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 (25 mg/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.
Francalancia
N A
NA
Department of Surgery, University of Pittsburgh, Pa.
Wang
S C
SC
Thai
N L
NL
Aeba
R
R
Simmons
R L
RL
Yousem
S A
SA
Hardesty
R L
RL
Griffith
B P
BP
D000199:Actins / Q000235:genetics*; D000818:Animals; D016572:Cyclosporine / Q000627:therapeutic use*; D006084:Graft Rejection / Q000175:diagnosis / Q000235:genetics*; D007165:Immunosuppression Therapy; D007376:Interleukin-2 / Q000235:genetics*; D015847:Interleukin-4 / Q000235:genetics*; D016040:Lung Transplantation / Q000276:immunology*; D008297:Male; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011914:Rats, Inbred BN; D011917:Rats, Inbred Lew; D013997:Time Factors; D014158:Transcription, Genetic
D000199:Actins; D007376:Interleukin-2; D012333:RNA, Messenger; D015847:Interleukin-4; D016572:Cyclosporine