双重荧光标记法检测人视网膜色素上皮细胞吞噬的功能

目的:检测人视网膜色素上皮(humanretinalpig-mentepithelium,HRPE)细胞特异性及非特异性吞噬动力学,比较二者的不同之处。方法:用双重荧光标记法检测HRPE细胞吞噬动力学,即分别用红色染料硫氰酸罗达明(sulforho-damine,SR)、绿色荧光染料异硫氰酸酯荧光素(FITC)标记HRPE细胞和视杆细胞外节膜盘(rodoutersegments,ROS)。用1×1010个/L的FITC-ROS及自发绿色荧光的乳胶微球(leatexbeads,LB)于37℃孵育培养的正常HRPE细胞,在孵育的不同时间(5min￣48h)去除孵育物,终止吞噬反应。用配有特殊三通广谱吸收波长,长工作距离镜头的荧光显微镜实时,活体观察并记录结合及吞噬的数量。用扫描电镜及激光共聚焦扫描显微镜证明HRPE细胞对LB及ROS结合与吞噬。结果:孵育0.25h时ROS已结合于HRPE细胞表面,0.5h时可见极少数的ROS被HRPE细胞摄入细胞内;之后的孵育过程中被结合及被吞噬的ROS数目不断增加,至孵育18h对ROS的结合达到饱和,但摄入过程继续进行,至孵育24h,对ROS的吞噬达到饱和。当HRPE细胞与LB孵育时,至1.5h结合方启动,6h摄入开始;在观察的48h内被结合及被吞噬的LB数目随时间延长而呈线性增加。结论:HRPE细胞的特异性及非特异性吞噬动力学明显不同,HRPE细胞对ROS的结合及吞噬比其对LB的结合及吞噬发生得更早,而且结合及吞入过程均具有时间饱和性。     

人视网膜色素上皮细胞吞噬过程中MERTK基因mRNA表达水平与蛋白激酶C关系的研究

目的 探讨人视网膜色素上皮(hRPE)细胞吞噬过程中MERTK基因mRNA表达水平与蛋白激酶C(PKC)的相互作用.方法 对照实验研究.采用1×1010个/L视细胞外节膜盘(ROS),于37℃下孵育体外培养的正常hRPE细胞,在孵育的不同时间终止吞噬反应.用双重荧光标记法,检测hRPE细胞的吞噬能力.用液闪记数γ-32P放射活性法,检测不同吞噬时间点的PKC活性.以逆转录聚合酶链反应(RT-PCR)法,检测MERTK基因的mRNA水平变化情况.以PKC激活剂和拮抗剂处理hRPE细胞后,再行MERTK基因mRNA表达水平检测.采用SPSS 13.0统计学软件进行数据分析,实验组与对照组hRPE细胞吞噬ROS能力、PKC活性比较采用Student't检验,MERTK基因mRNA表达水平比较采用单因素方差分析.结果 ROS孵育后的hRPE细胞结合及吞入ROS的数量逐渐增多,至24 h时,hRPE细胞吞噬ROS的数量达到高峰,为(2.85±0.11)×106个,与对照组(0.00±0.00)×106个比较,差异有统计学意义(t=47.64,P＜0.05).ROS孵育后的hRPE细胞胞质PKC活性降低,至24 h时,PKC活性降至最低,细胞质的PKC活性为(151.13±17.67)nmol·g-1·min-1,细胞膜的PKC活性为(152.45±64.83)nmol·g-1·min-1;对照组细胞质的PKC活性为(329.63±14.26)nmol·g-1·min-1,细胞膜的PKC活性为(467.67±68.87)nmol·g-1·min-1;两组间PKC活性比较,差异有统计学意义(细胞质:t=89.66,P＜0.05;细胞膜t=10.31,P＜0.05).在hRPE细胞与ROS不同时段的孵育过程中,MERTK基因mRNA均呈现出高表达状态,孵育至90 min时,MERTK基因mRNA表达灰度值为1.8853±0.0077,与对照组灰度值0.7246±0.0062相比,差异有统计学意义(F=16 060.2167,P＜0.05);孵育至24 h时,MERTK基因mRNA表达灰度值为0.5946±0.0082,与对照组灰度值0.3343±0.0064比较,差异有统计学意义(F=919.8421,P＜0.05).上调hRPE细胞的PKC活性后,再行不同时段的ROS孵育,短时与长时孵育的MERTK基因mRNA表达灰度值均低于对照组(短时孵育:F=17 142.2331,长时孵育:F=1886.4614;P＜0.05).拮抗hRPE细胞的PKC活性后,再行不同时段的ROS孵育,30 min内MERTK基因mRNA表达灰度值为4.4670±0.0092至5.7034±0.0095范围,均高于对照组灰度值0. 9117±0.0021(F=199 012.9138,P＜0.05).结论 PKC的低活性和MERTK基因mRNA的高表达可促进hRPE细胞吞噬ROS的过程.PKC活性和MERTK基因mRNA表达水平作为上游调控信号,通过两条不同的信号通路,以负相关的方式调节hRPE细胞吞噬ROS的功能.     

