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.

Figure 1.

Intracellular cytokine staining of graft-infiltrating cells isolated from syngeneic, untreated (rejecting), and YTA3.1+DST pretreated (nonrejecting) heart allografts 5 days after transplantation. Total number of graft-infiltrating cells (A) and graft-infiltrating T cells staining positive for CD4 (black bar) and CD8 (white bar) (B). Absolute number of CD4⁺ and CD8⁺ T cells staining positive for IFN-γ (C), IL-2 (D), IL-4 (E), and IL-10 (F). Numbers given are ×10 and represent the mean of three experiments ±SD. n.a., not applicable. The flow-cytometry data of one set of experiments are shown in (G). Mean fluorescence intensity (MFI) for staining with anti-CD4 or anti-CD8 antibody is given on the x axis, and MFI for staining with anti-cytokine antibody or isotype control on the y axis, for which the percentages of positive cells are indicated.

Figure 2.

Competitive RT-PCR analysis of cytokine gene expression in syngeneic, untreated (rejecting), and YTA3.1+DST pretreated (nonrejecting) heart allografts 5 days after transplantation. The expression of C-β, IFN-γ, IL-2, IL-4, and IL-10 (A–E) is given in femtogram (fg) of the amount of co-amplified competitor construct. The numbers represent the mean of three experiments ±SD.

Figure 3.

Competitive RT-PCR for the expression of C-β, IFN-γ, IL-2, IL-4, and IL-10 (A–E) in graft-infiltrating cells isolated heart grafts transplanted into recipients pretreated with YTA3.1+DST, before (−, left bar) and after (+, right bar) restimulation with PMA/ionomycin. The result is given in femtogram (fg) of the amount of co-amplified competitor construct. The numbers represent the mean of three experiments ±SD.