Abstract
PurposeTo investigate the protective effects of interleukin-1 receptor antagonist (IL-1ra) gene transfer in a rat model of corneal graft rejection.MethodsWe constructed a recombinant plasmid (pcDNA3.1-hIL-1ra) with high IL-1ra expression in eukaryotic cells. Using a Wistar-SD rat model of corneal graft rejection, we examined the effects of IL-1ra in vivo after cationic polymer jetPEI-mediated nonviral gene delivery. Four groups were included: negative controls (group I, n = 20), pcDNA3.1-hIL-1ra corneal stromal injection (group II, n = 34), pcDNA3.1-hIL-1ra anterior chamber injection (group III, n = 34), and 500 µg/ml IL-1ra protein subconjunctiva injection (group IV, n = 20). IL-1ra expression after transfection was evaluated by real-time polymerase chain reaction (RT-PCR) and western blotting. The rejection indices of corneal grafts were analysed in the different groups. The expression levels of transforming growth factor β1 (TGF-β1), inflammatory chemokines including RANTES, interleukin-1 (IL-1) and the numbers of CD4+ and CD8+ T cells in the grafts were determined by biochemical assays at different time points after corneal transplantation.ResultsVarious degrees of inflammatory cell infiltration and graft neovascularisation were observed by histopathology. After injecting the pcDNA3.1-hIL-1ra plasmid into the cornea, IL-1ra mRNA and protein expression was detected in the corneal stroma and reached a peak on day 3. The graft survival curves indicated that the corneal transparency rates of grafts in the IL-1ra gene-treated group and the IL-1ra protein-treated group were higher compared with the untreated group (P<0.05). During the period of acute rejection, TGF-β1, RANTES, IL-1α and IL-1β levels in the grafts in the IL-1ra treatment groups were lower than the control group (P<0.05). CD4+ and CD8+ T cell counts were reduced significantly in the corneal grafts of groups II, III and IV compared with group I (P<0.05).ConclusionInterleukin-1 receptor antagonist (IL-1ra) gene transfer treatment inhibits graft rejection after corneal transplantation through the downregulation of immune mediators.
Highlights
The cornea is avascular and, as such, is an ‘‘immune privileged’’ site
Jie [9] demonstrated that the subconjunctival injection of large doses of Interleukin-1 receptor antagonist (IL-1ra) could prolong the survival of rat allografts following high-risk corneal transplantation
Reagents/Equipment The following reagents were purchased from the indicated companies: cationic polymer jetPEI in vivo (Poly-plus Transfection, France); rat IL-1a and IL-1b ELISA kits (Bioscience, USA); TRIzol extraction reagent (Invitrogen, USA); total RNA extraction kit (TakaRa, USA); mouse anti-human IL-1ra antibody (Cytoskeleton, USA); biological microscope (Olympus BH-2, Japan); surgical microscope (Topcon-300, Japan); slit-lamp microscope (Topcon, Japan); ultra-microtome (LKB-V, Sweden); gel imaging system (BioRad, USA); and PCR instrument (PE, USA)
Summary
The cornea is avascular and, as such, is an ‘‘immune privileged’’ site. the corneal transplant success rate is the highest among organ transplants [1,2]. Immune responses are the primary cause for graft failure in penetrating keratoplasty [3]. Interleukin-1 (IL-1) is the key cytokine participating in corneal transplant rejection [4]. Antagonising the biological activity of IL-1 could effectively prolong graft survival. Recent studies have indicated [5,6,7,8] that IL-1ra may be effective for treating the rejection of corneal transplants. Jie [9] demonstrated that the subconjunctival injection of large doses of IL-1ra could prolong the survival of rat allografts following high-risk corneal transplantation. IL-1ra clearly inhibits immune and inflammatory reactions, as a natural protein, the cytokine is not stable for clinical applications, and its subconjunctival injection may increase patient suffering
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