Autoantibodies from patients with kidney allograft vasculopathy stimulate a proinflammatory switch in endothelial cells and monocytes mediated via GPCR-directed PAR1-TNF-α signaling.

Non-HLA-directed regulatory autoantibodies (RABs) are known to target G-protein coupled receptors (GPCRs) and thereby contribute to kidney transplant vasculopathy and failure. However, the detailed underlying signaling mechanisms in human microvascular endothelial cells (HMECs) and immune cells need to be clarified in more detail. In this study, we compared the immune stimulatory effects and concomitant intracellular and extracellular signaling mechanisms of immunoglobulin G (IgG)-fractions from kidney transplant patients with allograft vasculopathy (KTx-IgG), to that from patients without vasculopathy, or matched healthy controls (Con-IgG). We found that KTx-IgG from patients with vasculopathy, but not KTx-IgG from patients without vasculopathy or Con-IgG, elicits HMEC activation and subsequent upregulation and secretion of tumor necrosis factor alpha (TNF-α) from HMECs, which was amplified in the presence of the protease-activated thrombin receptor 1 (PAR1) activator thrombin, but could be omitted by selectively blocking the PAR1 receptor. The amount and activity of the TNF-α secreted by HMECs stimulated with KTx-IgG from patients with vasculopathy was sufficient to induce subsequent THP-1 monocytic cell activation. Furthermore, AP-1/c-FOS, was identified as crucial transcription factor complex controlling the KTx-IgG-induced endothelial TNF-α synthesis, and mircoRNA-let-7f-5p as a regulatory element in modulating the underlying signaling cascade. In conclusion, exposure of HMECs to KTx-IgG from patients with allograft vasculopathy, but not KTx-IgG from patients without vasculopathy or healthy Con-IgG, triggers signaling through the PAR1-AP-1/c-FOS-miRNA-let7-axis, to control TNF-α gene transcription and TNF-α-induced monocyte activation. These observations offer a greater mechanistic understanding of endothelial cells and subsequent immune cell activation in the clinical setting of transplant vasculopathy that can eventually lead to transplant failure, irrespective of alloantigen-directed responses.

Induced antibodies directed to the angiotensin receptor type 1 provoke skin and lung inflammation, dermal fibrosis and act species overarching.

OBJECTIVE: To determine contributions and functions of autoantibodies (Abs) directed to the angiotensin receptor type 1 (AT1R), which are suggested to be involved in the pathogenesis of AT1R Abs-related diseases such as systemic sclerosis (SSc). METHODS: C57BL/6J mice were immunised with membrane-embedded human AT1R or empty membrane as control. Mice deficient for CD4+ or CD8+ T cells and B cells were immunised with membrane-embedded AT1R or an AT1R peptide proposed to be a dominant T cell epitope. A monoclonal (m)AT1R Ab was generated by hybridoma technique and transferred into C57BL/6J and AT1Ra/b knockout mice. The induced phenotype was examined by histology, immunohistochemistry, immunofluorescence, apoptosis assay and ELISA. In vitro, Abs responses towards AT1R were measured in cells of different origins and species. RESULTS: AT1R-immunised mice developed perivascular skin and lung inflammation, lymphocytic alveolitis, weak lung endothelial apoptosis and skin fibrosis accompanied by Smad2/3 signalling, not present in controls or mice deficient for CD4+ T and B cells. The AT1R peptide 149-172 provoked lung inflammation. Application of the mAT1R Ab induced skin and lung inflammation, not observed in AT1Ra/b knockout mice. In vitro, AT1R Abs activated rat cardiomyocytes and human monocytes, enhanced angiotensin II-mediated AT1R activation in AT1R-transfected HEK293 cells via AT1R binding and mAT1R Ab-activated monocytes mediated the induction of profibrotic markers in dermal fibroblasts. CONCLUSION: Our immunisation strategy successfully induced AT1R Abs, contributing to inflammation and, possibly, to fibrosis via activation of AT1R. Therefore, AT1R Abs are valuable targets for future therapies of SSc and other AT1R Ab-related diseases.