Ligation Of HLA Class Research Articles

Ligation of HLA Class I Molecules Induces YAP Activation through Src in Human Endothelial Cells.

Ab cross-linking of HLA class I (HLA I) molecules on the surface of endothelial cells (EC) triggers proliferative and prosurvival intracellular signaling, which is implicated in the process of chronic allograft rejection, also known as transplant vasculopathy. Despite the importance of Ab-mediated rejection in transplantation, the mechanisms involved remain incompletely understood. In this study, we examined the regulation of yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in human ECs challenged with Abs that bind HLA I. In unstimulated ECs, YAP localized mainly in the cytoplasm. Stimulation of these cells with Ab W6/32 induced marked translocation of YAP to the nucleus. The nuclear import of YAP was associated with a rapid decrease in YAP phosphorylation at Ser127 and Ser397, sites targeted by LATS1/2 and with the expression of YAP-regulated genes, including connective tissue growth factor (CTGF), and cysteine-rich angiogenic inducer 61 (CYR61). Transfection of small interfering RNAs targeting YAP/TAZ blocked the migration of ECs stimulated by ligation of HLA I, indicating that YAP mediates the increase in EC migration induced by HLA I ligation. Treatment of intact ECs with Src family inhibitors induced cytoplasmic localization of YAP in unstimulated ECs and, strikingly, blocked the nuclear import of YAP induced by Ab-induced HLA I activation in these cells and the increase in the expression of the YAP-regulated genes CTGF and CYR61 induced by HLA I stimulation. Our results identify the Src/YAP axis as a key player in promoting the proliferation and migration of ECs that are critical in the pathogenesis of transplant vasculopathy.

OR2 Ligation of HLA class II molecules promotes endothelial cell permeability through activated Src and ERK signals

Aim Microvascular inflammation, increased vascular permeability, and accumulation of intravascular mononuclear cells are characteristics of acute (AMR) and chronic (cAMR) antibody-mediated rejection. In solid organ transplantation, donor specific antibodies (DSA) are associated with AMR, and early or late graft loss. However, the mechanisms underlying how DSA against HLA class II (HLA II) activate the endothelium and mediate inflammation and vascular permeability are poorly understood. Methods We utilized recombinant Class II Transactivator (CIITA) sub-cloned with pAd/PL-DEST vector to achieve expression of HLA II on vascular endothelium, and then evaluated the effects of HLA II antibodies (anti-HLA II) on endothelial cell (EC) permeability. Immunoblotting was used to determine the activation of signaling pathways and confocal microscopy was used to determine vascular endothelial - Cadherin (VE-Cad) phosphorylation and internalization. Results Ligation of HLA II by anti-HLA II activated Src and ERK, induced stress fiber formation, and increased EC permeability. Anti-HLA II also stimulated VE-Cad phosphorylation at Tyr685 as well as its internalization, thereby disassembling intercellular junctions. Class II mediated stress fiber formation and EC permeability was abrogated by pharmacological inhibition of ERK (U0126), Src (PP2) or MLC2 (ML-7). Both pharmacological and siRNA inhibition of Src, but not ERK, inhibited class II-induced phosphorylation of VE-cCd at Tyr685 and consequent VE-Cad internalization. Conclusions In conclusion, our data show that HLA class II DSA promotes actin stress fiber formation via a Src/ERK/MLC2 dependent pathway. Further, class II crosslinking with antibodies induces rapid endocytosis of VE-Cad via Src-dependent tyrosine phosphorylation of VE-Cad, thereby disrupting the endothelial barrier function and contributing to vascular permeability.

Ligation of HLA class ii molecules on endothelial cells induces p-selectin cell surface expression and myeloid cell recruitment

Aim A hallmark of chronic antibody-mediated rejection is the presence of leukocytes infiltrates within the allograft. We tested the hypothesis that ligation of HLA class II on EC mediates myeloid cell recruitment by inducing p-selectin presentation through Weibel Palade body exocytosis. Methods Class II expression was induced by adenoviral transfection of EC with CIITA. Western blots were used to quantitate protein phosphorylation. Monocyte adherence was measured by counting CFSE-labeled monocytes (MonoMac 6) recruited to HLA-class II Ab stimulated EC in the presence/absence of Fc receptor blockade with human IgG. Transendothelial cell migration was determined using a transwell assay. P-selectin expression was detected by Cell ElISA. Results Class II ligation by antibody (Ab) against a monomorphic epitope determinant on class II antigens significantly increased monocyte transendothelial migration in a Fc-independent manner. Class II Ab-mediated myeloid cell transendothelial migration was attenuated by blockading with recombinant PSGL-1 (rPSGL-1). Class II Ab caused a 1.54 ±± 0.11 fold increases in monocyte adherence that was inhibited by pretreatment with rPSGL-1. In addition, pretreatment of EC with Src inhibitor PP2 and ERK inhibitor U0126 resulted in decreased myeloid cell recruitment (Fig. 1). Furthermore, PP2 and U0126 diminished class II induced p-selectin cell surface expression (Fig. 2). Conclusions We provide evidence that class II DSA promotes a MAPK-mediated monocyte adherence by upregulating EC p-selectin expression, stimulating monocyte adherence and infiltration into allografts, leading to transplant vasculopathy. Figure options Download full-size image Download as PowerPoint slide Figure options Download full-size image Download as PowerPoint slide

