Mucosal proinflammatory cytokine production correlates with endoscopic activity of ulcerative colitis

Proinflammatory cytokines are believed to be involved in the pathogenesis of ulcerative colitis (UC). The aim of this study was to clarify the profiles of proinflammatory cytokine production in patients with UC in terms of disease intractability, endoscopic findings, and host response to lipopolysaccharide (LPS) stimulation. Colonic mucosal tissues were obtained from patients with active UC (n = 15, including 4 patients with intractable disease) and inactive UC (n = 7), non-inflammatory bowel disease (IBD) colitis (n = 11), and controls (n = 20). Organ culture was performed, and the amounts of four cytokines (described below) in the culture media were determined by enzyme-linked immunosorbent assay (ELISA). LPS stimulation enhanced interleukin (IL)-1β, IL-8, and IL-6 production in colonic specimens from all groups, but enhanced tumor necrosis factor (TNF)-α production only in active UC specimens. Levels of IL-6, IL-8, and TNF-α were significantly higher in active UC than in non-IBD colitis, and the production of all three of these cytokines was correlated to the endoscopic grade of inflammation. The production of these cytokines was also significantly higher in patients with intractable disease receiving corticosteroids than in patients with non-intractable disease receiving corticosteroids. These results suggest that enhanced production of mucosal proinflammatory cytokines may be implicated in the pathogenesis of UC. (Received Jan. 30, 1998; accepted Aug. 28, 1998)     

Intestinal anti-inflammatory activity of the Serpylli herba extract in experimental models of rodent colitis

IntroductionNowadays, there is an increasing interest for alternative options in the treatment of inflammatory bowel diseases (IBDs) that combine efficacy and an adequate safety profile.MethodsThe intestinal anti-inflammatory effects of Serpylli herba, the officinal drug in the European Pharmacopeia composed by the aerial parts of wild thyme (Thymus serpyllum), were evaluated in the trinitrobenzenesulfonic acid (TNBS)-induced rat colitis and dextran sodium sulfate (DSS)-induced mouse colitis, which are well characterized experimental models with some resemblance to human IBD.ResultsS. herba extract exerted an intestinal anti-inflammatory effect in both experimental models of colitis, as evidenced both histologically, since it facilitated the tissue recovery of the damaged colon, and biochemically as showed by the improvement of the different inflammatory markers evaluated, including myeloperoxidase activity, glutathione content, and leukotriene B₄ levels as well as the expression of the inducible proteins iNOS and COX-2. This beneficial effect was associated with the reduction in the expression of different cytokines, like TNFα, IL-1β, IFNγ, IL-6 and IL-17, the chemokine MCP-1, and the adhesion molecule ICAM-1, thus ameliorating the altered immune response associated with the colonic inflammation.ConclusionS. herba extract displays an anti-inflammatory effect on different models of rodent colitis that could be attributed to its immunomodulatory properties.     

双歧杆菌对实验性结肠炎中趋化因子的影响

目的观察双歧杆菌在三硝基苯磺酸（TNBS）／乙醇诱导的实验性结肠炎的作用,并探讨其作用机制。方法40只SPF级SI）大鼠随机均分为正常组（N组）、模型组（M组）、双歧杆菌干预组（双歧杆菌预防性应用组：A组;双歧杆菌治疗组：B组）。TNBS／乙醇制备大鼠结肠炎模型。A组造模前7d始予双歧杆菌0．1mL,B组造模后予双歧杆菌0．1mL,双歧杆菌悬液浓度：1x10。cfu／mL,正常组与模型组生理盐水2mL,连续灌胃14d。观察各组大鼠结肠炎疾病活动指数（DAI）,评价结肠大体形态损伤指数（CMDI）和组织学损伤指数（TDI）,免疫组织化学法检测结肠组织CCL20、CCR6的表达,EL／SA法检测血清中CCL20、CCR6、TNF-α和IL-10含量。结果双歧杆菌干预组DAI、CMDI和TDI评分明显低于模型组,血清TNF—d、CCL20、CCR6及结肠组织CCL20、CCR6的表达明显降低（P均〈0．01）,而血清IL-10水平升高（P〈0．01）。A组与B组差异无统计学意义。结论双歧杆菌可能通过降低组织及血清CCL20及其受体CCR6表达,调节促炎因子与抗炎因子的平衡,对实验性结肠炎发挥治疗作用,且提前应用能提高疗效,发挥更好的作用。     

