Regulation of the ASC expression in response to LPS stimulation is related to IL-8 secretion in the human intestinal mucosa

Expression of the caspase-activating adaptor ASC was reported to be associated with the production of IL-8, a primary mediator of mucosal inflammation, in vitro. However, the significance of the ASC-mediated IL-8 production in primary tissues has remained poorly understood. Primary intestinal mucosa isolated from surgically resected ileum or colon was incubated with several concentrations of LPS or left untreated. ASC expression was up-regulated at 2 h after stimulation with low doses of LPS (1-10 ng/ml), and was associated with IL-8 secretion, and then was down-regulated later. In contrast, ASC expression remained at the basal level in mucosal tissue treated with a high dose of LPS (1000 ng/ml). Interestingly, in mucosa from several cases of Crohn's disease, ASC was highly expressed without stimulation, and IL-8 was stably secreted with no regulation by LPS. These findings revealed that ASC expression correlates with IL-8 secretion and may play an important role in maintaining mucosal homeostasis.     

不同镇痛方法对患者IL-6、IL-8、IL-10的影响

目的评价不同镇痛方法对盆腔手术患者血浆中细胞因子变化的影响,并与传统术后镇痛法进行比较,探讨术后充分镇痛对免疫功能的影响。方法根据镇痛方法不同,将60例行子宫切除患者随机分为3组：第1组患者为术后根据临床需要,临时给予哌替啶50mg肌肉注射（Ⅰ组,n=20）;第Ⅱ组患者为罗哌卡因复合芬太尼硬膜外镇痛组（Ⅱ组,n=20）;第Ⅲ组患者为芬太尼静脉镇痛组（Ⅲ组,n=20）;观察麻醉前30min、手术后30min、2h、24h、48h和72h六个时点患者血清中白细胞介素-6（IL-6）、白细胞介素-8（IL-8）、白细胞介素-10（IL-10）水平的变化。结果3组患者术后血清IL-6、IL-8、IL-10水平与麻醉前值比较均升高（P〈0．01）,一般在术后24h达峰值。比较血清IL-6、IL-8、IL-10浓度变化,Ⅱ和Ⅲ组抑制这3种细胞因子释放的能力明显强于Ⅰ组（P〈0．05）,与Ⅲ组比较,Ⅱ组更为明显（P〈0．05）。结论硬膜外局麻药复合阿片受体激动药镇痛模式可更有效地降低术后炎性直激反应。     

Internalization of Clostridium difficile cytotoxin into cultured human lung fibroblasts

In cultured human lung fibroblasts treated with Clostridium difficile cytotoxin, the latency before appearance of the cytopathogenic effect was dose-related with a minimum of 45 min. At 37°C, the toxin was accessible on all cells to inactivation with trypsin or neutralization with antitoxin during the first tenth of the latency. At 0°C, the toxin was accessible considerably longer. The cytopathogenic effect was reversibly prevented by the lysosomotropic agents chloroquine and ammonium chloride, which had to be added within one-fifth of the latency to protect all cells. In the presence of chloroquine, but not of ammonium chloride, the time period during which the toxin remained amenable to neutralization with antitoxin was prolonged. The protective effect of ammonium chloride was not influenced by dropping the extracellular pH to 4.5, but that of chloroquine was abolished. The expression of the intoxication was not affected by inhibitors of the DNA, RNA or protein synthesis. Inhibitors of the energy metabolism prevented the cytopathogenic effect when added before the last phase of the latency. The results suggest that expression of the cytopathogenic effect requires internalization of the toxin, and that metabolic energy but no macromolecular synthesis is needed for the action of the toxin after this internalization.     

