EARLY INTRAGRAFT EXPRESSION OF CHEMMOKINE GENES DURING ACUTE REJECTION OF ALLOGENEIC CARDIAC GRAFTS

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119 OBJECTIVES: Chemokines recruit leukocytes to sites of inflammation and are likely to have an important role in allograft rejection. We have previously demonstrated increased chemokine gene expression and protein production at day 3 post-transplant during rejection of allogeneic skin grafts in mice. This expression occurs prior to detectable activation of allo-specific T cells, suggesting that this early post-transplant inflammatory response is mediated by innate immune mechanisms. The goal of the current study was to test the presence of early intra-allograft chemokines in a vascularized graft model. METHODS: C57BL/6 (H-2b) mice received a heterotopic heart graft from syngeneic or allogeneic DBA/2 (H-2d) or A/J (H-2a) donors. Some groups of recipients received 100 ug of monoclonal anti-NK 1.1, anti-CD4, and/or anti-CD8 antibody the day of and the day after transplantation. Grafts were removed 1-8 days post-transplant for RNA isolation and Northern, blot analysis. Graft cryosections were stained with anti-chemokine antibodies for immunohistology analysis. Grafts were digested with collagenase and the cells stained with anti-T cell and anti-NK cell antibodies and analyzed by flow cytometry. RESULTS: By contrast to allogeneic skin grafts, allogeneic heart grafts had early (day +2 post-transplant) and high expression of the IFN-γ induced chemokine IP-10 and KC but not MIP-1α or MIP-1β. The increased chemokine gene expression occurred prior to detectable alloantigen T cell priming and suggested that T cells did not mediate early increases in intra-allograft chemokine gene expression. Antibody staining and flow cytometry analysis of cells infiltrating allogeneic cardiac grafts indicated the presence of NK cells (>60% of infiltrating CD45+ cells) within 24-48 h post-transplant. From 30-50% of the graft infiltrating NK 1.1+ cells expressed the CD69 activation marker during this time period post-transplantation. The presence of NK cells was absent in allogeneic cardiac grafts from recipients treated with an anti-NK 1.1 mAb. Intra-allograft expression of MIP-1α, MIP-1β and RANTES, as well as IP-10, was observed late (day +8) during the rejection process. CONCLUSIONS: The early intragraft inflammation during primary allograft rejection of A/J and DBA/2 hearts by C57BL/6 recipients may be dependent upon the activities of host NK cells. These results suggest a model where early graft infiltration by NK cells establish inflammatory sites which accelerate and direct alloantigen-specific T cell migration.