An interleukin 12 p40-IgG2b fusion protein abrogates T cell mediated inflammation: anti-inflammatory activity in Crohn's disease and experimental colitis in vivo

BACKGROUND AND AIMS: Interleukin-12 (IL-12), a p35/p40 heterodimer, plays a pivotal role in the immune response in Crohn's disease (CD). Since IL-12 p40 dimers act as IL-12 antagonists, we assayed p40 dimer proteins to modulate chronic intestinal inflammation. METHODS: We generated a fusion protein consisting of the IL-12(p40) subunit fused to the constant region of IgG2b. IL-12(p40)-IgG2b was tested in a murine 2,4,6,-trinitrobenzene sulphonic acid (TNBS) colitis model and in lamina propria mononuclear cells (LPMNC) from patients with CD in vitro. RESULTS: Dimeric IL-12(p40)-IgG2b fusion protein bound specifically to the IL-12 receptor. In concentrations <10(-7) M, it acted as an IL-12 antagonist as it inhibited interferon gamma (IFN-gamma) secretion, suppressed proliferation, and increased apoptosis of LPMNC from patients with CD. However, in concentrations >10(-6) M, IL-12(p40)-IgG2b increased IFN-gamma secretion and lymphocyte proliferation thereby acting as an IL-12 agonist. In TNBS colitic mice, IL-12(p40)-IgG2b decreased mortality (10% v 68%), prevented body weight loss, reduced tumour necrosis factor alpha, and increased IL-10 secretion. CONCLUSIONS: The IL-12(p40)-IgG2b fusion protein has dichotomic properties as a specific IL-12 antagonist and selective repressor of mucosal inflammation at low concentration and as an IL-12 agonist at high concentration.     

Exacerbation of experimental colitis by nonsteroidal anti-inflammatory drugs is not related to elevated leukotriene B4 synthesis.

The ability of nonsteroidal anti-inflammatory drugs to exacerbate experimental colitis, and the possible contributions of the "shunting" of arachidonate via the 5-lipoxygenase pathway, were investigated using a rat model in which colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid in a vehicle of 50% ethanol. Twice daily treatment with indomethacin (0.1-1 mg/kg SC) during the first week after trinitrobenzene sulfonic acid/ethanol administration resulted in dose-dependent increases in the severity of colitis and in the incidence of mortality. Mortality was not observed in vehicle-treated colitic rats or in normal rats treated with indomethacin. Similar exacerbation of colitis was observed in rats treated with naproxen (5 mg/kg). Whereas treatment with a 5-lipoxygenase inhibitor, PF-5901 (100 mg/kg PO), resulted in a significant reduction of the severity of colitis, concomitant administration of PF-5901 and indomethacin (0.5 mg/kg SC) did not inhibit the exacerbative effects of the indomethacin in this model. In separate studies, administration of indomethacin was found to significantly increase colonic myeloperoxidase activity (a measure of tissue granulocyte numbers) and suppress colonic prostaglandin E2 synthesis, while not significantly affecting colonic leukotriene B4 synthesis. The effect on myeloperoxidase activity was seen during the period 21-24 hours after trinitrobenzene sulfonic acid ethanol administration, but not during the period 45-48 hours after induction of colitis. In in vitro studies using samples of inflamed colon and in vivo studies in which colonic eicosanoid production was measured by colonic dialysis, inhibition of prostaglandin E2 synthesis was not accompanied by significant changes in leukotriene B4 synthesis. These results suggest that inhibitors of colonic prostaglandin synthesis can markedly exacerbate colitis, and that this effect is unrelated to alterations in colonic leukotriene B4 synthesis. Endogenous prostaglandins may exert anti-inflammatory effects during the acute stages of colitis.     

