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础作用。其结构和功能的改变可导致光感受器和视力的丧失。RPE细胞作为眼后极内一种重要的免疫调节细胞，我们进行了广泛的研究。我们对RPE细胞的研究可分为三大类：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。本项目主要研究细胞因子与免疫系统细胞和眼微环境细胞相互作用的途径。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）和与视网膜血管炎患者相关的可能的生物标志物。tlr是先天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、突发的反应性，旨在将病原体限制在感染部位。这种反应性的爆发被细胞因子、趋化因子和粘附分子的释放所突出。在视网膜内，不受控制的炎症爆发本身会导致细胞损伤。因此，极有可能产生下调力来限制免疫病理损伤。我们假设RPE细胞主要通过TLR信号传导，其次通过自身刺激产生ifn - β，这是这种限制力的关键组成部分。本研究的总体目的是确定ifn - β产生的过程，并确定ifn - β通过其抗病毒作用、免疫抑制作用和抗增殖作用保护视网膜的机制。用Poly I:C处理或用RNA病毒感染的RPE细胞产生ifn - β。动力学研究表明，ifn - β水平在48小时内持续增加，这与IRF-7基因表达的上调有关，IRF-7是ifn - β产生的已知正反馈分子。微阵列分析显示，在ifn - β处理的细胞中，22283个基因中有480个基因上调或下调幅度大于2倍。我们假设视网膜中TLR信号传导过程中ifn - β的诱导是一种免疫抑制因子，用于限制免疫病理损伤。RPE细胞的细胞因子激活导致趋化因子CXCL9和CXCL10以及粘附分子ICAM-1的产生。用ifn - β预处理RPE细胞可抑制ICAM-1的产生并消除CXCL9的产生。这种处理没有改变CXCL10的产量。抗ifn - β抗体阻断ifn - β的抑制作用。Real time PCR分析显示，ifn - β处理抑制了sICAM-1和CXCL9基因的表达。这些结果表明，RPE细胞来源的ifn - β在下调视网膜中CXCL9和ICAM-1表达中起关键作用，并表明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。

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 hypothesize that IFN-beta produced by RPE cells primarily by TLR signaling and secondarily by auto-stimulation is a critical component of that limiting force. The general aim of this study is to identify processes by which IFN-beta is produced and to characterize mechanisms by which IFN-beta can protect the retina through its antiviral actions, immunosuppressive actions and anti-proliferative actions. RPE cells treated with Poly I:C or infected with an RNA virus produce IFN-beta. Kinetic studies revealed that IFN-beta levels continue to increase over a 48 h period and this was associated with the up-regulation of IRF-7 gene expression, a known positive feedback molecule for IFN-beta production. Microarray analysis revealed that in IFN-beta treated cells, 480 genes out of 22,283 genes were up or down-regulated by greater than 2 fold. We hypothesize that IFN-beta induction during TLR signaling in the retina is an immunosuppressive factor produced to limit immunopathologic damage. 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. Retinal vasculitis is a major component of ocular inflammation that plays a role in retinal tissue damage in patients with idiopathic uveitis and Behcets disease. We found that levels of selected soluble adhesion molecules and cytokines were altered in the serum of patients with retinal vasculitis. Type 1 IFN, IFN-alpha and IFN-beta were not detectable in normal individuals but were detected in up to 39% of the serum from Behcets patients and 47% of uveitis patients. Our in vitro findings further demonstrated that the retinal vascular endothelial cell could be activated throuth TLR3 to produce sE-selectin, sICAM-1 and IFN-beta. Further analysis of innate immune signaling may prove to be a novel target for future studies on pathogenic mechanisms and therapeutic approaches in retinal vasculitis.