BACKGROUND: The role of natural killer (NK) cells and macrophages (Møs) in the absence of T cell immunity in discordant xenograft (xg) rejection was investigated. METHODS: Guinea pig hearts were transplanted into athymic nude rats receiving no treatment or antixenoantibody (xAb), anticomplement (C), antinatural killer (NK) cell therapy (antiasialo GM1 [anti-ASGM1]) or their combinations. For anti-xAb therapy, natural xAbs were absorbed/neutralized by pretransplant guinea pig blood transfusion (pGPBT), followed by administration of the malononitriloamide MNA715. Cobra venom factor (CVF) was administered as anti-C therapy. FACScan analysis and a standard cytotoxicity assay determined NK cell number and cytotoxicity, respectively. ELISA and the CH50 assay measured titers of xAb and C activity, respectively. Rejected Xgs were examined by light microscopy and by immunohistochemistry. RESULTS: All hyperacutely (15+/-4 min) rejected Xgs from untreated rats showed deposits of C3 and IgM without cellular infiltrates. Combined anti-xAb and anti-C (pGPBT/MNA715/CVF) treatment significantly prolonged the survival of Xgs (3.7+/-0.6 days, P<0.001 vs. control group) showing NK cell and Mø infiltration without deposition of xAbs or C3. NK cell depletion (day -12) followed by exposure of recovering NK cells to the guinea pig antigens failed to induce specific NK cell nonresponsiveness and to further prolong xg survival in combined anti-xAb/anti-C group. In contrast, adding of continuous and repetitive depletion of NK cells significantly further prolonged xg survival to 7.4+/-0.5 days (P<0,001 vs. the anti-xAb/anti-C group) with rejected Xgs densely infiltrated by activated Møs without involvement of NK cells, C or xAbs. CONCLUSIONS: When xAb and C are suppressed in the discordant guinea pig-into-nude rat model, NK cells play an important role in xg rejection. When also NK cells are suppressed, activated Møs seem to reject discordant Xgs. The induction of specific NK cell nonresponsiveness fails in the guinea pig-into-rat combination.