The antiangiogenic activity of cleaved high molecular weight kininogen is mediated through binding to endothelial cell tropomyosin

Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn2+-dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin.     

The antiplatelet activity of Escherichia coli lipopolysaccharide is mediated through a nitric oxide/cyclic GMP pathway.

In this study, Escherichia coli LPS dose-dependently (100-500 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human and rabbit platelets stimulated by agonists. LPS also dose-dependently inhibited the intracellular Ca2+ mobilization in human platelets stimulated by collagen. In addition, LPS (200 and 500 microg/ml) significantly increased the formation of cyclic GMP but not cyclic AMP in platelets. LPS (200 microg/ml) significantly increased the production of nitrate within a 10-min incubation period. Furthermore, LPS also dose-dependently inhibited platelet aggregation induced by PDBu (30 nmol/l), a protein kinase C activator. These results indicate that the antiplatelet activity of E. coli LPS may be involved in the activation of a nitric oxide/cyclic GMP pathway in platelets, resulting in inhibition of platelet aggregation. Therefore, LPS-mediated alteration of platelet function may contribute to bleeding diathesis in septicemic and endotoxemic patients.     

The experimental production of macrocytic anemia by operations of the intestinal tract

1. The literature concerning attempts to produce macrocytic anemia of theliver-deficiency type in animals by operations on the gastrointestinal tract hasbeen reviewed. Operations on the stomach have failed consistently to produce suchan anemia, but success has been achieved by operations on the small intestinewith the creation of blind loops or intestinal stenosis.

2. The technic we have used to produce macrocytic anemia in the rat is describedin detail. The essentials are that the blind loop should fill with peristalsis andthat it should not be too low down in the small intestine.

3. Anemia does not usually develop until an interval of several weeks or monthsafter the operation. It is then macrocytic in type and acute in course.

4. The anemia is probably dependent on stagnation in the blind loop and achange in the bacterial flora of the small intestine.

     

复合膳食纤维对实验性溃疡性结肠炎大鼠肠黏膜屏障功能的影响

目的 观察添加复合膳食纤维(DFC)的肠内营养(EN)对实验性结肠炎大鼠肠黏膜屏障功能的影响.方法 将96只SD大鼠用乙酸灌肠法制成结肠炎模型后随机分成3组,C组给予不含DFC的整蛋白型肠内营养粉剂(商品名:能全素);T1组和T2组分别给予含整蛋白型肠内营养 粉剂和DFC的EN.DFC由可溶性膳食纤维(SDF)和不溶性膳食纤维(IDF)按一定比例配制,T1组和T2组中,SDF和IDF的比例分别为1:2和1:3.检测EN后第1、3、5、7天大鼠血浆D-乳酸浓度和血浆二胺氧化酶(DAO)活性并取肠道标本观察肠黏膜变化情况.结果 第3、5、7天的T1组与T2 组血浆D-乳酸浓度和DAO活性较C组下降(P<0.05),T1组与T2组间差异无统计学意义(P>0.05).各时间点T1组与T2组结肠损伤组织学评分均较C组下降(P<0.05),第5、7天T2组较T1组评分下降幅度大(P<0.05).结论 结肠炎大鼠用含有DFC的EN可降低肠道通透性,对肠黏膜屏障有一定的保护作用,不同比例的SDF和IDF 对肠黏膜保护效果不同,适量增加IDF的比例可以增加这种保护效果.     

Modulating activity of interferon-gamma on endotoxin-induced cytokine production in cancer patients.