复方清下汤对脓毒症大鼠细胞因子白介素-1、白介素-6调控及肺损伤的作用

目的盲肠结扎穿孔导致大肠埃希菌腹膜炎进而建立脓毒症肺损伤大鼠模型,检测炎性反应时,细胞因子的调控变化,探讨肺水肿的形成机制。经复方清下汤处理后检测上述变化。方法将健康SD大鼠随机分为4组,每组10只:假手术组(SHAM组),只翻动盲肠,不做其他处理;脓毒血症肺损伤组(模型组),盲肠结扎穿孔诱发急性肺损伤(ALI)模型;盲肠结扎穿孔+复方清下汤组(造模后立即灌胃给药,造模后8 h再次灌胃1次,剂量:10 ml/kg);盲肠结扎穿孔+头孢哌酮舒巴坦组(抗生素舒普深)(造模后立即静脉注射1次,造模后8 h再次静脉注射1次,剂量:0.2 g/kg),造模24 h后收集标本。分别观察大鼠的一般状态,留取下腔静脉血清进行白介素-1(IL-1)、白介素-6(IL-6)的测定。镜下观察肺组织病理形态学改变,测量肺湿/干比值的变化。结果与SHAM组比较,模型组IL-1、IL-6水平明显升高(P0.01),肺间质和肺泡内水肿,伴大量红细胞渗出(出血)和纤维素沉积,肺泡间隔毛细血管内皮细胞高度肿胀。肺湿/干比值明显增加(P0.01),抗生素及中药处理组与模型组比较,IL-1、IL-6水平明显降低(P0.01),肺湿/干比值明显降低(P0.01),肺组织镜下表现:中药处理组及抗生素组组较模型组肺泡间隔变窄,毛细血管内皮细胞肿胀减轻,出血减轻,纤维素渗出明显减少。结论实验应用放免检测、显微镜观察以及称量肺湿/干比值等手段,进一步证实了脓毒血症大鼠肺损伤时血清中主要的炎性细胞因子IL-1、IL-6过度表达的情况,并从病理学角度,证实炎性介质的过度表达是造成脓毒症肺损伤的重要原因。经复方清下汤处理的动物模型得到相反结论,为治疗脓毒血症大鼠肺损伤提供一个可能的新的手段。     

Interleukin-6 triggers human cerebral endothelial cells proliferation and migration: the role for KDR and MMP-9

Interleukin-6 (IL-6) is involved in angiogenesis. However, the underlying mechanisms are unknown. Using human cerebral endothelial cell (HCEC), we report for the first time that IL-6 triggers HCEC proliferation and migration in a dose-dependent manner, specifically associated with enhancement of VEGF expression, up-regulated and phosphorylated VEGF receptor-2 (KDR), and stimulated MMP-9 secretion. We investigated the signal pathway of IL-6/IL-6R responsible for KDR's regulation. Pharmacological inhibitor of PI3K failed to inhibit IL-6-mediated VEGF overexpression, while blocking ERK1/2 with PD98059 could abolish IL-6-induced KDR overexpression. Further, neutralizing endogenous VEGF attenuated KDR expression and phosphorylation, suggesting that IL-6-induced KDR activation is independent of VEGF stimulation. MMP-9 inhibitor GM6001 significantly decreases HCEC proliferation and migration (p<0.05), indicating the crucial function of MMP-9 in promoting angiogenic changes in HCECs. We conclude that IL-6 triggers VEGF-induced angiogenic activity through increasing VEGF release, up-regulates KDR expression and phosphorylation through activating ERK1/2 signaling, and stimulates MMP-9 overexpression.     

Bmx regulates LPS-induced IL-6 and VEGF production via mRNA stability in rheumatoid synovial fibroblasts

Discordant cytokine production is characteristic of chronic inflammatory conditions like rheumatoid arthritis (RA), and anti-cytokine therapeutics are becoming routinely used to treat RA in the clinic. Fibroblasts from rheumatoid synovium have been shown to contribute to cytokine production in inflamed joints; likewise these cells also produce cytokines in response to inflammatory mediators signalling through Toll like receptors (TLRs). Tyrosine kinase activity is essential to LPS-induced cytokine production, and we have previously implicated a role for the Tec kinase, Bmx, in inflammatory cytokine production. Here we show that Bmx kinase activity in RASF is increased following LPS stimulation and that Bmx is involved in the regulation of LPS-induced IL-6 and VEGF production via mRNA stabilisation. This is an important insight into the regulation of VEGF, which is involved in a wide range of different pathologies, and may lead to more effective design of novel anti-inflammatory/angiogenic therapeutics for conditions such as RA.     

The involvement of IL-6 and IL-8 in acute invasive gastroenteritis of children

The involvement of the proinflammatory cytokines, interleukin 8 (IL-8) and 6 (IL-6), was studied during the first 72 h of acute invasive gastroenteritis. Study population included 33 infants and young children aged six months to six years and seven age-matched controls. As a group, patients with acute invasive gastroenteritis had an increased serum level of IL-8 and IL-6 as compared with healthy controls (p < 0.002 and p < 0.001, respectively). Subjects were then divided into two groups based on stool cultures (proven and non-proven bacterial cultures). Patients with bacterial-proven acute invasive gastroenteritis tended to have increased IL-8 serum concentrations (p < 0.07) as compared with those with non-proven bacterial etiologies and IL-6 levels were only detected in subjects with positive bacterial cultures (p < 0.05). When dividing each sub-group into early and late blood drawing with respect to disease onset, no statistical differences were found in each group but subjects with bacterial-proven etiologies had significant higher IL-6 levels as compared with non-proven etiologies at the two time points (p < 0.019 and p < 0.015, respectively).In conclusion, the proinflammatory cytokines, IL-6 and IL-8, are involved in acute invasive gastroenteritis. The difference in IL-6, and to a lesser degree IL-8, between proven and non-proven bacterial etiologies, needs further investigation.     

