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The Mechanisms of Monocyte Recruitment by Graft Endothelium in Response to HLA I Antibody Binding

  • Author(s): Valenzuela, Nicole M.
  • Advisor(s): Reed, Elaine F
  • et al.
Abstract

Successful organ transplantation is challenged by the recipient's immune responses against the donor's polymorphic HLA molecules. The humoral response produces antibodies against mismatched HLA class I and class II molecules, which have a significant negative impact on long-term graft survival. Antibody-mediated rejection is characterized by intragraft macrophages, which may be elicited by HLA I antibody-induced endothelial cell activation. Given that the presence of intragraft macrophages significantly correlates with poor graft outcome, we investigated the mechanisms of HLA I antibody leading to recruitment of monocytes by endothelial cells, to determine effective therapeutic strategies to reduce infiltration into the graft. First, we determined the effect of endothelial cell activation by crosslinking of HLA I. Using an in vitro system with human endothelial cells stimulated with murine monoclonal HLA I antibodies, we found that HLA I ligation triggers endothelial exocytosis via calcium signaling, leading to increased cell surface P-selectin, which was required for increased monocyte adherence. We confirmed these results using an in vivo murine model of antibody-mediated rejection, where monocyte infiltration was significantly increased in allografts treated with donor specific MHC I antibodies. Macrophage accumulation in the graft could be prevented by therapy with the P-selectin antagonist rPSGL-1. Next, we explored the contribution of antibody-Fc receptor interactions to recruitment. Adhesion was stimulated by HLA I antibodies through P-selectin, irrespective of the isotype of HLA I antibody. Further, HLA I antibodies of an isotype with a high affinity for human Fc receptors elicited significantly more monocyte binding, and predominantly utilized FcgRI. Firm adherence to ICAM-1 on the endothelium required monocyte Mac-1 but not LFA-1. Finally, we investigated the activation of monocytes to a pro-adhesive state. Monocytes required intact calcium, Src and PI3 kinase signaling to adhere to HLA I antibody-treated endothelial cells, but not Rho kinase. Crosslinking of the P-selectin receptor PSGL-1, or of Fc receptors, triggered stable adhesion to ICAM-1. Deposition of low titer HLA I antibody on cytokine-activated endothelial cells enhanced monocyte binding, likely due to antibody-Fc receptor interactions. To conclude, we have elucidated the mechanisms of how HLA I antibody binding to endothelium promotes monocyte recruitment.

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