Regulation of human IL-18 gene expression: interaction of PU.1 with GC-box binding protein is involved in human IL-18 expression in myeloid cells

Interleukin-18 (IL-18) is a pro-inflammatory cytokine which participates in host defense against a variety of infections as well as in chronic inflammation including autoimmune diseases. However, little is known about human IL-18 regulation at the gene level. We have previously demonstrated that sodium butyrate, a bacterial fermentation product, induces IL-18 production via the proximal region of the promoter. In this study we investigated the molecular mechanisms for basal and sodium butyrate-induced expression of IL-18 in human myeloid cells. Two regulatory regions, a consensus binding site for PU.1 and a GC-rich region, are required for basal IL-18 promoter activity in human myeloid cells. PU.1 bound to the PU.1 consensus binding site in electrophoretic mobility shift assays, and overexpression of PU.1 led to activation of the IL-18 promoter through this site. Mutation analysis revealed that the GC-rich region, but not PU.1 site, participates in sodium butyrate-induced transactivation. Furthermore, DNA pull-down experiments and the critical spacing of the two binding sites suggest that formation of a protein complex involving both cis elements and the respective binding proteins might be crucial for human IL-18 expression.     

Escherichia coli-induced expression of IL-1 alpha, IL-1 beta, IL-6 and IL-8 in normal human renal tubular epithelial cells

The aim of the present study was to investigate whether the IL-1 family cytokines, in addition to IL-6 and IL-8, could be induced in normal human cortical epithelial cells in response to bacterial stimuli. Human renal tissue was obtained from 9 patients undergoing elective tumour nephrectomy. Renal cortical epithelial cells of tubular origin were prepared from the unaffected tissue. The proximal tubular cells were stimulated for 2, 6 and 24 h with a heat-inactivated pyelonephritogenic Escherichia coli strain DS-17. Cultured unstimulated tubular cells served as controls. IL-1 alpha, IL-1 beta, IL-1 receptor antagonist, IL-6, IL-8, IL-10, TNF-alpha, G-CSF and GM-CSF were analysed using immunohistochemistry at the single cell level. The nonstimulated cells were found to express low levels of IL-6 and IL-8 (mean value < 3% of total cells). In contrast, E. coli exposure resulted in significantly increased incidences of IL-6 and IL-8 expressing cells (mean values approximately 18% of total cells) peaking within two hours of stimulation (P < 0.008 and P < 0.02 versus non-stimulated cells, respectively). A gradual decrease was thereafter observed at 6 and 24 h, respectively, although persistently higher compared to controls. A different kinetic response was found for IL-1 alpha, IL-1 beta and IL-1 receptor antagonist-expressing cells, which peaked 24 h after E. coli stimulation (mean values 3--10%) (P < 0.008, P < 0.02, P < 0.02 versus non-stimulated cells, respectively). Low levels of TNF-alpha and GM-CSF were found in 3 of the 9 donated epithelial cells, peaking at 2 h, and IL-10 and G-CSF producing cells in 1 patient each. In conclusion we found that heat-inactivated pyelonephritic E. coli induced a proinflammatory cytokine response in the normal human proximal tubular cells including the IL-1 family, IL-6 and IL-8.     

GM-CSF, IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, ICAM-1 and VCAM-1 gene expression and cytokine production in human duodenal fibroblasts stimulated with lipopolysaccharide, IL-1 alpha and TNF-alpha.

The role of mucosal fibroblasts in intestinal inflammatory reactions is not known. In this study, we demonstrate that fibroblasts grown from histologically normal human duodenal biopsy tissues expressed mRNA genes for granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) when stimulated with lipopolysaccharide (LPS) or IL-1 alpha. The increased mRNA expression of GM-CSF, IL-1 alpha, IL-1 beta, IL-6 and IL-8 in response to IL-1 alpha and LPS stimulation was time- and dose-dependent. In contrast, IL-10 was weakly expressed when fibroblasts were stimulated with LPS, IL-1 alpha or tumour necrosis factor-alpha (TNF-alpha), but the expression was enhanced in the presence of cycloheximide combined with optimal concentrations of LPS, IL-1 alpha or TNF-alpha, IL-1 alpha was a more potent stimulator than LPS for GM-CSF, IL-6, IL-8 and IL-10 expression, but not for IL-1 alpha and IL-1 beta. Increased GM-CSF, IL-6 and IL-8 gene expression was associated with the production of cytokine proteins in culture supernatant, but IL-1 alpha and IL-1 beta remained undetectable. Dexamethasone suppressed both gene expression and protein production of GM-CSF, IL-6 and IL-8 when fibroblasts were exposed to IL-1 alpha. TNF-alpha stimulated the release of GM-CSF, IL-6 and IL-8 and, combined with IL-1 alpha, cytokine production was enhanced synergistically. Finally, both LPS and IL-1 alpha up-regulated ICAM-1 and VCAM-1 gene expression. These findings implicate duodenal fibroblasts in the initiation and/or regulation of intestinal inflammation.     

