Immunoregulatory Defects In Inflammatory Bowel Disease

这里报道的研究集中在两个主要发现上。首先，以前在UC中发现的非恒定NKT细胞实际上能够对NKT细胞刺激剂硫脂家族中的一种自身糖脂产生反应。这一发现强烈表明UC中的NKT细胞对自身抗原有反应，UC是一种经典的自身免疫性疾病。第二，UC中NKT细胞携带IL-13R&2受体：1)受硫脂刺激上调；2)IL-13与NKT细胞相互作用，以自分泌方式诱导NKT细胞杀伤作用增强。因此，该受体对NKT细胞介导的病理效应至关重要。 UC固有层NKT细胞对硫脂的反应表现为三种方式：诱导NKT细胞产生细胞因子，诱导NKT细胞的细胞毒作用，增强NKT细胞IL-13R&2受体的表达，但不增加IL-13R；1受体的表达。这些发现与我们先前观察到的UC中NKT细胞对高水平表达CD1d的B细胞在没有外源抗原的情况下的刺激反应是一致的，因为在后一种情况下，B细胞上的CD1d可能装载了与硫脂交叉反应的内源性自身糖脂。溶血硫脂诱导的反应也与我们以前的研究一致，因为它们表明上述转基因B细胞可以刺激UC中的NKT细胞，介导以产生IL-13而不是IL-4(或各种Th1细胞因子)为特征的非典型Th2反应，并对上皮细胞表现出细胞毒作用()。从目前的研究中获得的一个全新的观察结果是，如上所述，硫脂上调IL-13R&2受体不仅导致携带该受体的细胞数量的增加，而且可能更重要的是使单个细胞上受体的表达强度大大增加。这意味着硫脂对NKT细胞的刺激导致了一个正反馈电路，在这个电路中，通过TCR识别抗原而诱导的细胞毒作用最终可以通过增加一个也介导细胞毒作用的受体的表达来增强。 硫脂刺激NKT细胞导致IL-13R；2而不是IL-13R；1上调的观察结果促使我们确定该受体是否可以作为UC中NKT细胞的替代标记物。为了研究这种可能性，我们转向恶唑酮结肠炎模型，这是一种由IL-13和NKT细胞驱动的结肠炎，并表明给予与假单胞菌外毒素(IL-13-PE)相连的IL-13通过靶向和消除NKT细胞及其IL-13分泌来消融恶唑酮结肠炎。由于已有文献证明IL-13-PE的细胞毒作用依赖于与IL-13R&2受体()的结合，本研究提供了涉及UC类型炎症的NKT细胞携带该受体的有力证据。其他研究表明，用-GalCer-四聚体检测到的携带TCR的不变细胞也携带IL-13R；2支持这一结论。 在对UC患者的平行研究中，我们首次发现患者循环中的CD4+T细胞具有容易检测到的携带IL-13R；2的细胞，而CD患者几乎检测不到这种水平。此外，用IL-13-PE耗尽细胞会导致承载受体的细胞和产生IL-13的细胞共同耗尽。这些发现与UC患者循环中携带IL-13R；2的细胞有关，这是患者炎症组织中发现的先兆。在后一种情况下，我们发现大多数皮损固有层单核细胞(约70%)携带IL-13R；2，而携带IL-13R；1受体的细胞比例很低。此外，承载受体的细胞还带有NKT细胞标记CD161，大多数(如果不是全部)是产生IL-13的细胞。最后，我们发现，UC固有层细胞的IL-13-PE耗竭导致IL-13的产生和细胞毒活性大大降低，就像循环细胞的情况一样，再次验证了IL-13R和2是UC的NKT细胞标记物的概念。这些研究提供了明确的证据，表明带有NKT细胞标志物的IL-13产生细胞在活动期炎症的UC粘膜中不仅仅是一小部分细胞亚群。由于疾病的弥漫性，UC没有适当的病变；相反，它们构成了炎症区域的大部分细胞。这一事实极大地增加了UC主要是由于这些细胞及其产生的IL-13的可能性。

The studies reported here center around two major findings. The first is that the non-invariant NKT cells previously identified in UC are in fact capable of responding to a self-glycolipid, a member of the sulfatide family of NKT cell stimulants. This finding strongly suggests that NKT cells in UC are responding to a self antigen and that UC qualifies as a classical autoimmune disease. The second is that NKT cells in UC bear the IL-13Rα2 receptor that: 1) is up-regulated by sulfatide stimulation; and 2) is the receptor through which IL-13 interacts with NKT cells to induce enhancement of NKT cell cytotoxicity in an autocrine fashion. This receptor is therefore critical to NKT cell-mediated pathologic effects. The response of UC lamina propria NKT cells to sulfatide was shown in three ways: it induced NKT cell cytokine production, induced NKT cell cytotoxic function and augmented NKT cell expression of the IL-13Rα2 receptor, but not the IL-13Rα1 receptor. These findings were consistent with and help explain our previous observation that NKT cells in UC respond to stimulation by a B cell stably expressing high levels of CD1d in the absence of exogenous antigen, since in the latter case it is possible that the CD1d on the B cell was loaded with an endogenous self-glycolipid that cross-react with sulfatide. The responses elicited by the lyso-sulfatide are also consistent with our previous studies in that they showed that NKT cells in UC can be stimulated by the aforementioned transfected B cell to mediate an