视网膜色素上皮细胞吞噬功能与MERTK-Ras-肌球蛋白通路关系的研究

目的：：研究视网膜色素上皮( retinal pigment epithelium, RPE)细胞吞噬功能与 MERTK 受体及其下游信号通路Ras-MEK-MLCK-肌球蛋白的关系。方法：用视细胞外节膜盘( rod outer segments,ROS)于37℃孵育离体培养的3~5代C57小鼠RPE细胞,在孵育0、30、60、120、180、240 min终止吞噬反应。双重荧光标记法检测RPE细胞吞噬动力学;以MERTK及Ras抗体、磷酸化MEK及MLCK抗体应用Western-Blot方法检测不同孵育时间(30、60、120、180 min ) MERTK、Ras、MEK 及 MLCK的激活状态;以瞬时转染质粒方法抑制Ras及MERTK基因表达后,再次以 Western-Blot 方法检测对应时间MERTK-Ras通路激活状态及吞噬功能的变化。结果：C57小鼠RPE细胞吞入ROS发生在孵育30min,并在3h达到饱和。在吞噬过程中,随着孵育时间的延长, RPE细胞MERTK、Ras、MEK和MLCK的蛋白表达水平不断增多(与对照组相比,P<0.05)。 Ras及MERTK干扰后的RPE细胞与ROS共孵育(30、60、120、180min)的全过程中,MERTK、Ras、MEK 和 MLCK 的蛋白表达及 ROS 的吞入数量始终维持较低水平,仅在孵育180 min 见到少量ROS吞入;与未转染 RPE 细胞相比, siRas-RPE 细胞及siMERTK-RPE细胞与ROS共孵育120、180min时,MERTK、Ras、MEK及MLCK的蛋白表达量均明显减少(P<0.05)。结论：Ras-MEK-MLCK-肌球蛋白通路是鼠RPE细胞吞噬过程中MERTK受体激活的下游信号通路。     

视网膜色素上皮细胞吞噬功能与信号转导系统的关系

视网膜色素变性(RP)是一种严重的致盲性眼病,其确切病因不明。除了遗传因素外,近年的研究表明,视网膜色素上皮(RPE)细胞吞噬功能异常是导致视觉功能障碍的主要原因。本文简单介绍视网膜色素上皮细胞特异性吞噬功能,详细分析了IP3、PKC、cAMP、cGMP、PTK及Ca2+对RPE细胞吞噬的调节作用,综述了RP发病机制中涉及RPE细胞吞噬功能与信号转导系统的研究进展。     

