synoviocytes and an indirect effect by inhibition of the production of LIF-inducing cytokines.     

Modulation of proinflammatory cytokine production in tumour necrosis factor-alpha (TNF-α)-transgenic mice by treatment with cells engineered to secrete IL-4, IL-10 or IL-13

TNF-α is one of the major proinflammatory cytokines involved in the pathogenesis of chronic inflammatory joint disease, in human rheumatoid arthritis as well as in murine models of this disease. It was previously described that a highly destructive chronic spontaneous inflammatory arthritis develops in mice expressing a human TNF-α transgene modified with the 3′ untranslated region of β-globin. The present study investigates in this mouse model the effects of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 administered in vivo on proinflammatory cytokine expression. Groups of TNF-α-transgenic mice were engrafted with xenogeneic transfected Chinese hamster ovary (CHO) fibroblasts secreting murine IL-4, IL-10 or IL-13. In vivo treatments consisted of 3 or 4 weekly engraftments, starting when the mice were 4 weeks old. Control groups of transgenic mice were engrafted with β-galactosidase gene-transfected CHO cells or injected with medium. A significant decreased expression of TNF-α transgene, endogenous mouse TNF-α and IL-1 mRNA was observed in splenocytes of mice treated for 3 or 4 weeks with CHO/IL-4 and CHO/IL-13, and, to a lesser extent, with CHO/IL-10, compared with controls. Finally, attenuation of histological scores of arthritides was statistically significant only in the group of CHO/IL-4-treated mice after 3 weeks of treatment (P < 0.05), and was not significant in any other group. These results show that IL-4, IL-10 or IL-13, administered by gene therapy, can decrease the mRNA steady state levels of both endogenous and transgenic cytokines in human TNF-α transgenic mice. In addition, IL-4 can slightly attenuate the development of arthritides in this model.     

Hypoxia augments cytokine (transforming growth factor-beta (TGF-β) and IL-1)-induced vascular endothelial growth factor secretion by human synovial fibroblasts

Vascular endothelial growth factor (VEGF) is abundant in synovium and synovial fluids, where it probably contributes to vascular permeability and angiogenesis in arthritic joints. To investigate the probable sources of VEGF in synovium, we compared the ability of several cytokines (TGF-β, platelet-derived growth factor (PDGF), IL-1, tumour necrosis factor (TNF), basic fibroblast growth factor (bFGF) that are associated with arthritis and angiogenesis, to stimulate secretion of VEGF protein by human synovial fibroblasts. TGF-β was the strongest inducer of VEGF secretion; six times more VEGF was secreted when cells were stimulated by TGF-β than when stimulated by PDGF or IL-1 for 24 h. TNF-α and bFGF did not stimulate any secretion of VEGF. The stimulatory effects of TGF-β and IL-1 on VEGF secretion were additive. Hypoxic culture alone also stimulated VEGF secretion, but more importantly, hypoxic culture conditions doubled the rate of VEGF secretion stimulated by the cytokines TGF-β and IL-1. When dermal and synovial fibroblasts were stimulated identically by hypoxia and cytokines (TGF-β and IL-1), synovial fibroblasts secreted four times more VEGF than did dermal fibroblasts. Thus in rheumatoid arthritis, the capacity of synovial fibroblasts in the hypoxic environment to secrete large amounts of VEGF in response to cytokines such as TGF-β probably contributes significantly to angiogenesis in the synovium.     

CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts

Chronic periodontitis is a destructive inflammatory disease linked with unbalanced production between matrix metalloproteinases (MMPs), such as interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) and their endogenous tissue inhibitors of MMPs (TIMPs). In addition to aberrant MMP-1 and MMP-3 expression, periodontal lesions are characterized by dense infiltrations of activated T lymphocytes which may interact with CD40-expressing gingival fibroblasts in the connective tissue via the CD40L-CD40 pathway. In this study we investigated whether CD40 cross-linking influenced MMP production by gingival fibroblasts. Therefore, we analysed the CD40L-induced MMP production by these fibroblasts in the presence of cytokines that are increased in periodontal lesions, such as IL-1β, tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We show that CD40 ligation on gingival fibroblasts resulted in a decrease of their MMP-1 and MMP-3 production, while MMP-2 and TIMP-1 production were unaffected as determined by Western blot. This down-regulatory effect of CD40 engagement on MMP-1 and MMP-3 production by gingival fibroblasts was also present when MMP production was up-regulated by IL-1β and TNF-α or down-regulated by IFN-γ. These results suggest that CD40 ligation on gingival fibroblasts leads to a restraining of MMP-1 and MMP-3 production by gingival fibroblasts and thereby may be an important mechanism in the retardation of further periodontal tissue damage.     

