ABSTRACT
Current classification of chronic kidney disease (CKD) into stages using indirect systemic measures (estimated glomerular filtration rate (eGFR) and albuminuria) is agnostic to the heterogeneity of underlying molecular processes in the kidney thereby limiting precision medicine approaches. To generate a novel CKD categorization that directly reflects within kidney disease drivers we analyzed publicly available transcriptomic data from kidney biopsy tissue. A Self-Organizing Maps unsupervised artificial neural network machine-learning algorithm was used to stratify a total of 369 patients with CKD and 46 living kidney donors as healthy controls. Unbiased stratification of the discovery cohort resulted in identification of four novel molecular categories of disease termed CKD-Blue, CKD-Gold, CKD-Olive, CKD-Plum that were replicated in independent CKD and diabetic kidney disease datasets and can be further tested on any external data at kidneyclass.org. Each molecular category spanned across CKD stages and histopathological diagnoses and represented transcriptional activation of distinct biological pathways. Disease progression rates were highly significantly different between the molecular categories. CKD-Gold displayed rapid progression, with significant eGFR-adjusted Cox regression hazard ratio of 5.6 [1.01-31.3] for kidney failure and hazard ratio of 4.7 [1.3-16.5] for composite of kidney failure or a 40% or more eGFR decline. Urine proteomics revealed distinct patterns between the molecular categories, and a 25-protein signature was identified to distinguish CKD-Gold from other molecular categories. Thus, patient stratification based on kidney tissue omics offers a gateway to non-invasive biomarker-driven categorization and the potential for future clinical implementation, as a key step towards precision medicine in CKD.
Subject(s)
Disease Progression , Glomerular Filtration Rate , Kidney , Precision Medicine , Renal Insufficiency, Chronic , Transcriptome , Humans , Precision Medicine/methods , Renal Insufficiency, Chronic/pathology , Renal Insufficiency, Chronic/urine , Renal Insufficiency, Chronic/diagnosis , Renal Insufficiency, Chronic/physiopathology , Middle Aged , Female , Male , Kidney/pathology , Kidney/physiopathology , Aged , Biopsy , Adult , Neural Networks, Computer , Case-Control Studies , Gene Expression Profiling , Unsupervised Machine LearningABSTRACT
BACKGROUND AND AIMS: Nephropathy involves pathophysiological changes to the glomerulus. The primary glomerular endothelial cells (GEnCs) have emerged as an important tool for studying glomerulosclerotic mechanisms and in the screening process for drug-candidates. The success of the studies is dependent on the quality of the cell model. Therefore, we set out to establish an easy, reproducible model of the quiescent endothelial monolayer with the use of commercially available extracellular matrices (ECMs). METHODS: Primary hGEnCs were seeded on various ECMs. Cell adhesion was monitored by an impedance sensing system. The localization of junctional proteins was assessed by immunofluorescence and the barrier function by passage of fluorescent dextrans and magnitude of VEGF response. RESULTS: All ECM matrices except recombinant human laminin 111 (rhLN111) supported comparable cell proliferation. Culturing hGEnCs on rhLN521, rhLN511 or fibronectin resulted in a physiologically relevant barrier to 70kDa dextrans which was 82% tighter than that formed on collagen type IV. Furthermore, only hGEnCs cultured on rhLN521 or rhLN511 showed plasma-membrane localized zonula occludens-1 and vascular endothelial cadherin indicative of proper tight and adherens junctions (AJ). CONCLUSION: We recommend culturing hGEnCs on the mature glomerular basement membrane laminin - rhLN521 - which, as the only commercially available ECM, promotes all of the characteristics of the quiescent hGEnC monolayer: cobblestone morphology, well-defined AJs and physiological perm-selectivity.
