Chemoattractant cytokines, chemokines, are likely to play a critical role in directing leukocytes to graft sites and in amplifying intragraft inflammation during rejection. Since second-set graft rejection occurs at an accelerated rate, we hypothesized that chemokine genes would be expressed earlier during secondary allograft rejection than during rejection of primary allografts. We have tested this hypothesis by using Northern blot analysis to compare intragraft expression levels of genes encoding interleukin (IL) 1beta and six chemokines during rejection of C57BL/6 skin grafts on naive and C57BL/6-sensitized BALB/c recipients. Expression levels of IL-1beta, interferon-gamma inducible protein, macrophage inflammatory protein-1 (MIP-1) alpha, and MIP-1beta genes were 10- to 17-fold higher on day 5 after transplantation in C57BL/6 grafts on C57BL/6-presensitized recipients than in C57BL/6 grafts on unprimed recipients. Intragraft expression of the chemokines regulated upon activation, normal T cell expressed and secreted (RANTES), during primary C57BL/6 graft rejection was virtually undetectable at day 7 after primary transplantation, but was expressed at high levels by day 5 after secondary transplantation. In third-party CBA/Ca allografts on unsensitized and C57BL/6-presensitized BALB/c mice, similar levels of IL-1beta, MIP-1alpha, and MIP-1beta expression were observed. High levels of RANTES and interferon-gamma inducible protein expression, however, were observed at day 5 after transplantation in CBA/Ca grafts on C57BL/6-presensitized recipients and correlated with accelerated rejection of the third-party grafts. Although T cells from C57BL/6-presensitized recipients did not express increased reactivity to CBA/Ca stimulator cells in vitro, serum antibodies from these recipients demonstrated reactivity to cells from CBA/Ca and A/J mice. When compared with transfer of unprimed cells, transfer of C57BL/6-primed lymphoid cells to sublethally irradiated BALB/c mice engrafted with C57BL/6 grafts resulted in increased intragraft proinflammatory cytokine gene expression. Deletion of T cells before transfer abrogated the increased intragraft expression of proinflammatory cytokine genes. Collectively, these results indicate that the accelerated expression of chemokine genes during second-set rejection of allogeneic skin grafts is mediated by immune T cells.
