Monocyte recruitment to HLA class I antibody-activated endothelial cells is dependent upon MTOR

Abstract

Antibody-mediated rejection (AMR) persists as a major issue affecting solid organ transplants. The purpose of this study was to investigate the proximal signaling events that regulate mTOR-mediated leukocyte recruitment following HLA I stimulation in endothelial cells (EC). For confocal microscopy experiments, EC were treated with rapamycin for 24 hours, stimulated with HLA antibodies, incubated for 1 h with sheep anti-ICAM-1 antibody, followed by 30 min incubation with secondary FITC-labeled goat anti-sheep IgG and then fixed. Cells were imaged by a Zeiss confocal microscope. EC were treated with l mTOR inhibitors (RAD001 or Rapamycin) or U0126, PKC, Rho or ROCK inhibitors, then stimulated with HLA I antibodies. Phosphorylation of downstream effectors of the mTOR and Rho pathways were measured by Western blot. Recruitment of primary monocytes to EC was determined by static adherence assays. Using flow-based adherence assays, we previously discovered that mTOR inhibition impairs the ability of EC to support firm adhesion of tethered monocytes by affecting late stage ICAM-1 clustering, which has been attributed to Rho signaling. As demonstrated by confocal microscopy, this impairment was due to a decrease in association of actin stress fibers with ICAM-1, which disrupts ICAM-1 clustering. We determined by Western blot that mTOR operates upstream of RhoA, as inhibition of Rho did not disrupt phosphorylation of proteins downstream of the mTOR pathway, though inversely, mTOR inhibition blocked phosphorylation of downstream effectors of Rho. mTOR in turn regulates ERM phosphorylation, which is crucial for ICAM-1 clustering. RhoA pathway inhibition of EC blocked HLA-I antibody-induced recruitment of primary monocytes in static adherence assays. Our findings connect mTOR-mediated class I signaling to cytoskeletal remodeling involving RhoA and ERM proteins, suggesting that mTOR inhibition reduces leukocyte recruitment to activated endothelium by impairing firm adhesion.