IMMUNOREGULATORY DEFECTS IN INFLAMMATORY BOWEL DISEASE

项目1：在之前的研究中，我们发现慢性结肠炎 与Th1型细胞反应相关的可由直肠 增白剂2，4，6-三硝基苯磺酸的使用 酸(TNBS)。我们在此报告口服半抗菌素 直肠给药前有效的结肠蛋白(HCP) 抑制后者诱发结肠炎的能力。CD4+T细胞来源 经抗CD3/抗CD28刺激后，喂饲HCP的小鼠可获得5-10倍的 增加转化生长因子-β的产生和分泌增加的IL-4 和IL-10，但干扰素-γ水平低于T细胞 用卵清蛋白喂养的对照动物。此外，喂食六氯酚的小鼠的结肠 显著增加转化生长因子-β的分泌，降低IL-12的分泌 免疫组织化学研究和分离的单个核细胞的表达 饲喂HCP的动物分泌较少的IL-12异源二聚体。最后，也是大多数 重要的是，口服HCP的抑制作用是 同时全身注射抗转化生长因子-β或 IL-12提示IL-12与转化生长因子-β的相互作用 三七总皂甙诱导结肠炎耐受的研究。这些研究表明 转化生长因子-β的产生第一次可以取消实验 肉芽肿性结肠炎，即使在这样的结肠炎被确立之后，因此， 转化生长因子-β水平的调节可能与治疗有关。 人类炎症性肠病。项目2：在进一步研究 免疫调节TNBS-结肠炎的机制研究 TH1 T细胞存在下CD40L-CD40相互作用的研究 在这种实验性疾病中产生IL-12。我们发现， 抗CD40L抗体对TNBS结肠炎小鼠的治疗作用 固有层诱导阻止干扰素-γ的产生 以及疾病的临床和组织学证据。在……里面 相比之下，Th2型细胞因子IL-4的分泌在 抗CD40L治疗。在进一步的研究中，我们表明，预防 疾病活动性的原因是抑制IL-12的分泌和 重组IL-12p70异源二聚体注入TNBS+ 抗CD40L处理的小鼠逆转了抗CD40L的作用 身患重病。这些研究表明，CD40L-CD40相互作用是 对TNBS-结肠炎的发展至关重要。项目3：进一步 研究，我们调查了人类炎症性肠病(IBD) (溃疡性结肠炎和克罗恩病)与改变有关 淋巴因子分泌图谱。在最初的研究中，我们发现Lp T 炎性克罗恩病粘膜细胞表现为干扰素-γ升高 分泌与对照LP T细胞相比，尤其是在刺激时 通过CD2/CD28途径。相比之下，IL-4和IL-5的产生通过 克罗恩病患者LP T细胞较正常对照组减少 LP T细胞。此外，克罗恩氏LP T细胞产生IL-2的能力 减少了。在进一步的研究中，我们发现来自溃疡病的LP T细胞 TCR/CD3/CD28或CD2/CD28刺激的结肠炎黏膜 产生更多的IL-5。如此增加的IL-5产量是 与IL-4分泌增加无关，与克罗恩病相反 疾病、溃疡性结肠炎LP T细胞产生IL-2和干扰素-γ 是很正常的。综上所述，这些研究提供了强有力的证据 两种主要IBD的免疫病理过程特点 与非常不同的细胞因子分泌模式有关：在克罗恩病中 在溃疡性结肠炎中存在Th1细胞因子模式 Th2细胞因子模式占优势。这些不同的模式可能决定 炎症过程的类型存在，并可能影响类型 需要以细胞因子为基础的治疗。

Project 1: In previous studies we showed that a chronic colitis associated with a Th1 type cell response can be induced by the rectal administration of the hatptenizing reagent 2,4,6-trinitrobenzene sulfonic acid (TNBS). We report here that oral administration of haptenized colonic proteins (HCP) before rectal administration of TNBS effectively suppresses the ability of the latter to induce colitis. CD4+ T cells from HCP-fed animals stimulated with anti-CD3/anti-CD28 had a 5-10-fold increase in production of TGF-beta and secreted increased amounts of IL-4 and IL-10 but lower levels of IFN-gamma in comparison to T cells from ovalbumin-fed control animals. In addition, the colons of HCP-fed mice showed strikingly increased TGF-beta secretion but decreased IL-12 expression by immunohistochemical studies and isolated mononuclear cells from HCP-fed animals secreted less IL-12 heterodimer. Finally, and most importantly, the suppressive effect of orally administered HCP was abrogated by the concomitant systemic administration of anti-TGF-beta or rIL-12 suggesting a reciprocal relationship between IL-12 and TGF-beta on tolerance induction in TNBS-induced colitis. These studies suggest for the first time that TGF-beta production can abrogate experimental granulomatous colitis even after such colitis is established, and thus, that regulation of TGF-beta levels may have relevance to the treatment of human inflammatory bowel disease. Project 2: In further studies of the immunologic mechanisms regulating TNBS-colitis we investigated the role of the CD40L-CD40 interaction in the presence of TH1 T cells and the production of IL-12 in this experimental disease. We found that the administration of anti-CD40L antibodies to mice in whom TNBS-colitis is being induced prevented interferon gamma production by lamina propria CD4+ T cells and also clinical and histological evidence of disease. In contrast, the secretion of IL-4, a Th2-type cytokine, was increased after anti-CD40L treatment. In further studies we showed that the prevention of disease activity was caused by an inhibition of IL-12 secretion and that the injection of recombinant IL-12p70 heterodimer into TNBS + anti-CD40L treated mice reversed the effect of anti-CD40L and resulted in severe disease. These studies show that the CD40L-CD40 interaction is critical for the development of TNBS-colitis. Project 3: In a further study, we investigated whether human inflammatory bowel disease (IBD) (ulcerative colitis and Crohn's disease) is associated with altered lymphokine secretion profiles. In initial studies, we showed that LP T cells from inflamed Crohn's disease mucosa manifest increased IFN-gamma secretion compared with control LP T cells, particularly when stimulated via the CD2/CD28 pathway. In contrast, IL-4 and IL-5 production by Crohn's disease LP T cells was decreased compared with that of control LP T cells. In addition, IL-2 production by Crohn's LP T cells was decreased. In further studies, we showed that LP T cells from ulcerative colitis mucosa stimulated via either the TCR/CD3/CD28 or CD2/CD28 produced increased amounts of IL-5. Such increased IL-5 production was not associated with increased IL-4 secretion and, in contrast to Crohn's disease, ulcerative colitis LP T cell production of IL-2 and IFN-gamma was normal. Taken together, these studies provide strong evidence that the immunopathologic process characteristic of the two major forms of IBD is associated with very different cytokine secretion patterns: in Crohn's disease a Th1 cytokine pattern is present whereas in ulcerative colitis a Th2 cytokine pattern prevails. These different patterns may determine the type of inflammatory process present and may influence the kind of cytokine-based treatment needed.