atypical Th2 response characterized by production of IL-13 but not IL-4 (or various Th1 cytokines) and to exhibit cytotoxicity for epithelial cells ( ). A totally new observation obtained from the present study was that, as eluded to above, sulfatide upregulation of the IL-13Rα2 receptor not only results in enhancement of the number of cells bearing the receptor, but perhaps more importantly to a great increase in the intensity of receptor expression on individual cells. This observation implies that stimulation of NKT cells by sulfatide leads to a positive feedback circuit in which induction of cytotoxicity by TCR-recognition of antigen can ultimately be enhanced by increased expression of a receptor that also mediates cytotoxity. The observation that sulfatide stimulation of NKT cells results in upregulation of the IL-13Rα2 but not the IL-13Rα1 prompted us to determine if this receptor could server as a surrogate marker of NKT cells in UC. To investigate this possibility we turned to the oxazolone-colitis model, a colitis driven by IL-13 and NKT cells, and showed that administration of IL-13 linked to pseudomonas exotoxin (IL-13-PE) ablates oxazolone-colitis by targeting and eliminating NKT cells and its IL-13 secretion. Since it is well documented that the cytotoxic effect of IL-13-PE depends on binding to the IL-13Rα2 receptor ( ), this study provided strong evidence that NKT cells involved in a UC type inflammation bear this receptor. Additional studies showing that invariant TCR-bearing cells detected with α-GalCer-tetramer also bear IL-13Rα2 supported this conclusion. In parallel studies of humans with UC we first showed that circulating CD4+ T cells in patients have easily detectable cells bearing the IL-13Rα2 whereas patients with CD have barely detectable levels. Furthermore, depletion of cells with IL-13-PE led to co-depletion of receptor-bearing cells and IL-13-producing cells. These findings relating to IL-13Rα2-bearing cells in the circulation of UC patients presaged findings in the inflamed tissues of patients. In the latter case, we found that the majority of lesional lamina propria mononuclear cells (approximately 70%) bear IL-13Rα2 whereas the percentage of cells bearing the IL-13Rα1 receptor was low. In addition, the receptor-bearing cells also bear the NKT cell marker, CD161 and most (if not all) are IL-13-producing cells. Finally, we showed that IL-13-PE depletion of UC lamina propria cells led to greatly reduced IL-13 production and cytotoxic activity, as in the case of circulating cells, again validating the concept that IL-13Rα2 is an NKT cell marker in UC. These studies provide unequivocal evidence that IL-13-producing cells bearing NKT cell markers are more than a minor sub-population of cells in actively inflamed UC mucosa there are no proper lesions in UC as the disease is diffuse; on the contrary, they make up the majority of cells in the inflamed areas. This fact greatly increases the likelihood that UC is primarily due to these cells and the IL-13 they produce.