Tumor cell IL-6 gene expression is regulated by IL-1 alpha/beta and TNF alpha: proposed feedback mechanisms induced by the interaction of tumor cells and macrophages.

In the present report, we show that progressive growth of the immunogenic C57BL/6J sarcoma, MCA/76-9, was accompanied by an increase in serum interleukin-6 (IL-6) activity. The possible pathways leading to the induction of IL-6 release by the tumor cells are described. It was shown that macrophage products IL-1 alpha, IL-1 beta, and to a lesser extent, TNF alpha, induced the tumor cells in vitro to transcribe the IL-6 gene and release the gene product. IL-1 induced significantly more IL-6 mRNA and bioactivity than TNF alpha, although both cytokines induced a cumulative increase of bioactivity in the supernates over a period of 24 h. The tumor cells were shown to express receptors for IL-1 alpha, which could be blocked with anti-IL-1 receptor antibody. Given the previous reports that tumor-associated macrophages expressed both IL-1 alpha/beta and TNF alpha, the data suggest, first, that the mutual interaction of tumor cells and macrophages in situ may contribute to the observed increase in circulating IL-6 activity, and second, that the release of IL-6 in vivo may serve to regulate both anti-tumor immune responses and suppressor mechanisms.     

The significance of strong histone deacetylase 1 expression in the progression of prostate cancer

Song Y H, Shiota M, Tamiya S, Kuroiwa K, Naito S & Tsuneyoshi M
(2011) Histopathology 58 , 773–780 The significance of strong histone deacetylase 1 expression in the progression of prostate cancer Aims: Histone deacetylases (HDACs) play important roles in many types of cancer. Recently, it has been reported that HDAC1 expression in prostate cancer is significantly higher than in benign prostate cell lines and tissues. The expression of HDAC1 in association with the clinicopathological data was investigated to define its functional and pathological roles in prostate cancer. Methods and results: HDAC1 expression was examined immunohistochemically in 148 patients with prostate cancer. Strong expression of HDAC1 in benign prostate glands, high‐grade prostatic intraepithelial neoplasia (PIN) and prostate cancer was observed in 17/148 (11%), 19/71 (27%) and 69/148 (47%) patients. Strong HDAC1 expression was correlated with high Gleason score (P = 0.025) and high pT stage (P = 0.012). Patients with strong HDAC1 expression had higher biochemical recurrence rates (P = 0.0010). Furthermore, strong HDAC1 expression had a significant impact on patient biochemical recurrence rates in multivariate analysis (P = 0.004). Conclusions: These results indicate that overexpression of HDAC1 contributes to progression and poor prognosis in prostate cancer. The findings may play an important role in the emergence of effective new approaches for therapy and prognostic markers of prostate cancer.     

组蛋白去乙酰化酶1在人气管上皮损伤修复过程中的表达

目的 检测组蛋白去乙酰化酶1(HDAC1)在人气管上皮损伤修复中的表达及其变化,探讨影响气管干细胞增殖分化的分子调控机制.方法 5-氟尿嘧啶(5-FU)造成人离体气管上皮损伤,应用免疫组织化学和Western blotting方法检测修复过程中各时间点气管上皮HDAC1的表达.结果 去除5-FU后0h,气管上皮脱落,仅残留少量G0期细胞,HDAC1表达阴性; 3～6h,细胞数量增多,为扁平状,未见明显HDAC1阳性细胞;12h,上皮细胞由扁平变立方并连接成片,HDAC1阳性细胞数明显增多,并可见数个HDAC1阳性细胞围绕着底部1个HDAC1阴性细胞和多个HDAC1阳性细胞间夹着数个HDAC1阴性细胞分布的现象;24h,细胞数继续增多并变复层,HDAC1阳性细胞数最多,累及全层;48h,接近假复层结构,HDAC1阳性细胞数略减少,阳性细胞分布集中于假复层结构顶部即靠近管腔侧;正常气管上皮HDAC1表达阴性.Western blotting分析显示,HDAC1表达量的变化趋势与免疫组织化学结果一致.结论 随着气管干细胞分化细胞数增多,HDAC1阳性细胞数明显增多,提示HDAC1可作为气管干细胞由增殖转向分化的分子开关.     

