Pillai R G, Beutelspacher S C, Larkin D F P, George A J T
Department of Immunology, Division of Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.
Br J Ophthalmol. 2008 Feb;92(2):259-64. doi: 10.1136/bjo.2007.115923. Epub 2007 Nov 9.
Allograft rejection is the commonest cause of corneal transplant failure and is significantly higher in high-risk patients. Corneal tissue is reported to produce chemokines in response to stress/inflammation. Expression of chemokines is central to the recruitment of leucocytes during inflammatory events. This study was designed to evaluate the effects of surgical trauma or storage conditions on chemokine expression.
Murine corneas were manipulated by incubation in different conditions for up to 24 h, by the addition of endotoxin or by surgical trauma. The ex vivo production of chemokines was assessed using a real-time reverse-transcriptase PCR (RT-PCR) assay to measure mRNA encoding MIP-1alpha, MIP-1beta and MIP-1gamma, MCP1, IP-10, lymphotactin, fractalkine, RANTES, eotaxin, MIG, MIP2 and the cytokine MIF. The expression of RANTES was also determined by ELISA, and the ability of supernatant from corneas on chemotaxis of cells was also determined. Finally, we compared the survival of corneal grafts that had (or had not) been treated with endotoxin.
We found that on incubation in corneal storage medium, expression of mRNA for the majority of these chemokines greatly increased. Upregulation of chemokine mRNA expression was also seen following the mechanical trauma of suture insertion and exposure of the cornea to endotoxin. In the case of mechanical trauma, functional activity of the chemokines was demonstrated using a chemotaxis assay. Orthotopic transplantation of LPS-treated corneas, in which chemokine expression was elevated, resulted in increased infiltration by leucocytes and more rapid rejection of allogeneic grafts.
Our results indicate that ex vivo storage and manipulation of murine corneas can influence the expression of chemokines in corneas, and can result in earlier graft rejection. This may be of importance when considering procedures for manipulation and ex vivo storage of donor corneas prior to transplantation, as well as the surgical procedure itself.
同种异体移植排斥是角膜移植失败最常见的原因，在高危患者中发生率显著更高。据报道，角膜组织在应激/炎症反应时会产生趋化因子。趋化因子的表达对于炎症事件中白细胞的募集至关重要。本研究旨在评估手术创伤或保存条件对趋化因子表达的影响。
将小鼠角膜在不同条件下孵育长达24小时，添加内毒素或进行手术创伤来处理。使用实时逆转录聚合酶链反应（RT-PCR）测定法评估趋化因子的体外产生，以测量编码MIP-1α、MIP-1β和MIP-1γ、MCP1、IP-10、淋巴细胞趋化因子、fractalkine、RANTES、嗜酸性粒细胞趋化因子、MIG、MIP2和细胞因子MIF的mRNA。还通过酶联免疫吸附测定（ELISA）确定RANTES的表达，并测定角膜上清液对细胞趋化性的能力。最后，我们比较了经（或未经）内毒素处理的角膜移植物的存活情况。
我们发现，在角膜保存介质中孵育时，这些趋化因子中大多数的mRNA表达大幅增加。在缝合插入的机械创伤以及角膜暴露于内毒素后，也观察到趋化因子mRNA表达上调。在机械创伤的情况下，使用趋化性测定法证明了趋化因子的功能活性。趋化因子表达升高的经脂多糖（LPS）处理的角膜原位移植，导致白细胞浸润增加和同种异体移植物更快排斥。
我们的结果表明，小鼠角膜的体外保存和处理可影响角膜中趋化因子的表达，并可导致移植物更早排斥。在考虑移植前供体角膜的处理和体外保存程序以及手术程序本身时，这可能具有重要意义。
