Rapamycin treatment depresses intragraft expression of KC/MIP-2, granzyme B, and IFN-gamma in rat recipients of cardiac allografts

Abstract

Rapamycin (RPM) treatment prevents accelerated rejection of cardiac allografts in sensitized rats. The prominent feature of this brisk 24-h rejection, which includes a panoply of both cellular and humoral host immune responses, is a massive infiltration of rejecting grafts with neutrophils. In this study we tested the hypothesis that RPM-mediated therapeutic effects on accelerated rejection may be linked to decreased expression of protein encoded by gro/melanoma-growth stimulatory activity gene (KC) and macrophage inflammatory protein-2 (MIP-2) genes, the operational rat homologues of the human intercrine-alpha cytokines with proinflammatory IL-8-like neutrophil activation/chemotactic properties. The induction of these genes was then correlated with mRNA profiles encoding for Th1-selective IFN-gamma and CTL-specific granzyme B proteins. Northern blot analysis of RNA from cardiac allografts of sensitized untreated recipients, revealed maximal levels of KC and MIP-2 mRNA at 3 to 6 h after transplantation. In contrast, IFN-gamma mRNA, which was at most very weakly expressed at 3 h, peaked between 6 to 12 h. As with IFN-gamma, granzyme B transcripts were undetectable at 3 h, but peaked around the time of actual graft rejection at 24 h. RPM therapy abrogated accelerated rejection and prolonged cardiac allograft survival to ca. 46 days. This effect was associated with markedly reduced expression of KC and MIP-2 mRNA in the first 24 h as well as at 7 and 34 days after transplantation. Moreover, RPM completely blocked intragraft appearance of granzyme B and IFN-gamma mRNA in long term cardiac allografts. Immunohistologic analysis has revealed that accelerated rejection was associated with extensive neutrophil infiltration, which peaked at 18 to 24 h. At this time, leukocytes and endothelium were intensely stained for IL-8 and IFN-gamma antibodies. In contrast, the allografts from RPM-treated hosts showed essentially no neutrophil infiltration and minor, focal staining for IL-8 and IFN-gamma. This study demonstrates an association between the early expression of genes for proinflammatory IL-8-dependent neutrophil chemotactic activity, and later expression of genes associated with activation/effector activity of CTL and NK cells. It also documents a novel effect of RPM in vivo, which results in the suppression of intragraft IL-8-like and CTL-dependent mRNA/protein production and diminished neutrophil infiltration; these may contribute to the striking efficacy of RPM therapy in sensitized graft recipients.