MiR-200b attenuates IL-6 production through IKKβ and ZEB1 in human gingival fibroblasts

Objective

MicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we have analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF).

Materials and methods

Total RNA and protein were extracted from HGF after stimulation by interleukin-1β (IL-1β; 1 ng/ml) or tumor necrosis factor-α (TNF-α; 10 ng/ml) and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1β, inhibitor of nuclear factor kappa-B kinaseβ (IKKβ), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot.

Results

IL-1β and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKβ and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKβ and ZEB1 and increased E-cadherin mRNA and protein levels in HGF.

Conclusions

These results suggest that miR-200b attenuates inflammatory response via IKKβ and ZEB1 in periodontal tissue.

     

外周注射福尔马林诱导前扣带皮层中胶质细胞激活和细胞因子表达增高

为了观察外周注射福尔马林对大鼠前扣带皮层(ACC)中星形胶质细胞标记物胶质纤维酸性蛋白(GFAP)和S100B,小胶质细胞标记物CD14以及与胶质细胞密切相关的炎症前细胞因子白介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)表达的影响,本研究结合行为学检测和RT-PCR方法,观察了大鼠单侧足底皮下注射福尔马林后的行为学变化,并检测了在不同时间点ACC中GFAP、S100B、CD14、IL-1β和TNF-α在基因水平的表达变化。结果显示:福尔马林急性疼痛刺激的大鼠出现典型的双时相自发性疼痛反应。ACC中GFAP和S100B mRNAs表达水平在刺激后30min、1h增高,2h恢复正常;CD14 mRNA表达水平在15min开始增高,1h达到高峰,6h恢复正常;IL-1β和TNF-αmRNAs表达水平在刺激后30min、1h、2h增高,6h恢复正常。上述结果表明,福尔马林外周急性伤害性刺激能够诱导ACC内短暂性的星形胶质细胞、小胶质细胞激活及IL-1β、TNF-α表达增高,提示激活的胶质细胞及表达上调的炎症前细胞因子可能参与伤害性信息的调制。     

BCG-PPD、H37Rv-PPD诱导入巨噬细胞死亡及TNF-α、IL-1β和IL-10的表达差异

目的 本实验对不同毒力结核杆菌的衍生蛋白(PPD)对人巨噬细胞(THP-1)的影响及其与TNF-αt、IL-1 β及IL-10的差异性进行研究.方法 用H37Rv-PPD和BCG-PPD分别在3 h、8 h、15 h及24 h四个时间点刺激分化成熟的THP-1细胞,再应用Hochest染色法,荧光镜下观察细胞的转归差异(凋亡及坏死情况),同时取上清用ELISA法测TNF-α、IL-1β及IL-10的浓度.结果 BCG-PPD刺激下细胞核以椭圆凋亡小体多见,而H37Rv-PPD刺激下细胞核则多呈坏死状,以坏死多见;BCG-PPD刺激下上清中TNF-α及IL-10的表达量低于H37Rv-PPD刺激组,但BCG-PPD刺激下IL-1β的表达量却高于后者.结论 提示高毒力菌株衍生蛋白(H37Rv-PPD)引起THP-1坏死的原因可能与TNF-α的过度表达有关,而凋亡少见可能与IL-10抑制凋亡作用有关,而低毒力菌株衍生蛋白诱导凋亡与IL-1β有关.可能菌株毒力差异就存在于菌株的蛋白成分之中,且与上述几种细胞因子密切相关.     

MiR-200b attenuates IL-6 production through IKKβ and ZEB1 in human gingival fibroblasts

Objective

MicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we have analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF).

Materials and methods

Total RNA and protein were extracted from HGF after stimulation by interleukin-1β (IL-1β; 1 ng/ml) or tumor necrosis factor-α (TNF-α; 10 ng/ml) and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1β, inhibitor of nuclear factor kappa-B kinaseβ (IKKβ), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot.

Results

IL-1β and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKβ and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKβ and ZEB1 and increased E-cadherin mRNA and protein levels in HGF.

Conclusions

These results suggest that miR-200b attenuates inflammatory response via IKKβ and ZEB1 in periodontal tissue.