细胞内Ca2+及MERTK基因在人视网膜色素上皮细胞吞噬功能中的作用

目的 观察细胞内Ca2+及MERTK基因在人视网膜色素上皮（RPE）细胞的吞噬功能中所起的作用及二者的相互关系。方法 用视细胞外节膜盘（ROS）于37℃孵育培养的RPE细胞,在孵育不同终止吞噬反应。双重荧光标记法检测 RPE细胞吞噬动力学;钙离子荧光探针负载法在荧光显微镜下测定RPE细胞内Ca2+的变化;半定量逆转录聚合酶链反应（RT PCR）法检测相应时间点MERTK 基因表达的变化及施加Ca2+激活剂 （A23187载体）或拮抗剂（verapamile）后MERTK 基因表达的变化。结果 RPE细胞与ROS孵育过程中,ROS结合于RPE细胞表面发生在15 min时,RPE细胞吞噬ROS在24 h时达到饱和。细胞内Ca2+在孵育15 min 时升高,并在24 h内保持高水平;MERTK 基因表达在RPE细胞与ROS孵育5 min时增强,并在全部孵育过程中呈现出高表达状态;以A23187载体开放钙通道升高RPE细胞内Ca2+后,MERTK 基因信使核糖核酸（mRNA）水平升高,并呈剂量依赖性。经A23187预处理后的孵育实验中所检测到的MERTK mRNA水平除3 h 这一时间点外均高于对照组; verapamile拮抗RPE细胞内Ca2+后,MERTK 基因表达明显下降并呈剂量依赖性;以verapamile预处理后继续与ROS孵育,所检测到的MERTK基因在24 h内均低于对照组。结论 MERTK基因及细胞内Ca2+发挥着维持RPE细胞吞噬过程的重要作用,MERTK作为上游调控信号启动下游的细胞内Ca2+信号来维持RPE细胞对ROS的摄入过程。     

视网膜色素上皮细胞吞噬功能与信号转导系统的关系

视网膜色素变性(RP)是一种严重的致盲性眼病，其确切病因不明。除了遗传因素外，近年的研究表明，视网膜色素上皮(RPE)细胞吞噬功能异常是导致视觉功能障碍的主要原因。本简单介绍视网膜色素上皮细胞特异性吞噬功能，详细分析了IR、PKC、cAMP、cGMP、PTK及Ca^2 对RPE细胞吞噬的调节作用，综述了RP发病机制中涉及RPE细胞吞噬功能与信号转导系统的研究进展。     

视网膜下液致视网膜色素上皮细胞和神经胶质细胞增殖过程中PKC活性的变化

目的:研究视网膜下液(SRF)能否引起体外培养的人视网膜色素上皮(RPE)和神经胶质(RG)细胞发生增殖及在其增殖过程中是否出现蛋白激酶 C 的激活和转位, 来探讨RPE 和 RG 细胞增殖与其细胞中 PKC 信号系统变化的关系以及 PKC 抑制剂的作用。方法:实验对象为体外培养的 RPE 和 RG 细胞;刺激因素为提取 PVR 分级是 B、C 级两级患者的 SRF 和来自角膜移植后的供体眼球所提供的正常玻璃体成分;PKC 特异激活剂佛波酯(PMA)为阳性对照;DMEM 培养液作为空白对照。用 3H- 胸腺嘧啶脱氧核苷(3H- TdR)掺入法测定各自的RPE 和 RG 细胞增殖情况。用 B、C 级 SRF、正常玻璃体成分、PMA、DMEM 培养液分别在不同的时间刺激 RPE 和 RG细胞, 通过细胞裂解和离心获取细胞质和细胞膜蛋白粗提液,用同位素 32P 标记和液体闪烁计数法检测细胞质和细胞膜 PKC 活性水平。选用 PKC 抑制剂地喹氯铵预处理各组细胞后,再分别观察各组 RPE 和 RG 细胞中 PKC 活性表达水平及增殖情况。结果:用 B、C 级 SRF 和 PMA 处理过的 RPE 细胞出现高增殖;SRF 和 PMA 都可以激活 RPE 和 RG 细胞质中的 PKC,并使其由胞质向胞膜转位,但 SRF 作用于 RPE 和 RG 细胞时,胞膜上 PKC 活性峰值出现的时间较 PMA 明显延长。其中,B 级 SRF 作用于 RPE 和 RG 细胞时,胞膜上 PKC 活性峰值出现的时间较 C 级长且峰值低,增殖程度也低;正常玻璃体成分和 DMEM 培养液组没有出现 PKC 活性变化和高增殖。用 PKC 特异抑制剂预处理各组细胞后,没有出现PKC 活性和细胞增殖的改变,组间无显著性差异,P>0.05。结论:SRF 可促进 RPE 和 RG 细胞增殖,RPE 和 RG 细胞中的 PKC 是以激活和转位方式参与细胞增殖的过程;使用PKC 特异抑制剂可阻止此过程发生。     

玻璃体条件培养液对人视网膜色素上皮细胞生长活性及细胞周期的影响

目的:探讨在含有人玻璃体的条件培养液作用下体外培养的人视网膜色素上皮(HRPE)细胞形态学、细胞生长活性及细胞周期的改变.方法:将HRPE细胞培养在含100,250,500,1 000mL/L人玻璃体的DMEM条件培养液(VCM).用倒置显微镜观测细胞的形态学改变,分别用MTT、流式细胞术检测细胞的生长活性及细胞周期的改变.结果:不同体积分数的VCM使HRPE细胞的形态发生了改变,HRPE细胞出现了多核及更早的脱色素,类似成纤维细胞表型的生长状态.含100,250及500mL/L玻璃体体积的VCM处理后HRPE细胞的增长活性与对照组相比存在显著性差异(P均＜0.01),其中含250mL/L玻璃体的VCM刺激HRPE细胞增生的能力最强.VCM促使更多的HRPE细胞进入了DNA合成期(S期,6%～13%),含250与500mL/L玻璃体体积的VCM使HRPE细胞出现了异倍体的改变.结论:一定体积分数的VCM使HRPE细胞的S期比例增加,细胞增生能力增强.     

三氟啦嗪对视网膜色素上皮细胞cAMP-PKA信号系统的影响

目的 :检测TFP对培养的豚鼠RPE细胞cAMP浓度和PKA活性的影响。探讨CaM被抑制时 ,与cAMP -PKA信号系统相关性及在RPE细胞增殖调控中的作用。从信号传导角度探讨TFP抑制RPE细胞迁移和增殖的机制。方法 :1 应用12 5I -cAMP试剂盒 ,采用放免法测定 10umol/LTFP作用不同时间RPE细胞内cAMP浓度的变化。 2 应用液闪法测定 10umol/LTFP作用不同时间后 ,RPE细胞PKA活性的变化。结果 :1 TFP对豚鼠RPE细胞cAMP浓度的影响 :统计结果表明 ,与对照组cAMP值 ( 0 410 9± 0 0 931pmol/5 0ul)相比 ,TFP作用 5分钟内即引起cAMP快速的升高达 ( 0 80 5 1± 0 5 5 3pmol/5 0ul) (P <0 0 1) ,直到 3小时仍维持较高的水平 ( 0 72 85±0 0 2 94pmol/5 0ul) (P <0 0 5 )。 2 TFP对豚鼠RPE细胞PKA活性的影响 :统计结果表明 ,与对照组PKA活性值 ( 4 5 5 73±2 41cpm/mgprotiein)相比 ,TFP作用 5min内即引起PKA活性快速上升达 ( 135 6 5± 1 81cpm /mg protein) (P <0 0 1) ,直至3h仍维持较高水平 ( 6 5 95± 5 0 1cpm/mg protein) (P <0 0 5 )。结论 :1 在TFP抑制RPE细胞增殖过程中 ,cAMP -PKA信号系统呈上调趋势。 2 在RPE细胞增殖抑制过程中 ,Ca2 + -CaM系统与cAMP -PKA信号系统存在着拮抗关系。 3 在TFP引起cA     

烟焦油对体外人视网膜色素上皮细胞的损害

目的:研究烟焦油对体外培养的人视网膜色素上皮细胞(human retinal pigmentepithelium,HRPE)超微形态结构的影响,以及趋化因子MCP-1的细胞外表达水平。方法:将体外培养的HRPE分为空白对照组、二甲亚砜(dime-thylsulfoxide,DMSO)对照组和焦油实验组,其中焦油实验组中焦油浓度分别为1,10,50mg/L,经48h培养后,与空白对照组及DMSO对照组进行比较,使用倒置显微镜及透射型电子显微镜观察HRPE的形态以及超微结构变化,并用ELISA法选择检测各组培养上清中细胞因子MCP-1的表达量。结果:倒置显微镜显示焦油实验组随着焦油浓度的增大,发生变性坏死的HRPE细胞数量逐渐增多,并且细胞的形态发生明显改变;电镜显示焦油实验组随着焦油浓度的增大,RPE细胞形态扁平,微绒毛减少,胞质稀疏、减少,细胞核细长、核染色质稀疏,染色质浓缩,可见空泡,细胞呈萎缩状态;ELISA结果显示随着焦油浓度的增大,RPE分泌至胞外的MCP-1逐渐减少,差异有统计学意义(P<0.05)。结论:烟焦油可改变视网膜色素上皮细胞正常的形态和生理功能,造成色素上皮细胞的严重损害。     

ROS ingestion by RPE cells is turned off by increased protein kinase C activity and by increased calcium

