Monocyte Recruitment to HLA Class I Antibody-Activated Endothelial Cells Is Dependent Upon mTOR.

Abstract

Aim: A hallmark of antibody (Ab)-mediated rejection is the presence of intravascular monocytes in the allograft. The purpose of this study was to identify the role of the mTOR signaling pathway in HLA class I Ab-mediated monocyte recruitment to endothelial cells (EC). Methods: Static and flow-based (ibidi) adhesion assays were used to assess monocyte (MM6) recruitment to HLA-activated primary human aortic endothelial cells. mTOR inhibition was characterized using pharmacological inhibitors (KU 0063794 and sirolimus) at different time points and doses in endothelial cells and monocytes. Results were verified using siRNA knock-down (ko) of rictor and raptor, components of the mTOR pathway. Cell-based ELISA was performed to quantitate Weibel-Palade Body exocytosis; protein phosphorylation was measured by Western blot and quantitated by densitometry. An established murine cardiac transplant model was used to determine the effect of sirolimus treatment on acute AMR. Briefly, Rag1 ko animals were transplanted with an H-2d mismatched heart transplant and received passive transfer of anti-donor Dd+Kd IgG antibodies. Results: Pretreatment of EC for 24 hours with sirolimus or Ku reduced monocyte adherence to EC by > 80%, whereas 2- hour mTOR inhibition resulted in partial inhibition of monocyte binding. siRNA ko of mTOR using siRNA yielded comparable results significantly decreasing monocyte adherence. siRNA against raptor and rictor revealed that both mTORC1 and mTORC2 are involved in HLA-mediated monocyte recruitment. Inhibition of mTOR had no effect on P-selectin induction. Instead, flow-based adhesion assays revealed that mTOR inhibition impairs the ability of EC to support firm adhesion of tethered monocytes by affecting late stage ICAM-1 clustering and its downstream signaling effects. mTOR inhibition in monocytes using sirolimus and Ku significantly impaired monocyte binding to activated endothelium. A reduction in endothelial damage and infiltrating mononuclear cells was observed in vivo in recipients of heart allografts treated with sirolimus. Conclusion: We found that mTOR blockade prevents monocyte recruitment to HLA-activated EC through mTORC1 and mTORC2 signaling pathways. In EC, mTOR inhibition prevents ICAM-1 clustering and blunts firm adhesion of monocytes. Our results provide novel insights into the mechanisms underlying monocyte recruitment in the setting of AMR. DISCLOSURE:Salehi, S.: Grant/Research Support, Pfizer. Reed, E.: Grant/Research Support, Pfizer.