A novel human glassy-cell carcinoma cell line producing IL-6 and IL-8 from uterine cervix

Summary A novel human cell line, TOM-2, was established from a rare uterine cervical cancer, glassy cell carcinoma (GCC). TOM-2 is the second established GCC cell line so far reported. The cells were intermediately or poorly differentiated with dysplastic nuclei and polygonal shape and secreted two tumor markers and cytokines, i.e., CA-125 and SCC, interleukin (IL)-1α, -6, and -8, and TNF-α. Growth of TOM-2 was so strongly dependent on population density that it was not possible to determine the plating efficiency. In mass culture, the following characteristics were observed: doubling time, 83 h; mode of chromosome number, 79; human papillomavirus type 18 DNA, detectable; tumorigenicity, easily transplantable into subcutis of nude mice; chemosensitivity in vitro, considerably sensitive to Cisplatin and 5-FU but not to 9 other antineoplastic agents. This novel cell line will be useful for developing new therapeutic strategies for the rare cancer, GCC.     

IL-8 promotes cell proliferation and migration through metalloproteinase-cleavage proHB-EGF in human colon carcinoma cells

Interleukin-8 (IL-8) has been reported to promote tumor cell growth in colon cancer cells after binding to its receptors, which are members of the G-protein coupled receptor (GPCR) family. Recent studies demonstrated that stimulation of GPCR can induce shedding of epidermal growth factor (EGF) ligands via activation of a disintegrin and metalloprotease (ADAM), with subsequent transactivation of the EGF receptor (EGFR). In this study, we investigated mechanisms of cell proliferation and migration stimulated by IL-8 in a human colon carcinoma cell line (Caco2). IL-8 increased DNA synthesis of Caco2 in a dose dependent manner and this was inhibited by ADAM, EGFR kinase, and MEK inhibitors. IL-8 transiently induced EGFR tyrosine phosphorylation after 5-90 min and this was completely inhibited by ADAM inhibitor. Neutralizing antibody against HB-EGF as a key ligand for EGFR also blocked transactivation of EGFR and cell proliferation by IL-8. Since IL-8-induced cell migration was further suppressed by the ADAM inhibitor and the HB-EGF neutralizing antibody, our data indicate that IL-8 induces cell proliferation and migration by an ADAM-dependent pathway, and that HB-EGF plays an important role as the major ligand for this pathway.     

Involvement of protein kinase Cdelta in iron chelator-induced IL-8 production in human intestinal epithelial cells

We have shown that the bacterial iron chelator, deferoxamine (DFO), triggers inflammatory signals, including the production of CXC chemokine IL-8, in human intestinal epithelial cells (IECs) by activating ERK1/2 and p38 kinase pathways. In the present study, we show that PKCdelta, one of the novel protein kinase C (PKC) isoforms, involves in signal transduction pathways leading to DFO-induced IL-8 production. Pretreatment of human intestinal epithelial HT-29 cells with rottlerin showed remarkable inhibition of DFO-induced IL-8 production. In contrast, other PKC inhibitors such as G?6976, G?6983, GF109203X, and staurosporine revealed less or no inhibitory effects on DFO-induced IL-8 production, suggesting a potential role of PKCdelta. Accordingly, DFO caused phosphorylation of PKCdelta in the Thr505 and Ser643 residues in HT-29 cells. Transfection of dominant-negative PKCdelta vector inhibited DFO-induced PKCdelta phosphorylation as well as IL-8 promoter activity. In addition, suppression of endogenous PKCdelta by siRNA significantly reduced DFO-induced IL-8 production. Collectively, these results suggest that PKCdelta plays a pivotal role in signaling pathways leading to iron chelator-induced IL-8 production in human IECs.     

The adenylate cyclase response to parathyroid hormone in fetal lung fibroblasts is enhanced by cortisol

Parathyroid hormone (PTH, <10^–8 M) stimulated adenylate cyclase in fibroblasts, but not epithelial cells, isolated from fetal rat lung. In contrast to osteosarcoma cells (UMR 106), the response of fibroblasts to PTH was increased by pretreatment with cortisol (< 10^–8–10^–7 M).     

