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Critical Role of Macrophage Fc?R Signaling and Reactive Oxygen Species in Alloantibody-Mediated Hepatocyte Rejection.

ABSTRACT: Humoral alloimmunity negatively impacts both short- and long-term cell and solid organ transplant survival. We previously reported that alloantibody-mediated rejection of transplanted hepatocytes is critically dependent on host macrophages. However, the effector mechanism(s) of macrophage-mediated injury to allogeneic liver parenchymal cells is not known. We hypothesized that macrophage-mediated destruction of allogeneic hepatocytes occurs by cell-cell interactions requiring Fc?Rs. To examine this, alloantibody-dependent hepatocyte rejection in CD8-depleted wild-type (WT) and Fc?-chain knockout (KO; lacking all functional Fc?R) transplant recipients was evaluated. Alloantibody-mediated hepatocellular allograft rejection was abrogated in recipients lacking Fc?R compared with WT recipients. We also investigated anti-Fc?RI mAb, anti-Fc?RIII mAb, and inhibitors of intracellular signaling (to block phagocytosis, cytokines, and reactive oxygen species [ROS]) in an in vitro alloantibody-dependent, macrophage-mediated hepatocytoxicity assay. Results showed that in vitro alloantibody-dependent, macrophage-mediated hepatocytotoxicity was critically dependent on Fc?Rs and ROS. The adoptive transfer of WT macrophages into CD8-depleted Fc?R-deficient recipients was sufficient to induce alloantibody-mediated rejection, whereas adoptive transfer of macrophages from Fc?-chain KO mice or ROS-deficient (p47 KO) macrophages was not. These results provide the first evidence, to our knowledge, that alloantibody-dependent hepatocellular allograft rejection is mediated by host macrophages through Fc?R signaling and ROS cytotoxic effector mechanisms. These results support the investigation of novel immunotherapeutic strategies targeting macrophages, Fc?Rs, and/or downstream molecules, including ROS, to inhibit humoral immune damage of transplanted hepatocytes and perhaps other cell and solid organ transplants.

SUBMITTER: Zimmerer JM

PROVIDER: S-EPMC6289737 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Critical Role of Macrophage FcγR Signaling and Reactive Oxygen Species in Alloantibody-Mediated Hepatocyte Rejection.

Zimmerer Jason M JM Liu Xin L XL Blaszczak Alecia A Avila Christina L CL Pham Thomas A TA Warren Robert T RT Bumgardner Ginny L GL

Journal of immunology (Baltimore, Md. : 1950) 20181105 12

Humoral alloimmunity negatively impacts both short- and long-term cell and solid organ transplant survival. We previously reported that alloantibody-mediated rejection of transplanted hepatocytes is critically dependent on host macrophages. However, the effector mechanism(s) of macrophage-mediated injury to allogeneic liver parenchymal cells is not known. We hypothesized that macrophage-mediated destruction of allogeneic hepatocytes occurs by cell-cell interactions requiring FcγRs. To examine th ...[more]

PMID: 30397035

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Project description:In response to microenvironmental cues, macrophages undergo a profound phenotypic transformation acquiring distinct activation phenotypes ranging from pro-inflammatory (M1) to anti-inflammatory (M2). To study how activation phenotype influences phagocytosis and production of reactive oxygen species (ROS) mediated by receptors for IgG antibodies (Fcγ receptors) and by CD13, human monocyte-derived macrophages were polarized to distinct phenotypes using IFN-γ (Mϕ-IFN-γ), IL-4 (Mϕ-IL-4), or IL-10 (Mϕ-IL-10). Phenotypically, Mϕ-IFN-γ were characterized as CD14+CD80+CD86+ cells, Mϕ-IL-4 as CD209highCD206+CD11b+CD14low, and Mϕ-IL-10 as CD16+CD163+ cells. Compared to non-polarized macrophages, FcγRI expression increased in Mϕ-IFN-γ and Mϕ-IL-10 and FcγRIII expression increased in Mϕ-IL-10. None of the polarizing cytokines modified FcγRII or CD13 expression. Functionally, we found that cytokine-mediated activation significantly and distinctively affected FcγR- and CD13-mediated phagocytosis and ROS generation. Compared to non-polarized macrophages, FcγRI-, FcγRII-, and CD13-mediated phagocytosis was significantly increased in Mϕ-IL-10 and decreased in Mϕ-IFN-γ, although both cytokines significantly upregulated FcγRI expression. IL-10 also increased phagocytosis of Escherichia coli, showing that the effect of IL-10 on macrophage phagocytosis is not specific for a particular receptor. Interestingly, Mϕ-IL-4, which showed poor FcγR- and CD13-mediated phagocytosis, showed very high phagocytosis of E. coli and zymosan. Coupled with phagocytosis, macrophages produce ROS that contribute to microbial killing. As expected, Mϕ-IFN-γ showed significant production of ROS after FcγRI-, FcγRII-, or CD13-mediated phagocytosis. Unexpectedly, we found that Mϕ-IL-10 can also produce ROS after simultaneous stimulation through several phagocytic receptors, as coaggregation of FcγRI/FcγRII/CD13 induced a belated but significant ROS production. Together, these results demonstrate that activation of macrophages by each cytokine distinctly modulates expression of phagocytic receptors, FcγR- and CD13-mediated phagocytosis, and ROS production.

Project description:It is now well accepted that radiation induced bystander effects can occur in cells exposed to media from irradiated cells. The aim of this study was to follow the bystander cells in real time following addition of media from irradiated cells and to determine the effect of inhibiting these signals. A human keratinocyte cell line, HaCaT cells, was irradiated (0.005, 0.05 and 0.5 Gy) with ? irradiation, conditioned medium was harvested after one hour and added to recipient bystander cells. Reactive oxygen species, nitric oxide, Glutathione levels, caspase activation, cytotoxicity and cell viability was measured after the addition of irradiated cell conditioned media to bystander cells. Reactive oxygen species and nitric oxide levels in bystander cells treated with 0.5Gy ICCM were analysed in real time using time lapse fluorescence microscopy. The levels of reactive oxygen species were also measured in real time after the addition of extracellular signal-regulated kinase and c-Jun amino-terminal kinase pathway inhibitors. ROS and glutathione levels were observed to increase after the addition of irradiated cell conditioned media (0.005, 0.05 and 0.5 Gy ICCM). Caspase activation was found to increase 4 hours after irradiated cell conditioned media treatment (0.005, 0.05 and 0.5 Gy ICCM) and this increase was observed up to 8 hours and there after a reduction in caspase activation was observed. A decrease in cell viability was observed but no major change in cytotoxicity was found in HaCaT cells after treatment with irradiated cell conditioned media (0.005, 0.05 and 0.5 Gy ICCM). This study involved the identification of key signaling molecules such as reactive oxygen species, nitric oxide, glutathione and caspases generated in bystander cells. These results suggest a clear connection between reactive oxygen species and cell survival pathways with persistent production of reactive oxygen species and nitric oxide in bystander cells following exposure to irradiated cell conditioned media.

Dataset Information

Critical Role of Macrophage Fc?R Signaling and Reactive Oxygen Species in Alloantibody-Mediated Hepatocyte Rejection.

Publications

Critical Role of Macrophage FcγR Signaling and Reactive Oxygen Species in Alloantibody-Mediated Hepatocyte Rejection.

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