title: Differential role for competitive reverse transcriptase-polymerase chain reaction and intracellular cytokine staining as diagnostic tools for the assessment of intragraft cytokine profiles in rejecting and nonrejecting heart allografts.
abstract: The early and reliable diagnosis of allograft rejection is a difficult task and the assessment of cytokine expression in the grafts can be a helpful parameter. We have compared competitive reverse transcriptase-polymerase chain reaction (RT-PCR) with intracellular cytokine staining by flow cytometry as tools to measure cytokine expression in rejecting and nonrejecting murine cardiac allografts. Both techniques gave comparable results for cytokine expression in rejecting allografts and syngeneic controls. Grafts from mice pretreated with anti-CD4 antibody and donor-specific blood transfusion showed a marked reduction in cytokine expression, as assessed by competitive RT-PCR, even though a cellular infiltrate was present in the graft. In contrast, the cytokine production measured by intracellular cytokine staining of the isolated graft-infiltrating cells was high and exceeded even that of the rejecting allografts. We conclude that intracellular cytokine staining of graft-infiltrating leukocytes by flow cytometry does not necessarily reflect accurately the cytokine milieu in the graft. This technique might therefore have a limited clinical application in contrast to competitive RT-PCR for the differentiation between graft acceptance and graft rejection.
authors.affiliation: Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom.
authors.forename: B M
authors.initials: BM
authors.lastname: Spriewald
authors.forename: M
authors.initials: M
authors.lastname: Hara
authors.forename: A
authors.initials: A
authors.lastname: Bushell
authors.forename: S
authors.initials: S
authors.lastname: Jenkins
authors.forename: P J
authors.initials: PJ
authors.lastname: Morris
authors.forename: K J
authors.initials: KJ
authors.lastname: Wood
mesh_terms: D000818:Animals; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D006084:Graft Rejection / Q000378:metabolism*; D006321:Heart / Q000187:drug effects; D016027:Heart Transplantation; D007425:Intracellular Membranes / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013194:Staining and Labeling; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014184:Transplantation, Homologous; D015854:Up-Regulation
chemical_list: D016207:Cytokines; D007476:Ionophores; D012333:RNA, Messenger; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate