内毒素诱导的大鼠葡萄膜炎的巨噬细胞中Toll样受体4的表达

目的 探讨在内毒素诱导的Wistar大鼠葡萄膜炎中Toll样受体4(TLR4)阳性细胞与虹膜组织中巨噬细胞的动态变化和分布.方法 实验研究.Wistar大鼠50只,用随机数字法随机分为5组,每组10只,分别为正常对照(0 h)组、6 h组、12 h组、24 h组及48 h组.除0 h组外其余各组均足垫部注射霍乱弧菌内毒素200μg,注射后于裂隙灯显微镜下观察双眼前节炎症反应变化.按实验分组于0、6、12、24、48 h处死大鼠.取虹膜一睫状体及脉络膜组织.通过葡萄膜铺片免疫组织化学方法检测TLR4和巨噬细胞的标记CD163的表达.人工计数虹膜中TLR4~+与CD163~+的细胞并计算细胞密度,计算圆形和多形性的CD163~+细胞占所有CD163~+细胞的百分比.进一步采用免疫荧光双标记检测TLR4和CD163共表达的情况.通过单因素方差分析分别对大鼠虹膜内阳性细胞密度以及圆形、多形性CD163~+细胞的百分比进行统计学检验.结果 正常大鼠虹膜睫状体组织不表达TLR4.6 h组有2只大鼠虹膜内可见少量TLR4~+细胞,12～48 h组所有大鼠虹膜内TLR4~+细胞明显增多(F=167.2,P＜0.001),虹膜内TLR4~+细胞密度分别为(506.1±39.5)个/mm~2(12 h组)、(492.3±54.5)个/mm~2(24 h组)及(663.8±150.2)个/mm~2 (48 h组).在注射LPS后12～48 h期间TLR4~+细胞形态无明显变化.0～48 h组大鼠虹膜内均有CD163~+细胞,0 h组圆形和多形性CD163~+细胞百分比为13%,12～48 h组其百分比约为80%,且圆形细胞主要位于虹膜基质层.免疫荧光双标记可见TLR4和CD163的共表达,TLR4位于细胞膜,CD163位于细胞质.5组大鼠脉络膜内均未见TLR4表达.结论 内毒素诱导的大鼠葡萄膜炎中虹膜内TLR4表达增高,部分虹膜固有巨噬细胞表达TLR4.TLR4可能在葡萄膜炎的发生发展中起一定作用.

Toll-like receptor 4 expression in macrophages in endotoxin-induced uveitis in Wistar rats

Objective To investigate the dynamics and distribution of toll-like receptor 4 (TLR4) in uvea-resident tissue macrophages during endotoxin-induced uveitis (EIU) in Wistar rats.Methods Fifty Wistar rats were randomly divided into five groups (n = 10 per group) based on the following time points:before LPS injection (0 h,control group) and 6,12,24,and 48 h after LPS injection.All the rats (except the control group) received a footpad injection of 200 μg of vibrio cholera lipopolysaccharide (LPS).The intensity of anterior segment inflammation was evaluated after the LPS injection.Ten rats each were killed before LPS injection and 6,12,24,48 h after injection.The iris-ciliary body complex and choroid from each eye were removed and cut into segments.Immunohistochemical localization of TLR4 and a resident tissue macrophage marker,cluster of differentiation 163 (CD163) ,was performed on whole mount isolated iris-ciliary body complexes and choroids.TLR4~+ and CD163~+ cells in the iris were counted,and the cell density (cells/mm~2) was calculated.For CD163~+ cells,the percent of round pleiomorphic cells in positive staining cells was calculated.The distribution patterns and the phenotypes of cells expressing these two proteins were further characterized by double-labeled immunofluorescence studies.Positive cell density and the percent of round-pleiomorphic CD163~+ cells were analyzed by one-way ANOVA followed by least significant difference procedure (LSD) tests for multiple comparisons.Results The iris-ciliary body complex did not express TLR4 in normal rats.Six h after the IPS injection,a small number of TLR4~+ cells were detected in the irides of two rats.The density of TLR4~+ cells in the iris was (506.1 ± 39.5) cells/mm~2(12 h),(492.3 ±54.5) cells/mm~2(24 h) and (663.8 ± 150.2) cells/mm~2(48 h),respectively.The number of TLR4~+ cells significantly increased 12,24 and 48 h after the injection (F = 167.2,P ＜0.001,ANOVA).No changes of morphology of TLR4~+ cells were detected 12-48 h after the injection.CD163 was expressed in the uvea in all rats.CD163~+ round tissue macrophages were present at all time periods (0-48 h).The proportion of these cells was 13% at 0 h and increased to approximately 80% at 12-48 h.These changes occurred mainly in the macrophages located in the stroma bordering the iris endothelial layer.Double-labeling immunofluorescence demonstrated the co-expression of TLR4 and CD163 in round stroma cells with TLR4 protein located at the cell membrane and CD163 protein in the cytoplasm.TLR4~+ cells could not be detected in choroid in any of the rats.Conclusions Iris tissue macrophages expressed TLR4 and TLR4~+ cells increased in the iris during EIU.It indicates that TLR4 may play an important role in the pathogenesis of acute anterior uveitis.