Modulation of IL-1 alpha, IL-1 beta, and 25K Mr non-IL-1 activity released by human mononuclear cells

To determine if the release of IL-1 alpha and IL-1 beta by cultured PBMC could be independently modulated by different exogenous stimuli, we examined the effect of LPS, IFN gamma, latex beads, and indomethacin on the release of IL-1 alpha and IL-1 beta. PBMC culture supernatants were fractionated by Sephacryl-S-200 column chromatography or HPLC (TSK G3000SW), and each fraction was tested for thymocyte mitogenic activity in the presence or absence of preincubation with anti-IL-1 alpha or anti IL-1 beta monoclonal antibody (mAb) and for the presence of IL-1 alpha or IL-1 beta protein by ELISA. In all experiments, thymocyte mitogenic activity not neutralizable by anti-IL-1 alpha or anti-IL-1 beta mAb was detected in the 25K Mr range, which ranged from 12 to 50% of the total thymocyte mitogenic activity released, depending on the stimuli. Cultured PBMC from 95% of individuals release thymocyte mitogenic activity in the absence of exogenous stimuli, which was increased 1.3-to 7-fold by lopopolysaccharide (LPS) (25-50 micrograms/ml). All of this increased activity was due to increased release of IL-1 beta and non-IL-1 thymocyte mitogenic activity, with no change in the total amount of IL-1 alpha released. Indomethacin (0.1 microgram/ml) induced release of increased thymocyte mitogenic activity of 1.3- to 1.4-fold over unstimulated cultures. All of this increased activity was due to increased release of IL-1 alpha and non-IL-1 activity with a concomitant decrease in IL-1 beta release. Interferon gamma (40-100 U/ml) increased the amount of IL-1 alpha and decreased IL-1 beta and non-IL-1 activity released, resulting in no overall change in the total amount of thymocyte mitogenic activity. Molecular weight fractionation of the PBMC culture supernatants revealed that thymocyte mitogenic activity eluting in the 25K Mr range was not due to IL-1 alpha or IL-1 beta. With certain culture conditions, thymocyte mitogenic activity was detected in the 30-40K Mr range. PBMC cultured with LPS and latex beads in the absence of serum released 30-40K Mr IL-1 alpha, as well as 17K Mr IL-1 alpha and 17K Mr IL-1 beta. PBMC cultured in 2% fetal calf serum (FCS) alone from some donors released only 30-40K Mr thymocyte mitogenic activity. Both IL-1 alpha and IL-1 beta protein was detected by ELISA in this Mr range but only the IL-1 alpha was bioactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

p38 MAPK is a critical regulator of the constitutive and the beta4 integrin-regulated expression of IL-6 in human normal thymic epithelial cells

Cytokines and adhesion receptors are key mediators in the dialog occurring between thymic epithelial cells (TEC) and thymocytes and regulating T cell maturation and epithelial embryonic differentiation. Among cytokines, IL-6 can be critical in the thymus, fostering proliferation, differentiation and/or survival of both TEC and thymocytes. We have previously reported in human normal TEC that clustering of the laminin receptor alpha6beta4 integrin induced by thymocyte contact or monoclonal antibody-mediated cross-linking regulates IL-6 gene expression via activation of NF-kappaB and NF-IL6 transactivators. Here we show that alpha6beta4 integrin activates p38 mitogen-activated protein kinase (MAPK) and that p38 is essential for IL-6 gene expression. In fact, beta4 cross-linking activated p38 and extracellular signal-regulated kinase (ERK) MAPK, Rac1, p21-activated protein kinase 1 (PAK1) and MAPK kinases (MKK) 3/MKK6. However, pharmacological blockade of p38 or ERK demonstrated that p38 inhibition abrogated both basal and beta4 integrin-induced production of IL-6 preventing NF-kappaB and NF-IL6 activation, whereas ERK inhibition reduced IL-6 production, hampering only NF-kappaB activation. Overall, our results indicate that p38 MAPK and alpha6beta4 integrin, expressed by TEC throughout their life, are critical regulators of the intrathymic availability of a cytokine controlling fate and functions of cells governing development and maintenance of thymic architecture and immune responses.     