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Aim The presence of HLA Ab in the allograft is associated with transplant vasculopathy (TV). Ligation of HLA class I elicits signaling events and induces the remodeling of actin cytoskeleton leading to cell survival, migration and proliferation in EC. However, the mechanism by which anti-HLA class II Ab contribute to TV development is not clear. We hypothesize that HLA II ligation induces signaling cascades mediating cytoskeleton reorganization. Methods To express HLA II, human aortic EC were infected with adenoviruses encoding Class II transactivator (Ad-CIITA). Expression of HLA II was determined by flow cytometric analysis using Ab against a monomorphic epitope on HLA II or locus specific Ab against HLA-DR, DQ and DP. Ad-CIITA infected EC were stimulated with anti-HLA II Ab and protein phosphorylation was examined by Western blot. Stress fiber formation was evaluated by fluorescence microscopy in EC stained with Texas Red-phalloidin. The FAK siRNA was utilized to deplete FAK expression. Results Infection with Ad-CIITA for 2d induced expression of HLA II in 80% of EC. Staining with locus specific Ab showed HLA-DR was expressed at the highest level with comparable levels of DP and DQ. Treatment of EC with HLA II Ab significantly increased the phosphorylation of FAK, Src, ERK and p70 S6 kinase (S6K). Ligation of HLA II also significantly augmented formation of stress fibers in EC compared to isotype control IgG [Fig. 1] Knockdown of FAK attenuated phosphorylation of Src, ERK and S6K and also abolished class II-mediated stress fiber formation. Download : Download full-size image Conclusions Ab ligation of HLA II molecules stimulates signal transduction and induces actin cytoskeleton remodeling in EC in a FAK dependent manner.

Antibody ligation of human leukocyte antigen class I molecules stimulates migration and proliferation of smooth muscle cells in a focal adhesion kinase–dependent manner

Chronic rejection manifests as transplant vasculopathy, which is characterized by intimal thickening of the vessels of the allograft. Intimal thickening is thought to result from the migration and proliferation of vascular smooth muscle cells (SMC) in the vessel media, followed by deposition of extracellular matrix proteins. The development of post-transplantation anti-human leukocyte antigen (HLA) antibodies (Ab) is strongly correlated with the development of transplant vasculopathy and graft loss. Here we demonstrate that cross-linking of HLA class I molecules on the surface of human SMC with anti-HLA class I Ab induced cell proliferation and migration. Class I ligation also increased phosphorylation of focal adhesion kinase (FAK), Akt, and ERK1/2 in SMC. Knockdown of FAK by siRNA attenuated class I-induced phosphorylation of Akt and ERK1/2, as well as cell proliferation and migration. These results indicate that ligation of HLA class I molecules induces SMC migration and proliferation in a FAK-dependent manner, which may be important in promoting transplant vasculopathy.

HLA Class I Antibody-Mediated Endothelial Cell Proliferation via the mTOR Pathway

Anti-HLA Abs have been shown to contribute to the process of transplant vasculopathy by binding to HLA class I molecules expressed by the endothelial and smooth muscle cells of the graft and transducing intracellular signals that elicit cell proliferation. The aim of this study was to determine the role of mammalian target of rapamycin (mTOR) in HLA class I-induced endothelial cell proliferation and to explore in depth the relationship between mTOR complexes and their downstream targets following ligation of HLA class I molecules by anti-HLA Abs. We used small interfering RNA technology to abrogate mTOR, rapamycin-insensitive companion of mTOR (rictor), or regulatory associated protein of mTOR (raptor) to study the function of these gene products to activate proteins involved in MHC class I-induced cell proliferation and survival. Knockdown of mTOR inhibited class I-mediated phosphorylation of proteins downstream of mTOR complex 1 and mTOR complex 2. Furthermore, knockdown of mTOR, rictor, or raptor blocked HLA class I-induced endothelial cell proliferation. Long-term pretreatment with the mTOR inhibitor rapamycin significantly blocked both mTOR-raptor and mTOR-rictor complex formation. Interestingly, rapamycin also blocked class I-induced Akt phosphorylation at Ser(473) and Bcl-2 expression. These results support the role of anti-HLA Abs in the process of transplant vasculopathy and suggest that exposure of the graft endothelium to anti-HLA Abs may promote proliferation through the mTOR pathway.