The activation of protein kinase C (PKC) by phorbol myristate acetate (PMA) rapidly inhibits the phagocytosis of rod outer segments (ROS) by cultured rat retinal pigment epithelial (RPE) cells. PMA, at a concentration between 3.3 and 10 nM, blocks ROS ingestion by 50%, but does not inhibit the binding of ROS. The Ca2+ ionophore, A23187, also inhibits ROS phagocytosis, with an IC50 of about 0.5-1.0 microM and interferes with the ability of RPE cells to bind ROS. The effects of both of these drugs are reversible after drug washout. When PMA and A23187 are applied to cells consecutively, the effects are additive. These results suggest either that PMA and A23187, act upon the same proteins in the pathway which controls ROS ingestion, or that A23187 affects phagocytosis at the ROS binding level, while PKC affects steps further along the ingestion path. The effect of this process is to shut down the ingestion of ROS, as is seen during the prolonged feeding of ROS to RPE cells in culture.     

Kinetic studies of rod outer segment binding and ingestion by cultured rat RPE cells

Retinal pigment epithelial (RPE) cells selectively phagocytize rod outer segments (ROS) by a process which may be mediated by specific cell surface receptors. We have studied the kinetics of this process using rat RPE cells grown in tissue culture. By cooling RPE cells to 17 degrees C, the binding and ingestion phases of phagocytosis can be separated. Maximum ROS binding with minimum ingestion occurs at 17 degrees C; above 17 degrees C the rate of ingestion increases markedly. Thus it is possible to measure the kinetics of ROS binding to RPE cells at 17 degrees C and of ROS ingestion at 37 degrees C. At 17 degrees C, ROS binding is saturable, both with respect to time and to ROS concentration. ROS ingestion saturates after 4 hr of incubation at 37 degrees C, after which the cells are refractory to further ROS ingestion for 1-2 hr. During this recovery period, rapid digestion of the internalized ROS takes place. Cycloheximide, when present at a concentration (2 x 10(-5) M) which inhibits protein synthesis by 92%, has no effect on ROS phagocytosis or on the recovery of ROS ingestion at 37 degrees C. This suggests that if receptors mediate the ingestion of ROS by RPE cells, they are not degraded after the ROS are internalized. Dystrophic rat (RCS-p+) RPE cells exhibit normal binding, but very limited ingestion of ROS at 37 degrees C. The rate and amount of ROS binding to these cells at 37 degrees C is comparable with that occurring to normal cells at 17 degrees C. These observations support the hypothesis that there are a limited number of receptors which are specific for ROS binding on the surface of normal and dystrophic rat RPE cells.     

Effect of glycoconjugates on rod outer segment phagocytosis by retinal pigment epithelial explants in vitro assessed by a specific double radioimmunoassay procedure

Rod outer segment (ROS) phagocytosis by explanted bovine retinal pigment epithelium (RPE) was evaluated by a procedure using an indirect double radioimmunoassay which distinguished between ROS attached to the RPE cell surface and those which had been ingested. This approach has been used to investigate the effect of a variety of glycoconjugates on the phagocytic process. Inclusion of the glycosaminoglycans (GAGs) chondroitin sulphate type-A (CS-A) and type-C (CS-C), hyaluronic acid (HA) or dermatan sulphate (DS) in the incubation medium significantly inhibited the ingestion phase of ROS phagocytosis, whereas the binding phase was inhibited to a lesser extent. The interphotoreceptor matrix (IPM), containing these GAGs as part of proteoglycans, also had an inhibitory effect on phagocytosis. The free monosaccharides mannose, fucose and galactose all stimulated the ingestion of ROS by RPE cells. These findings support the suggestion that glycoconjugates may have a physiological role in the photoreceptor renewal process.     

