Studies Of The Bioregulatory Aspects Of The Retinal Pigment Epithelial Cell

RPE细胞在维持神经视网膜的结构和生理完整性方面起着基本的作用。其结构和功能的改变会导致光感受器和视力的丧失。我们对RPE细胞作为眼后极重要的免疫调节细胞进行了广泛的研究。我们对RPE细胞的研究活动可分为三类：正常细胞功能研究、细胞因子相互作用研究和感染过程研究。该项目致力于研究细胞因子与免疫系统的细胞以及眼部微环境中的细胞相互作用的方式。这些研究表明，细胞因子介导的RPE细胞活化可能是眼免疫的一个基本组成部分，也是RPE细胞移植的一个重要方面。在过去的一年里，我们研究了RPE细胞中的Toll样受体(TLR)以及与视网膜血管炎患者相关的可能的生物标志物。TLR是天然免疫的重要组成部分，参与宿主对微生物病原体的防御。TLR信号提供了一种快速、强大、突发性的反应性，旨在限制感染部位的病原体。这种反应性的爆发突出表现为细胞因子、趋化因子和黏附分子的释放。在视网膜内，不受控制的炎症爆发本身就会导致细胞损伤。因此，很有可能还会产生一种下调的力量来限制免疫病理损害。我们发现，RPE细胞产生的干扰素-β主要是通过TLR信号，其次是自身刺激产生的，是这种限制力的关键组成部分。RPE细胞的细胞因子激活导致趋化因子CXCL9和CXCL10以及黏附分子ICAM-1的产生。用干扰素-β处理RPE细胞后，ICAM-1的产生受到抑制，CXCL9的产生被消除。这种处理并没有改变CXCL10的产量。抗干扰素-β抗体可阻断干扰素-β的抑制作用。实时荧光定量聚合酶链式反应分析显示，干扰素-β处理抑制了sICAM-1和CXCL9的基因表达。结果表明，RPE细胞来源的干扰素-β在下调视网膜CXCL9和ICAM-1表达中起关键作用，提示CXCL9的抑制是一种免疫抑制机制，可以保护视网膜免受过度炎症的影响。 最近我们发现，干扰素-β可以显著下调多种细胞，如内皮细胞、巨噬细胞、上皮细胞、神经母细胞瘤和神经胶质细胞中CXCL9基因的表达和蛋白合成。有趣的是，这种下调效应发生在CXCL9上，而不是CXCL10或CXCL11上。为了探讨干扰素-β治疗对CXCL9和sICAM-1产生的潜在体内影响，我们研究了实验性脑型疟疾的动物模型系统。已知CXCL9和ICAM-1在本病的发病机制中起关键作用。我们发现，干扰素-β治疗导致感染伯吉安卡疟原虫后死亡的小鼠的存活率增加。存活率的增加与脑内皮细胞上CXCL9和ICAM-1蛋白表达的减少有关。此外，脑组织中CXCL9和ICAM-1的基因表达也被干扰素-β处理显著抑制。这些研究清楚地表明，体内干扰素-β治疗导致显著的免疫抑制，与抑制CXCL9和ICAM-1有关。我们现在正在评估这些动物的视网膜变化，并评估其他眼部疾病。

The RPE cell plays a basic role in maintaining the structural and physiological integrity of the neural retina. Alterations in its structural and functional actions can result in loss of photoreceptors and vision. We have studied the RPE cell extensively as an important immunoregulatory cell within the posterior pole of the eye. Our research activities on RPE cells can be subdivided into three categories: normal cell function studies, cytokine interactions and infectious processes. This project has concentrated on studying the ways in which cytokines interact with cells of the immune system and with cells in the ocular microenvironment. These studies indicate that cytokine-mediated activation of RPE cells may be a basic component of ocular immunity and an important aspect of RPE cell transplantation. During the past year, we have studied the Toll-Like receptors (TLR) in RPE cells and possible biological markers associated with patients with retinal vasculitis. TLRs are crucial components of innate immunity that participate in host defense against microbial pathogens. TLR signaling provides a rapid, robust, burst of reactivity designed to limit pathogens at the site of infection. This burst of reactivity is highlighted by release of cytokines, chemokines and adhesion molecules. Within the retina an uncontrolled inflammatory burst can itself lead to cellular damage. Therefore, it is highly probable that a downregulatory force is also produced to limit immunopathologic damage. We identified that IFN-beta produced by RPE cells primarily by TLR signaling and secondarily by auto-stimulation is a critical component of that limiting force. Cytokine activation of RPE cells results in the production of the chemokines, CXCL9 and CXCL10 and the adhesion molecule, ICAM-1. Pretreatment of RPE cells with IFN-beta resulted in inhibition of ICAM-1 production and elimination of CXCL9 production. This treatment did not alter CXCL10 production. Anti-IFN-beta antibody blocked the inhibitory action of IFN-beta. Real time PCR analysis revealed that IFN-beta treatment inhibited gene expression of sICAM-1 and CXCL9. The results indicate a critical role for RPE cell derived IFN-beta in the down-regulation of CXCL9 and ICAM-1 expression in the retina and suggest that the inhibition of CXCL9 is an immuno-suppressive mechanism that protects the retina from excessive inflammation. Recently we identified that IFN-beta can significantly down-regulate CXCL9 gene expression and protein production in a variety of cells, such as, endothelial cells, macrophages, epithelial cell, neuroblastoma and glial cells. Interestingly, this down-regulatory effect occurs for CXCL9 but not for CXCL10 or CXCL11. In order to explore the potential in vivo effects of IFN-beta treatment on CXCL9 and sICAM-1 production, we studied the animal model system of experimental cerebral malaria. CXCL9 and ICAM-1 are know to be key components in the pathogenesis of this disease. We found that IFN-beta treatment resulted in an increase in survival of mice from death following infection with Plasmodium bergie ANKA. This increase in survival was associated with a decrease in the protein expression of CXCL9 and ICAM-1 on brain endothelial cells. Moreover, gene expression of CXCL9 and ICAM-1 in the brain was also significantly inhibited by IFN-beta treatment. These studies clearly demonstrate that IFN-beta treatment in vivo results in significant immuno-suppression associated with inhibition of CXCL9 and ICAM-1. We are now evaluating retinal changes in these animals and evaluating other ocular diseases.