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.
Highlights
Transplant rejection is the key limiting factor for the success of solid organ transplantation, which is determined by various immunologic and non-immunologic factors[1,2]
Induction of annexin V positivity by L243 was observed in various types of primary ECs, including human dermal microvascular endothelial cells (HDMVECs), human aortic endothelial cells (HAoECs), and human pulmonary microvascular endothelial cells (HPMVECs), but not in the EC line EA.hy[926] (Fig. 1g, Figure S2)
Data are presented as mean ± SEM. *P < 0.05. e human umbilical vein endothelial cells (HUVECs) were treated with L243 for 3 h and stained with Magic Red to determine cathepsin L activity
Summary
Transplant rejection is the key limiting factor for the success of solid organ transplantation, which is determined by various immunologic and non-immunologic factors[1,2]. Complement-independent effects of DSAs are mediated via ligation with endothelial HLA molecules to induce intracellular signal transduction cascades[8,11]. It has been well established that ligation of HLA class I (HLA I) antibodies causes activation[17] and leukocyte adhesion to ECs independent of complement[18,19] Others have demonstrated that HLA II antibodies, such as the monoclonal antibody (mAb) L243 can cause cell death in the absence of complement in various types of non-adherent blood cells, such as leukemia cells[22,23] and B cells[24]. We hypothesized that HLA II antibodies may cause complementindependent cell death in human ECs
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