Surface modification of retinal pigment epithelial cells: effects on phagocytosis and glycoprotein composition

Proteases have been used as a tool to investigate the role of cell-surface molecules of cultured retinal pigment epithelial cells (RPE) in the phagocytosis of rod outer segments (ROS). Proteolytic digestion of RPE cells by pronase, thermolysin and Staphylococcus aureus V8 protease (V8 protease) inhibited the phagocytosis of ROS without affecting the viability of the RPE cells. A particular feature of RPE cell proteolysis was that those macromolecules responsible for ROS ingestion were susceptible, while those macromolecules that mediated ROS binding were resistant to cleavage by all three proteases. By taking advantage of this phenomenon, ROS were used as affinity particles to obtain a plasma membrane-enriched fraction of RPE cells before and after proteolytic digestion. All three proteases partially or completely removed several glycoproteins from the cell surfaces. Removal of these glycoproteins was correlated with a loss in phagocytic ability by RPE cells. Two high-molecular-weight (MW) glycoproteins of MWs 160,000 and 214,000 were consistently removed by all proteases tested. Protease-treated RPE cells restored their phagocytic capabilities and normal glycoprotein composition within 24 hr after proteolytic treatment. These data suggest that glycoproteins located on the surfaces of RPE cells may be involved in mediating the phagocytosis of ROS by these cells.     

Retinal pigment epithelium of new-born rats is capable of phagocytosis of rod outer segments in vitro

The ability of retinal pigment epithelium (RPE) cells of rats at 0, 1, 3, 4, 5, 8, 10, 12 and 15 days after birth towards phagocytosis of rod outer segments (ROS) has been studied in short-term explant culture of the scleral part of the eye. The neural retina was removed before cultivation. The suspension of ROS isolated from the retina of adult rats was layered on top of RPE cells. The phagocytic activity was evaluated by visualization of phagosomes in the cytoplasm of RPE cells after 4-5 hr incubation, using electron microscopy. It has been shown that RPE cells at all the stages studied, including new-born rats, are capable of ROS phagocytosis.     

Nitric oxide as a second messenger in phagocytosis by cultured retinal pigment epithelial cells

PURPOSE: To investigate a possible role of the nitric oxide (NO)-cGMP signal transduction system in phagocytosis of rod outer segments (ROS) by cultured retinal pigment epithelial (RPE) cells. METHODS: Primary cultures of RPE cells from 10-day-old Brown Norway rats were used to study the phagocytosis of ROS by these cells. Phagocytosis of ROS was evaluated with or without an inhibitor of nitric oxide synthase (NOS), N(G)-nitro-L-arginine (L-NNA), and the reverse effects of L-NNA by L-arginine and 8-bromo-cGMP on phagocytosis were also studied. NO-associated cGMP production by RPE cells was monitored during phagocytosis using L-NNA. NOS activity was assayed in RPE cells and ROS to locate the source of NO. RESULTS: Phagocytosis of ROS was inhibited by L-NNA but not by D-NNA. L-NNA inhibited the ingestion in a dose-dependent manner, but not the binding of ROS. The inhibition was reversed by L-arginine and also by an NO donor, SIN-1. RPE cells challenged with ROS showed increased cGMP activity, which was significantly reduced by L-NNA and again restored by an overdose of L-arginine. NOS activity was found in RPE cells but not in ROS. CONCLUSIONS: Our data show that cGMP plays a role in the ingestion phase of ROS phagocytosis by RPE cells via a cGMP second-messenger system.     

Phagocytosis of light- and dark-adapted rod outer segments by cultured RPE cells: a reassessment

Rod outer segments (ROS) isolated from adult rat retinas are phagocytized by cultured rat retinal pigment epithelial (RPE) cells. Using a double immunofluorescent labeling procedure, we have compared the binding and ingestion of ROS isolated at different times of the day. After 2 hr of incubation, approximately 98% of the ROS are ingested, while 2% are still attached to the RPE cell surface, irrespective of the time of day or lighting conditions under which the ROS are isolated. These findings differ from those reported earlier, using a radioactive method for quantitating ROS phagocytosis (Hall, 1978).     

The phagocytosis of ROS by RPE cells is not inhibited by mannose-containing ligands

We have examined the ability of mannose and the mannose-rich ligands, mannan and mannosylated BSA, to inhibit the phagocytosis of rod outer segments (ROS) by cultured rat retinal pigment epithelial (RPE) cells. Mannose, at concentrations up to 0.25 M, had no effect on either the binding or the ingestion of ROS. At concentrations above 0.25 M, the cells were rounded and showed detachment from the substrate, and phagocytosis was markedly inhibited. Neither mannan (2 mg ml-1), nor mannosylated BSA(0.8 mg ml-1), affected the phagocytosis of ROS. These results suggest that the phagocytosis of ROS is probably not mediated by a mannose receptor on the surface of the RPE cells.