Interleukin-6 negatively regulates Porphyromonas gingivalis fimbria-stimulated fibronectin expression in human gingival fibroblasts

Our previous study demonstrated that fibronectin (FN) is a negative regulator of Porphyromonas gingivalis fimbria-induced pathogenesis in the initiation and development of chronic periodontal diseases. We show herein the regulatory action of interleukin-6 (IL-6) on FN expression in fimbria-treated human gingival fibroblasts. Interestingly, the decrease in FN expression in the cells treated with fimbriae at a high dose (8 microg of protein ml(-1)) was negated by treatment with anti-IL-6 antibody. Also, the increase in FN expression in cells treated with fimbriae at a low dose (1 microg of protein ml(-1)) was inhibited by exogenous IL-6. These results suggest that P. gingivalis fimbria-stimulated FN expression in human gingival fibroblasts is negatively regulated by endogenous IL-6.     

Serum and urine levels of interleukin-6 and interleukin-8 in children with acute pyelonephritis

Urinary tract infection (UTI) is a common clinical disorder in younger infants and children and may result in permanent renal damage. The inflammatory cytokines interleukin (IL)-6 and IL-8 play an important role in response to bacterial infection. This prospective study investigated the association between serum and urine IL-6 and IL-8 levels and acute pyelonephritis confirmed by (99m)Tc-dimercaptosuccinic acid (DMSA) scan. A total of 78 children aged 1-121 months with a diagnosis of first-time febrile UTI were included. The following inflammatory markers were assessed: fever; white blood cells count (WBC); C-reactive protein (CRP); and serum and urine IL-6 and IL-8. The patients were divided into the acute pyelonephritis group (n=42) and the lower UTI group (n=36) according to the results of DMSA scan. Fever, WBC and CRP levels were significantly higher in children with acute pyelonephritis than in those with lower UTI (all p <0.001). Significantly, higher initial serum and urine IL-6 and IL-8 levels were found in children with acute pyelonephritis than in those with lower UTI (all p <0.001). Serum and urine IL-6 in children with acute pyelonephritis were positively correlated with fever, CRP and leucocyturia. These results indicate that both serum and urine IL-6 and IL-8 levels, particularly IL-6, are useful diagnostic tools for early recognition of acute pyelonephritis in febrile children.     

游离脂肪酸对人牙周膜成纤维细胞增殖的影响

目的:探讨游离脂肪酸(FFAs)对人牙周膜成纤维细胞增殖的影响,研究游离脂肪酸在代谢综合征患者牙周病发病机制中的作用。方法:选用在牙周组织修复中起主要作用的人牙周膜成纤维细胞进行体外培养,对照组加入不含胎牛血清的DMEM,实验组分别加入不同浓度的游离脂肪酸进行刺激,在刺激24h-72h后,采用四甲基偶氮唑蓝比色(MTT)法检测人牙周膜成纤维细胞的增殖情况。结果:与对照组相比,游离脂肪酸可以抑制人牙周膜成纤维细胞的生长增殖(P<0.01),并且这种抑制作用具有浓度和时间依赖性,以培养72h后抑制作用最为明显(P<0.01)。结论:游离脂肪酸可以抑制牙周膜成纤维细胞的增殖,降低代谢综合征患者牙周组织的的修复能力,从而导致或加重牙周病的发生或发展。     

ATP-stimulated interleukin-6 synthesis through P2Y receptors on human osteoblasts

We investigated the effect of extracellular adenosine triphosphate (ATP) on the production of interleukin (IL)-6, whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as are involved in signal transduction systems in human osteoblastic SaM-1 cells. These human osteoblasts constitutively expressed P2X4, P2X5, P2X6, P2Y2, P2Y5, and P2Y6 purinergic receptors. ATP increased gene- and protein-expression of IL-6 in SaM-1 cells. The expression of the IL-6 mRNA was maximal at 1h, and the increase in IL-6 synthesis in response to ATP (10-100 microM) occurred in a concentration-dependent manner. Over the same concentration range of the nucleotide that was effective for IL-6 synthesis, ATP caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which increase was inhibited by pretreatment with suramin, a P2Y receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker, but not by the extracellular Ca(2+)-chelating agent EGTA. The pretreatment of SaM-1 cells with suramin or 2-APB also inhibited the increase in IL-6 synthesis in response to ATP. These findings suggest that extracellular ATP-induced IL-6 synthesis occurs through P2Y receptors and mobilization of Ca(2+) from internal stores in human osteoblastic cells.