Changes in cytokine mRNA levels in experimental corneal allografts after local clodronate-liposome treatment

PURPOSE. Corneal allograft rejection in rats can be prevented by subconjunc tival injections of liposomes containing dichloromethylene diphosphonate (c lodronate-LIP), which selectively eliminate macrophages. In this study, the effect of clodronate-LIP treatment on cytokine mRNA levels in corneal allo grafts was examined. METHODS, AO rats received corneal grafts of PVG rats. Rats were either not treated or injected subconjunctivally with clodronate-LIP on. the day of tr ansplantation and on postoperative days (PODs) 2, 4, 6, and 8. RNA was isol ated from the graft and rim of corneas at different times after transplanta tion and from normal controls. Interleukin <IL)-1 beta, IL-1 receptor antag onist (IL-IRA), IL-2, IL-4, IL-6, IL-10, IL-12p40, tumor necrosis factor (T NF)-alpha, TNF-beta/lymphotoxin (LT), interferon (IFN)-gamma, monocyte chem otactic protein 1 (MCP-1), and macrophage inflammatory protein 2 (MIP-2) mR NA levels were analyzed by semiquantitative reverse transcription-polymeras e chain reaction (RT-PCR). RESULTS. Corneal rejection, observed in all untreated rats by POD 12, was a ssociated with increased mRNA levels of all cytokines investigated in graft s and rims. Clodronate-LIP treatment prevented allograft rejection and stro ngly degreased the levels of IL-1 beta, IL-1RA, IL-2, IL-4, IL-6, IL-10, IF N-gamma, TNF-beta/LT, MCP-1, and MIP-2 mRNA in grafts and IL-1 beta, IL-2, IL-4, IL-6, and IFN-gamma mRNA in rims. Interleukin-12p40 mRNA levels were unaltered in clodronate-treated rats, except for a transient increase in gr afts at POD 3. TNF-alpha mRNA levels were increased by clodronate-LIP in gr afts and rims early after transplantation (PODs 3 and 7). Despite a normal appearance, long-term accepted corneal grafts (POD 100) contained mRNA for IL-10, IL-12p40, TNF-alpha, MCP-1, and MIP-2. CONCLUSIONS. Clodronate-liposome treatment markedly altered the mRNA levels of all cytokines investigated in corneal allografts. These results may exp lain in part the mechanism by which clodronate-LIP treatment prevents corne al allograft rejection.