Phosphorylation of S6 ribosomal protein at serine 235/236 is a marker of human heart transplantation

Antibody (Ab) ligation of HLA class I molecules on the surface of endothelial cells (EC) stimulates cell proliferation and survival pathways including activation of the PI3K/Akt pathway. We investigated phosphorylation of S6 ribosomal protein (p-S6RP), a downstream target of the PI3K/Akt/mTOR pathway in EC as a potential marker of Ab mediated-rejection(AMR) of heart allografts. Human aortic EC were treated with anti-HLA class I Ab and cell lysates were studied for phosphorylation of PI3K, Akt and S6 ribosomal protein by Western blot. Endomyocardial biopsies (n 65) from 35 heart transplant recipients (7 with AMR, 7 with acute cellular rejection (ACR)) were tested for the presence of p-S6RP using an anti-phospho serine 235/236 Ab. Diagnosis of AMR was based on histology and immunofluorescence or immunoperoxidase staining of paraffin-embedded tissue (CD68-positive intravascular macrophages). Diagnosis of ACR was based on ISHLT grading. The relationship between p-S6RP and rejection was analyzed by Fisher’s exact test.Treatment of EC with anti-class I Ab resulted in a time and dose dependent increase in phosphorylation of Akt, PI3K and the S6RP. To investigate these results in a relevant in vivo model we determined whether biopsy samples from transplant recipients diagnosed with AMR or ACR showed increased p-S6RP on the graft vasculature.Results showed show a significant association of p-S6RP staining with diagnosis of AMR (p 0.001). Four of 7 patients with ACR showed p-S6RPN staining (p NS). No association between p-S6RP staining with C4d deposition was observed. Study of the kinetics of p-S6RP staining showed that patients diagnosed with AMR display sustained positivity ( 144 days) compared to patients diagnosed with ACR ( 21days). These findings support the role of anti-class I Ab mediated signaling in AMR and indicate that phosphorylation of S6RP is a useful marker of AMR in cardiac allografts. Sustained p-S6RP is observed in patients with AMR supporting its role in chronic allograft vasculopathy.

Ligation of HLA class I molecules on endothelial cells induces phosphorylation of Src, paxillin, and focal adhesion kinase in an actin-dependent manner.

The development of chronic rejection is the major limitation to long-term allograft survival. HLA class I Ags have been implicated to play a role in this process because ligation of class I molecules by anti-HLA Abs stimulates smooth muscle cell and endothelial cell proliferation. In this study, we show that ligation of HLA class I molecules on the surface of human aortic endothelial cells stimulates phosphorylation of Src, focal adhesion kinase, and paxillin. Signaling through class I stimulated Src phosphorylation and mediated fibroblast growth factor receptor (FGFR) translocation to the nucleus. In contrast, Src kinase activity was not involved in class I-mediated transfer of FGFR from cytoplasmic stores to the cell surface. Inhibition of Src protein kinase activity blocked HLA class I-stimulated tyrosine phosphorylation of paxillin and focal adhesion kinase. Furthermore, HLA class I-mediated phosphorylation of the focal adhesion proteins and FGFR expression was inhibited by cytochalasin D and latrunculin A, suggesting a role for the actin cytoskeleton in the signaling process. These findings indicate that anti-HLA Abs have the capacity to transduce activation signals in endothelial cells that may promote the development of chronic rejection.

Ligation of HLA class I molecules on smooth muscle cells with anti-HLA antibodies induces tyrosine phosphorylation, fibroblast growth factor receptor expression and cell proliferation.

The development of transplant atherosclerosis, a manifestation of chronic rejection, is the major obstacle to long-term survival of cardiac and renal allografts. The incidence of transplant atherosclerosis is increased in transplant recipients producing antidonor HLA antibodies following transplantation, suggesting that anti-HLA antibodies play a role in the pathogenesis of the disease. We have postulated that anti-HLA antibodies mediate the development of transplant atherosclerosis by binding to class I molecules on the endothelium and smooth muscle of the graft and transducing signals which stimulate cell proliferation. In this report we demonstrate that anti-HLA class I antibodies transduce signals in smooth muscle cells stimulating increased tyrosine phosphorylation of intracellular proteins and up-regulation of fibroblast growth factor (FGF) receptors. Antibody binding to class I molecules on smooth muscle cells is also accompanied by increased responsiveness to basic FGF and augmented cell proliferation. These findings may explain the increased occurrence of transplant atherosclerosis in recipients producing anti-donor HLA antibodies.