Graft cytokine mRNA activity in rat single lung transplants by reverse transcription-polymerase chain reaction: Effect of cyclosporine

Intragraft levels of cytokine mRNA were studied in an orthotopic rat left lung transplant model. Three groups of rats were compared at 7 days after transplantation. Isogeneic (Lewis to Lewis), allogeneic (Brown-Norway to Lewis) untreated, and cyclosporine-treated (25mg/kg/day, intramuscularly) allogeneic animals underwent analysis of cytokine mRNA isolated from total RNA in freshly excised grafts. Reverse transcription-polymerase chain reaction amplification of interleukin (IL)-2, IL-4, and actin (control) mRNA was performed with custom-synthesized oligonucleotide amplimers targeted to known sequences of rat IL-2 and IL-4 cDNA. Semiquantitative analysis was performed by radioanalytic scanning of gel preparations. Sample specimens from the retrieved grafts were also graded histologically for rejection on a five-point scale. Rejection was most severe in the untreated allografts (p<0.003). IL-2 mRNA was significantly greater in the untreated allografts when compared with isografts (p<0.05) and cyclosporine-treated allografts (p<0.05). No significant differences in IL-4 mRNA between groups were observed. We conclude that semiquantitative analysis of cytokine mRNA by reverse transcription-polymerase chain reaction is a useful and sensitive method for the study of acute rejection in lung grafts and that this technique may become an important tool in future studies of cytokine-mediated responses in cyclosporine-treated allografts.