     

低氧对大鼠大脑皮层细胞表达IL-1β、IL-6和TNF-α mRNA的影响

目的:观察不同氧浓度环境下不同时间处理对大鼠大脑皮层细胞表达IL-1β、IL-6和TNF-α mRNA的影响。方法:原代培养新生SD大鼠大脑皮层细胞,设1%、4%两个低氧浓度和3、6 h两个低氧处理时间,处理结束后收集各组细胞,采用实时荧光定量PCR(RT-QPCR)方法检测各组细胞表达IL-1β、IL-6和TNF-αmRNA情况。结果:与相应的常氧对照组相比,1%和4%氧浓度环境处理细胞3 h和6 h时,IL-1β和TNF-αmRNA表达量均无明显变化(P>0.05);处理细胞3 h时,IL-6 mRNA表达量均无明显变化(P>0.05),但有上升的趋势,处理细胞6 h时IL-6 mRNA表达量均显著升高(P<0.05)。结论:1%和4%氧浓度环境虽均对大脑皮层细胞表达IL-1β和TNF-α mRNA无影响,但可诱导细胞表达IL-6 mRNA,且具有时间依赖性。     

Simultaneous measurement of antigen-stimulated interleukin-1 beta and gamma interferon production enhances test sensitivity for the detection of Mycobacterium bovis infection in cattle

In order to identify cytokines that may be useful as candidates for inclusion in diagnostic tests for Mycobacterium bovis infection in cattle, we compared the levels of gamma interferon (IFN-γ), interleukin 1β (IL-1β), IL-4, IL-10, IL-12, macrophage inflammatory protein 1β (MIP-1β), and tumor necrosis factor alpha (TNF-α) in whole-blood cultures from tuberculosis (TB) reactor animals or TB-free controls following stimulation with M. bovis-specific antigens (purified protein derivative from M. bovis [PPD-B] or ESAT-6/CFP-10). In addition to IFN-γ responses, the production of IL-1β and TNF-α was also statistically significantly elevated in TB reactor cattle over that in uninfected controls following stimulation with PPD-B or ESAT-6/CFP-10 peptides. Thus, we evaluated whether the use of these two additional readouts could disclose further animals not detected by measuring IFN-γ alone. To this end, receiver operating characteristic (ROC) analyses were performed to define diagnostic cutoffs for positivity for TNF-α and IL-1β. These results revealed that for ESAT-6/CFP-10-induced responses, the use of all three readouts (IFN-γ, TNF-α, and IL-1β) in parallel increased the sensitivity of detection of M. bovis-infected animals by 11% but also resulted in a specificity decrease of 14%. However, applying only IFN-γ and IL-1β in parallel resulted in a 5% increase in sensitivity without the corresponding loss of specificity. The results for PPD-B-induced responses were similar, although the loss of specificity was more pronounced, even when only IFN-γ and IL-1β were used as readout systems. In conclusion, we have demonstrated that the use of an additional readout system, such as IL-1β, can potentially complement IFN-γ by increasing overall test sensitivity for the detection of M. bovis infection in cattle.     

Proinflammatory Cytokines Regulate the Gene Expression of Hyaluronic Acid Synthetase in Cultured Rabbit Synovial Membrane Cells

To elucidate the mechanism of accumulation and fragmentation of hyaluronic acid (HA) under inflammatory conditions, we investigated the effect of proinflammatory cytokines on hyaluronic acid synthetase (HAS) mRNA expression using cultured rabbit synovial membrane cells. HASs mRNA levels were determined by real-time PCR. HAS2 mRNA expression was maximally enhanced 3.3- and 2.8-fold after 3-hour stimulation with IL-1β (1 ng/ml) and after 1-hour stimulation with TNF-α (10 ng/ml). HAS3 mRNA expression was increased by a maximum of 4.3 times after 3-hour stimulation with IL-1 β (10 ng/ml), whereas 1-hour stimulation with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) induced around a 2.5-fold increase in HAS3 mRNA. Although IFN-γ (1–100 ng/ml) alone showed little effect on HAS2 mRNA expression, the effect was synergized by combined with both IL-lβ and TNF-α, substantially increasing HAS2 mRNA expression.

These results suggest that proinflammatory cytokines regulate the HAS expression, and consequently may contribute to the accumulation and fragmentation of HA.     

IL-4, IL-10 and IL-13 down-regulate monocyte-chemoattracting protein-1 (MCP-1) production in activated intestinal epithelial cells

