HLA class II antibodies induce necrotic cell death in human endothelial cells via a lysosomal membrane permeabilization-mediated pathway

Abstract

Antibody-mediated rejection (AMR) is the major cause of allograft loss after solid organ transplantation. Circulating donor-specific antibodies against human leukocyte antigen (HLA), in particular HLA class II antibodies are critical for the pathogenesis of AMR via interactions with endothelial cells (ECs). To investigate the effects of HLA class II antibody ligation to the graft endothelium, a model of HLA-DR antibody-dependent stimulation was utilized in primary human ECs. Antibody ligation of HLA class II molecules in interferon-γ-treated ECs caused necrotic cell death without complement via a pathway that was independent of apoptosis and necroptosis. HLA-DR-mediated cell death was blocked by specific neutralization of antibody ligation with recombinant HLA class II protein and by lentiviral knockdown of HLA-DR in ECs. Importantly, HLA class II-mediated cytotoxicity was also induced by relevant native allele-specific antibodies from human allosera. Necrosis of ECs in response to HLA-DR ligation was mediated via hyperactivation of lysosomes, lysosomal membrane permeabilization (LMP), and release of cathepsins. Notably, LMP was caused by reorganization of the actin cytoskeleton. This was indicated by the finding that LMP and actin stress fiber formation by HLA-DR antibodies were both downregulated by the actin polymerization inhibitor cytochalasin D and inhibition of Rho GTPases, respectively. Finally, HLA-DR-dependent actin stress fiber formation and LMP led to mitochondrial stress, which was revealed by decreased mitochondrial membrane potential and generation of reactive oxygen species in ECs. Taken together, ligation of HLA class II antibodies to ECs induces necrotic cell death independent of apoptosis and necroptosis via a LMP-mediated pathway. These findings may enable novel therapeutic approaches for the treatment of AMR in solid organ transplantation.

Proteomics Reveals Extracellular Matrix Injury in the Glomeruli and Tubulointerstitium of Kidney Allografts with Early Antibody-Mediated Rejection

“…exposure to interferon gamma (IFNɣ)). [10][11][12] Ligation of HLA with α-HLA-I induces cell proliferation and activation of ERK and mTOR. [12][13][14] These effects are enhanced by IFNɣ and tumor necrosis factor alpha (TNFα).…”

“…15 Importantly, α-HLA-I-activated endothelial cells display cytoskeletal alterations and interact with leukocytes. [16][17][18][19] α-HLA-II induces necrosis in IFNɣ-treated endothelial cells, 10 and exerts prothrombotic effects in HGMECs. 9 The role of tubular cells in AMR is unclear.…”

Potential Novel Biomarkers in Chronic Graft-Versus-Host Disease

“…In addition, anti-HLA II antibodies have the ability to activate a cell death pathway and this should also be taken into consideration (52)(53)(54). A recent report extends this observation to DSA from patient allosera, revealing death of endothelial cells after DSA binding (55).…”

Autoantibodies Against Ro/SS-A, CENP-B, and La/SS-B are Increased in Patients with Kidney Allograft Antibody-Mediated Rejection

“…Antibodies against HLA and non-HLA antigens interact with proteins expressed by parenchymal cells, including endothelial and epithelial cells. [63][64][65][66][67] We leveraged our recent proteomics study of glomeruli and tubulointerstitium in grafts with AMR compared to ACR and ATN, 48 and built a protein-protein interaction network to study the connections between proteins significantly dysregulated in AMR kidneys, and protein targets of key antibodies identified in this study. We focused on targets of antibodies increased in AMR/mixed rejection and externally validated:…”