人IL-1RⅡ基因的克隆及其表达

克隆人IL 1RⅡ基因 ,构建其逆转录病毒载体 ,以探讨其在IL 1主导疾病中的作用。方法 :用RT PCR法从人外周血单个核细胞 (PBMC)中克隆人的IL 1RⅡ基因。将其克隆到原核表达质粒PET2 2b中 ,构建重组质粒PET2 2b IL 1RⅡ。将该重组质粒依次进行PCR、酶切鉴定及测序后 ,转化BL2 1菌 ,以IPTG诱导表达。表达产物用Westernblot鉴定。另外 ,将以酶切重组质粒PET2 2b IL 1RⅡ所获IL 1RⅡ基因的全长ORF ,克隆到逆转录病毒质粒中并转染 2 93细胞 ,用免疫组化染色法检查IL 1RⅡ基因的表达。结果 :用RT PCR法 ,从人PBMC中扩增出 12 0 3bp的cDNA ,测序证实为人IL 1RⅡ基因。Westernblot表明 ,重组质粒可表达IL 1RⅡ蛋白。免疫组化染色表明 ,IL 1RⅡ重组逆转录质粒可在 2 93细胞中高效表达IL 1RⅡ蛋白。结论 :成功地克隆了人IL 1RⅡ基因 ,构建了其原核表达载体和重组逆转录病毒载体 ,并在BL2 1菌中表达IL 1RⅡ重组蛋白 ,为进一步研究IL 1RⅡ基因在相关疾病中的作用创造了有利条件     

Altered gene expression in Staphylococcus aureus upon interaction with human endothelial cells

Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureus to human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction between S. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteria. To assess the bacterial response to the cellular environment, an expression library of S. aureus was used to identify genes whose expression was induced after 4 h of exposure to HUVEC. The identified genes could be divided into different categories based on the functions of the encoded proteins (transport, catabolism, biosynthesis, and DNA repair). Further analyses of five of the S. aureus transposon clones showed that HUVEC as well as human serum are stimuli for triggering gene expression in S. aureus.     

NK-mediated reduction of malignancy in human melanoma cells treated with theophylline

Theophylline-treated cells of the human melanoma line showed an increase in NK-sensitivity in vitro and a concomitant decrease in tumorigenicity and spontaneous metastasis in Balb/c nude mice. The MeWo cells were heterogeneous and contained related subpopulations which were cloned to produce two cell lines, one hypodiploid (Cd-16) and one hypotetraploid (Ct-1). Prolonged (3 months) or short-term (4 days) treatment of these cell lines with 1 mm theophylline markedly reduced the incidence and size of tumors in Balb/c nude mice early after s.c. injection and their ability to metastasize spontaneously to the lung was also reduced. The effect was much more pronounced with Cd-16 cells, which contain amplified DNA compared to Ct-1 cells which lack DNA amplification. Part of the tumor inhibition caused by theophylline was due to natural killer (NK) cells. Thus, in vivo treatment of nude mice with anti-asialo GM1, a procedure known to remove NK cells, partially reversed the inhibitory effects of theophylline on tumor formation and generation of metastasis by Cd-16 cells. Consistent with this observation theophylline treatment enhanced the in vitro NK sensitivity of Cd-16 cells four-fold whereas Ct-1 was enhanced only slightly. The data suggest that theophylline can act preferentially on certain tumor cell subpopulations to enhance their NK-sensitive phenotype and thereby inhibit their capacity to form tumors and to metastasize in nude mice.     

IL-1 alpha gene expression and protein production by fibroblasts from patients with systemic sclerosis.

We examined IL-1 alpha and IL-1 beta gene expression and protein production in human dermal fibroblasts from patients with systemic sclerosis (SSc) to investigate the abnormal function of SSc fibroblasts. Human dermal fibroblasts were biopsied from 13 patients with SSc, three patients with rheumatoid arthritis (RA) and five healthy normal controls (NC). Cells were cultured in serum-free media and total RNA was collected from second or third passage fibroblasts. In cultured SSc fibroblasts, IL-1 alpha and IL-1 beta mRNAs were constitutively expressed and intracellular pro-IL-1 alpha was present. These observations suggest that an autocrine effect of IL-1 alpha contributes to the fibrosis in SSc.