Several studies have demonstrated that intestinal epithelial cells play a major role in the initiation and perpetuation of intestinal inflammation by secreting proinflammatory cytokines and chemokines. MCP-1 is suggested to be a chemokine that plays a major part during intestinal inflammation in inflammatory bowel disease (IBD). Immunoregulatory cytokines such as IL-4, IL-10 and IL-13 have been described to exert anti-inflammatory properties on various cell types. The aim of our study was to determine the effect of Th2 cytokines on the production of MCP-1 by activated intestinal epithelial cells. We examined Caco-2 cells as well as intestinal epithelial cells which were isolated from surgical specimens. Production of the chemokine MCP-1 was determined under stimulated and non-stimulated conditions. IL-4, IL-10 and IL-13 were added to stimulated epithelial cells under various culture conditions. Supernatants were analysed for cytokine concentrations using ELISAs. Under stimulation with physiological agents like IL-1β or tumour necrosis factor-alpha (TNF-α), we observed markedly increased concentrations of MCP-1 in supernatants of Caco-2 cells and intestinal epithelial cells. IL-4, IL-10 and IL-13 all had the capacity to down-regulate the production of MCP-1 in Caco-2 cells as well as in freshly isolated epithelial cells. Caco-2 cells which were primed with Th2 cytokines 24 h before stimulation were subsequently decreased in their ability to be stimulated by IL-1β or TNF-α for MCP-1 production. As MCP-1 has been shown to play a major role during intestinal inflammation, the in vitro suppression of MCP-1 in enterocytes suggests the in vivo use of regulatory cytokines in patients with active IBD.     

Inhibition of interleukin-1 type I receptor expression in human cell-lines by an antisense phosphorothioate oligodeoxynucleotide

A 20 mer oligodeoxynucleotide designed to hybridize to specific 3′-untranslated sequences in the type 1 receptor IL-1r mRNA was identified which inhibited the expression of both IL-1r mRNA and protein in human A549 lung carcinoma cells and human dermal fibroblasts. The oligodeoxynucleotide exhibited an IC₅₀ value of approximately 100 nM in both cell lines and reduced IL-1r mRNA expression for up to 48 h. Multiple scrambled control oligonucleotides were without effect on IL-lr mRNA expression. Treatment of A549 cells with this oligodeoxynucleotide (but not scrambled controls) inhibited the IL-1 stimulated expression of the cell adhesion molecule ICAM-1 but was without effect on the TNF-α induction of this molecule. This study demonstrates that phosphorothioate oligodeoxynucleotides can selectively inhibit IL-lr expression leading to a reduction in IL-1 dependent gene expression.     

Opposite effects of interleukin-13 and interleukin-12 on the release of inflammatory cytokines,cytokine inhibitors and prostaglandin E from synovial fibroblasts and blood mononuclear cells

We examined the effects of interleukin-12 (IL-12) and interleukin-13 (IL-13) on cytokine, cytokine inhibitor and prostaglandin E (PGE) release from synovial fibroblasts and blood mononuclear cells (MNC). In resting synovial fibroblasts, we found that IL-13 is an inhibitor of IL-8 and PGE release. A significant decrease of PGE synthesis caused by IL-13 was also observed in tumor necrosis factor (TNF)-α-stimulated synovial fibroblasts, whereas IL-12 had no regulatory effects on these cells. In resting and cytokine-stimulated MNC, IL-13 markedly inhibited IL-1β, IL-8 and monocyte chemoattractant protein-1 (MCP-1) release and potently stimulated interleukin-1 receptor antagonist (IL-1ra) synthesis. In contrast, IL-12 stimulated the production of IL-1β and MCP-1 in TNF-α-stimulated MNC and inhibited IL-1ra synthesis in cytokine-stimulated cells. These findings identify novel biological actions of IL-12 and IL-13 on connective tissue and on blood mononuclear cells which indicate their regulatory functions as enhancer and suppressor of inflammatory processes, respectively.     

Interleukin-8 differentially modulates interleukin-4- and interleukin-2-induced human B cell growth

The effect of interleukin-8 (IL)-8 on human B cell growth, as determined by thymidine uptake and viable cell numbers was studied. IL-8 inhibited IL-4-induced growth of B cells costimulated with anti-μ antibodies (Ab) or Staphylococcus aureus Cowan strain I (SAC) in a dose-dependent fashion. In contrast, IL-8 did not inhibit IL-2-induced growth of B cells. The IL-8-mediated inhibition was specific, since it was blocked by anti-IL-8 mAb but not by control IgG1. Moreover, anti-tumor necrosis factor-α (anti-TNF-α) Ab blocked IL-8-mediated inhibition. On the other hand, TNF-α, but not other cytokines including IL-1β, IL-3, IL-5, IL-6, interferon-α (IFN-α) or IFN-γ, inhibited IL-4-mediated growth, and inhibition by TNF-α was blocked by anti-TNF-α Ab but not by control IgG. IL-4 had no effect on TNF-α binding by B cells while it decreased TNF-α production by B cells. IL-8 had no effect in binding of IL-4, IL-2 or TNF-α by B cells, however, it enhanced TNF-α production by B cells. These results indicate that IL-8 inhibited IL-4-induced human B cell growth by enhancement of endogenous TNF-α production.