Intravenous (IV) administration of purified lipopolysaccharide (LPS) from Salmonella abortus equi to cancer patients induces the formation of high amounts of endogenous cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). On repeated administration of LPS at 2-week intervals, a marked downregulation of the cytokine response was observed, especially between the first and the second challenge. This study sought to determine whether it would be possible to prevent this downregulation by pretreating patients with interferon-gamma (IFN-gamma), which is known to enhance cytokine production by monocytes and macrophages in vitro. Ten patients with disseminated cancer received a first injection of 4.0 ng LPS/kg. Thereafter, patients were divided into two groups. One group received two further LPS injections (4.0 ng/kg) at 2-week intervals. The second group was pretreated (-12 hours) with 50 micrograms IFN-gamma subcutaneously (SC) before the second and third LPS challenge. To prevent constitutional side effects such as fever and chills, patients received 1,600 mg ibuprofen orally before LPS injection. The results of the current study demonstrate that apart from TNF-alpha and IL-6, two other cytokines, interleukin-8 (IL-8) and granulocyte colony-stimulating factor (G-CSF) are produced in cancer patients in response to LPS. LPS application at 2-week intervals resulted in a transient attenuation of all cytokines (TNF-alpha, IL-6, IL-8, G-CSF) on the second challenge. In the case of TNF-alpha, IL-6, and G-CSF, pretreatment with IFN-gamma not only prevented the downregulation, but enhanced the production of these cytokines to levels higher than those obtained after the first LPS challenge. In contrast, the downregulation of IL-8 remained unaffected by IFN-gamma pretreatment. Further studies are warranted to determine whether the prevention of cytokine downregulation by IFN-gamma following repeated LPS injections is of clinical relevance in respect to the antitumor activity of LPS.     

Effect of aging on cytokine production in normal and experimental systemic lupus erythematosus-afflicted mice

Aging mice of strains susceptible to the induction of experimental systemic lupus erythematosus (SLE) develop a milder disease than young animals. To find out whether the decrease in susceptibility to disease is due to age-associated changes in cytokine profile, we first examined the secretion of cytokines by healthy mice aged 2-15 months. A gradual age-related decline in the levels of interleukin (IL)-2 and interferon (IFN) gamma, and an increase in IL-4, IL-10, IL-1, and tumor necrosis factor (TNF) alpha were observed. Experimental SLE was induced in 2- and 10-month-old mice by immunization with the monoclonal anti-DNA antibody bearing the 16/6 Id. Early increased production of pro-inflammatory cytokines (TNFalpha and IL-1), followed by a peak of the Th1-type cytokines (IL-2, IFNgamma) were observed in young mice. The Th2-type cytokines (IL-4, IL-10) peaked later. In contrast, only a mild increase in all of the above cytokines was determined in 10-month immunized mice. It thus appears that the decline in susceptibility to SLE induction in older mice may be related to changes in the capacity to produce cytokines.     

苦豆子总碱对大鼠溃疡性结肠炎细胞因子IL-10表达的影响

目的观察苦豆子总碱对大鼠溃疡性结肠炎(UC)的外周血和结肠组织中IL-10表达的影响。方法将SD大鼠随机分成6组(正常组、模型组、柳氮磺胺吡啶(SASP)、苦豆子总碱高剂量组、中剂量组、低剂量组),采用三硝基苯磺酸(TNBS)灌肠法,制备结肠炎大鼠模型。第2天开始灌胃,在第3周杀鼠后用酶联免疫吸附法(ELISA)检测IL-10在结肠黏膜组织和外周血清中的表达水平;同时分析结肠黏膜组织IL-10与DAI(疾病活动指数评分)/组织学损伤的相关性。结果(1)结肠部位和外周血清中IL-10的表达较对照组均显著降低,其中以结肠部位降低的幅度最大(P<0.01)。(2)苦豆子总碱高、中、低3个剂量组和SASP组都能显著性上调结肠部位和外周血清的IL-10的表达(P<0.05),且结肠部位的变化与外周血的相比差异明显。(3)结肠部位的DAI与结肠IL-10之间存在的负相关性(Pearsonr=-0.828,P<0.01),结肠部位的组织学损伤与结肠IL-10之间存在负相关性(Pearsonr=-0.819,P<0.01);所有检测为双侧,样本容量n=60。结论苦豆子总碱通过上调IL-10的表达减轻或改善实验性结肠炎大鼠的组织学损伤和症状。