Alloantibodies (alloAb) play a critical role in rejection after transplantation. Despite this, the cellular mechanisms contributing to alloAb-mediated allograft damage are not well understood. Using our well-characterized murine hepatocellular allograft model, we previously reported that alloAb-mediated damage to graft parenchymal cells occurs through Ab-dependent cell-mediated cytotoxicity (ADCC) mediated by macrophages (Mφ). However, the precise mechanisms by which Mφs mediate ADCC of allogeneic parenchymal cells is not known. We hypothesized that Mφ-mediated ADCC of allogeneic hepatocytes is contact-dependent and requires Fcγ receptors (FcγR). To examine this, serum alloAb and hepatocyte transplant survival were evaluated in Fcγ chain deficient (Fcγ KO; mice lack functional FcγRs), wild-type (C57BL/6, WT) low and high alloAb producing (CD8-depleted) recipients. WT high alloAb producers(CD8-depleted) developed hepatocyte rejection with median survival time (MST) of 14 days. Rejection was significantly delayed (MST=59 days, p<0.001) in Fcγ KO high alloAb producers (CD8-depleted). Low alloAb producers, WT and Fcγ KO recipients, developed rejection by CD8-dependent mechanisms and had similar MST of 10 days. Further investigations using an in vitro ADCC assay show that Mφ-mediated parenchymal cell damage is contact-dependent as Mφs separated from targets with a transwell assay did not mediate significant cytotoxicity (12±3%) as compared to Mφs co-cultured in direct contact with hepatocytes (57±8%, p=0.001). In vitro ADCC was also shown to be dependent on both FcγRI and FcγRIII as incubation with anti-FcγRI mAb (10±1%) and anti-FcγRIII mAb (9±2%, p<0.002 for both) significantly blocked Mφ ADCC. When combinational treatments were utilized (blocking FcγRI and III), Mφ ADCC was abrogated (0±0%, p<0.001). To investigate the specific ADCC mechanisms used in Mφ-mediated parenchymal cell damage, inhibitors aimed to block phagocytosis, cytokine, and reactive oxygen species (ROS) were incubated with Mφs prior to and during the co-culture with target hepatocytes. Results show that only inhibitors directed to block ROS (apocynin; 1.0 mM=0±0%; p<0.001) abrogated Mφ-mediated ADCC. These findings highlight specific Mφ-mediated mechanisms potentiating alloAb-dependent damage to graft parenchymal cells and suggest that targeting Mφ effector mechanisms may ameliorate alloAb mediated graft damage.

« Back to 2015 American Transplant Congress