An In situ Hybridisation method was developed and utilised to evaluate and pinpoint the cellular presence of IL2 and IL4 mRNA transcripts produced by Th 1 and Th2 subsets respectively of activated T cells in frozen tissue sections of Lewis heart allografts from rejecting DA recipients and from recipients tolerised by Cyclosporin, Blood Transfusion and Antibody therapy regimens. A panel of monoclonal antibodies was used on the tissue sections initially and confirmed the presence of the activated T cells known to express the cytokines being studied. The Polymerase Chain Reaction was used next on tissue homogenates from the same samples to establish the presence of IL2 and IL4 mRNA transcripts and showed that IL2 mRNA sequences were present in all rejecting samples, but not in all tolerised samples, whereas with the exception of some day 2 grafts, IL4 transcripts were present in all other rejecting grafts, and also in CsA and Transfused grafts but not in all Antibody treated grafts. As the presence of cytokine transcripts had been established by PCR, the ISH method was utilised to attempt to pinpoint specific cellular production of the IL2 and IL4 transcripts in the frozen sections of the tissue. All rejecting grafts and grafts from Transfused recipients were found to be positive for IL2 transcripts whereas CsA treated recipients grafts were negative, and Antibody treated recipients grafts found to be variable. IL4 transcripts were found in 50% of the rejecting grafts only, suggesting that ISH is not sensitive enough to record low levels of mRNA expression and that the use of both the ISH and PCR methods in tandem would be more beneficial in intragraft cytokine transcription detection
