Cell-associated human retinal pigment epithelium interleukin-8 and monocyte chemotactic protein-1: Immunochemical and in-situ hybridization analyses
Cell-associated human retinal pigment epithelium interleukin-8 and monocyte chemotactic protein-1: Immunochemical and in-situ hybridization analyses
复制标题
DOI：
10.1006/exer.1997.0380
复制
发表时间：
1997-12-01
影响因子：
3.4
通讯作者：
Elner, SG
中科院分区：
文献类型：
--
作者：
Elner, VM;Burnstine, MA;Elner, SG
人视网膜色素上皮（RPE）细胞响应于促炎细胞因子而分泌趋化因子，白细胞介素-8（IL-8）和单核细胞趋化蛋白-1（MCP-1）。在这项研究中，我们（1）检查了人RPE IL-8和MCP-1分泌的效率，（2）确定了人RPE细胞条件培养基和细胞提取物中分别归因于IL-8和MCP-1的中性粒细胞和单核细胞趋化活性的量，和（3）评估了免疫组织化学和原位杂交用于检测由精氨酸刺激的人RPE细胞产生的趋化因子的灵敏度。用不同生理浓度的白细胞介素-1 β刺激的人RPE细胞的条件培养基和提取物（IL-1 β）（0.2-20 ng ml（-1）），肿瘤坏死因子（肿瘤坏死因子-α）（0.2-20 ng ml（-1））或干扰素-γ（IFN-γ）（10-1000 U ml（-1）），以比较IL-8和MCP-1在24小时内的不同时间点的分泌和细胞相关水平。1均由促炎细胞因子处理的人RPE细胞有效分泌。在IL-1 β或TNF-α刺激后，观察到大量的剂量和时间依赖性的IL-8的RPE分泌，但细胞相关的IL-8仅在高剂量（20 ng/ml）IL-1 β刺激后才可检测到，并且占诱导的总IL-8的不到1%。在IL-1 β> TNF-α> IFN-γ刺激后也观察到剂量和时间依赖性的RPE细胞MCP-1分泌，平均4%的总MCP-1保留在RPE内。生物测定表明，中性粒细胞和单核细胞趋化活性的条件培养基刺激的RPE细胞，但不是在人RPE细胞提取物。特异性抗IL-8或抗MCP-1抗体对条件培养基诱导的趋化性的抑制表明IL-8和MCP-1负责大多数HRPE衍生的嗜中性粒细胞（> 60%）和单核细胞（53-57%）趋化活性，使用原位杂交，在IL-1 β、TNF-α刺激的RPE细胞和MCP-1刺激的RPE细胞中容易地检测到IL-8 mRNA。1 mRNA在IL-1 β> TNF-α>或IFN-γ刺激的细胞内容易显现。检测IL-8的免疫组织化学仅在暴露于高剂量IL-1 β（20 ng ml-1）8或24 h的RPE细胞中呈阳性，并且较弱。RPE细胞中MCP-1的免疫组织化学染色更强烈，并且在用IL-β、TNF-α或IFN-γ刺激的RPE细胞内可见。这项研究表明：（1）RPE细胞在用促炎细胞因子刺激时有效地分泌IL-8和MCP-1;（2）分泌的IL-8和MCP-1占人RPE嗜中性粒细胞和单核细胞趋化活性的大部分;（3）原位杂交容易检测细胞因子刺激的RPE细胞中的IL-8和MCP-1 mRNA;（4）免疫组化显示在细胞因子刺激的RPE细胞中细胞相关的MCP-1，但仅最小的细胞相关的IL-8。(C)出版社：Academic Press Limited。
Human retinal pigment epithelial (RPE) cells secrete chemokines, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in response to pro-inflammatory cytokines. In this study we (1) examined the efficiency of human RPE IL-8 and MCP-1 secretion, (2) determined the amount of neutrophil and monocyte chemotactic activity in human RPE cell conditioned media and cell extracts that is attributable to IL-8 and MCP-1, respectively, and (3) assessed the sensitivity of immunohistochemistry and in situ hybridization for detecting chemokine production by cytokine-stimulated human RPE cells. Conditioned media and extracts from human RPE cells stimulated with various physiologic concentrations of interleukin-1 beta (IL-1 beta) (0.2-20 ng ml(-1)), tumor necrosis factor (TNF-alpha) (0.2-20 ng ml(-1)) or interferon-gamma (IFN-gamma) (10-1000 U ml(-1)) were examined to compare secreted and cell associated levels of IL-8 and MCP-1 at various time points up to 24 hr.ELISA demonstrated that IL-8 and MCP-1 are both efficiently secreted by pro-inflammatory cytokine treated human RPE cells. Substantial dose-and time-dependent RPE secretion of IL-8 was observed following stimulation with IL-1 beta or TNF-alpha, but cell associated IL-8 was detectable only after high dose (20 ng ml(-1)) IL-1 beta stimulation and comprised less than 1% of the total IL-8 induced. Dose- and time-dependent RPE cell MCP-1 secretion was also observed following IL-1 beta > TNF-alpha > IFN-gamma stimulation, with an average of 4% of the total MCP-1 retained within RPE. Bioassays demonstrated neutrophil and monocyte chemotactic activity in conditioned media from stimulated RPE cells, but not in human RPE cell extracts. Inhibition of conditioned media-induced chemotaxis by specific anti-IL-8 or anti-MCP-l antibodies demonstrated that IL-8 and MCP-1 were responsible for the majority of HRPE-derived neutrophil (> 60%) and monocyte (53-57%) chemotactic activity, respectively.Using in situ hybridization IL-8 mRNA was readily detected within IL-1 beta > TNF-alpha stimulated RPE cells and MCP-1 mRNA easily visualized within IL-1 beta > TNF-alpha > or IFN-gamma stimulated cells. Immunohistochemistry to detect IL-8 was positive only in RPE cells exposed to high dose IL-1 beta (20 ng ml-l) for 8 or 24 hr and was weak. Immunohistochemical staining for MCP-1 in RPE cells was more intense and was visualized within RPE cells stimulated with IL-beta, TNF-alpha, or IFN-gamma. This study demonstrates that: (1) RPE cells efficiently secrete IL-8 and MCP-1 upon stimulation with pro-inflammatory cytokines; (2) secreted IL-8 and MCP-1 account for the majority of human RPE neutrophil and monocyte chemotactic activity; (3) in situ hybridization readily detects IL-8 and MCP-1 mRNA in cytokine stimulated RPE cells; and (4) immunohistochemistry demonstrates cell-associated MCP-1 in cytokine stimulated RPE cells, but only minimal cell-associated IL-8. (C) 1997 Academic Press Limited.