Detection of cytokine mRNA in vivo by polymerase chain reaction. Problems and solutions

The expression of cytokine mRNAs in murine sponge matrix graft infiltrates was studied using the polymerase chain reaction (PCR). In infiltrates of both C57B1/6 > C57B1/6 isografts and DBA/2 > C57B1/6 allografts, a similar pattern of cytokine expression was observed. On days 6, 8, and 10 postimplant, mRNAs encoding IL-4, IL-6, and IFN-gamma were detected. On days 8 and 10, mRNAs encoding IL-1 alpha, TNF, and lymphotoxin/TNF beta were also expressed. Surprisingly, no IL-2 mRNA was observed in isografts or allografts. Two modified PCR techniques were utilized to compare the level of expression of cytokine mRNAs in isografts versus allografts and to detect the expression of IL-2 mRNA in this system. A semiquantitative PCR protocol based on limiting cycles of amplification is described that was used to determine the amount of IL-4 mRNA expressed in isograft and allograft infiltrates relative to beta-actin mRNA. Using this method, no difference was found in the amount of IL-4 mRNA in infiltrates of day 8 isografts and allografts. The validation of this technique by analysis of samples with known relative mRNA levels is presented. A nested PCR protocol is described that provided greatly enhanced sensitivity over standard PCR analysis. Using this technique, IL-2 mRNA was detected in infiltrates from sponge allografts on all days tested, beginning on day 2 postimplant. No IL-2 mRNA was detected in isograft infiltrates or in peripheral blood from allograft-bearing animals. The pattern of cytokine mRNA expression observed in sponge matrix allografts is consistent with the presence of a weak alloreactive response superimposed upon an intense granulomatous process that occurs in both the isografts and allografts. This report demonstrates modified PCR techniques that can be used to resolve experimental problems associated with the analysis of cytokine mRNA expression in vivo.