Subject(s)
Endothelial Cells/physiology , Extracellular Matrix/chemistry , Capillary Permeability , Cell Adhesion , Cell Proliferation/drug effects , Cells, Cultured , Coated Materials, Biocompatible , Collagen Type IV/chemistry , Culture Media , Endothelial Cells/drug effects , Endothelium, Vascular/cytology , Fibronectins/chemistry , Humans , Kidney Glomerulus/blood supply , Laminin/chemistry , Microvessels/cytology , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
Stromal cells actively modulate the inflammatory process, in part by influencing the ability of neighboring endothelial cells to support the recruitment of circulating leukocytes. We hypothesized that podocytes influence the ability of glomerular endothelial cells (GEnCs) to recruit neutrophils during inflammation. To address this, human podocytes and human GEnCs were cultured on opposite sides of porous inserts and then treated with or without increasing concentrations of TNF-α prior to addition of neutrophils. The presence of podocytes significantly reduced neutrophil recruitment to GEnCs by up to 50% when cultures were treated with high-dose TNF-α (100 U/ml), when compared with GEnC monocultures. Importantly, this phenomenon was dependent on paracrine actions of soluble IL-6, predominantly released by podocytes. A similar response was absent when HUVECs were cocultured with podocytes, indicating a tissue-specific phenomenon. Suppressor of cytokine signaling 3 elicited the immunosuppressive actions of IL-6 in a process that disrupted the presentation of chemokines on GEnCs by altering the expression of the duffy Ag receptor for chemokines. Interestingly, suppressor of cytokine signaling 3 knockdown in GEnCs upregulated duffy Ag receptor for chemokines and CXCL5 expression, thereby restoring the neutrophil recruitment. In summary, these studies reveal that podocytes can negatively regulate neutrophil recruitment to inflamed GEnCs by modulating IL-6 signaling, identifying a potential novel anti-inflammatory role of IL-6 in renal glomeruli.
Subject(s)
Cell Communication/immunology , Endothelial Cells/immunology , Interleukin-6/immunology , Neutrophil Infiltration , Neutrophils/immunology , Podocytes/immunology , Cell Communication/genetics , Cell Line, Transformed , Endothelial Cells/cytology , Female , Gene Knockdown Techniques , Humans , Interleukin-6/genetics , Male , Neutrophils/cytology , Podocytes/cytology , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/immunologyABSTRACT
During kidney development, nephron epithelia arise de novo from fate-committed mesenchymal progenitors through a mesenchymal-to-epithelial transition (MET). Downstream of fate specification, transcriptional mechanisms that drive establishment of epithelial morphology are poorly understood. We used human iPSC-derived renal organoids, which recapitulate nephrogenesis, to investigate mechanisms controlling renal MET. Multi-ome profiling via snRNA-seq and ATAC-seq of organoids identified dynamic changes in gene expression and chromatin accessibility driven by activators and repressors throughout MET. CRISPR interference identified that paired box 8 (PAX8) is essential for initiation of MET in human renal organoids, contrary to in vivo mouse studies, likely by activating a cell-adhesion program. While Wnt/ß-catenin signaling specifies nephron fate, we find that it must be attenuated to allow hepatocyte nuclear factor 1-beta (HNF1B) and TEA-domain (TEAD) transcription factors to drive completion of MET. These results identify the interplay between fate commitment and morphogenesis in the developing human kidney, with implications for understanding both developmental kidney diseases and aberrant epithelial plasticity following adult renal tubular injury.
Subject(s)
Kidney , Nephrons , Humans , Mice , Animals , Kidney/metabolism , Cell Differentiation/genetics , Transcription Factors/metabolism , Signal Transduction , Epithelial-Mesenchymal TransitionABSTRACT
Modelling proximal tubule physiology and pharmacology is essential to understand tubular biology and guide drug discovery. To date, multiple models have been developed; however, their relevance to human disease has yet to be evaluated. Here, we report a 3D vascularized proximal tubule-on-a-multiplexed chip (3DvasPT-MC) device composed of co-localized cylindrical conduits lined with confluent epithelium and endothelium, embedded within a permeable matrix, and independently addressed by a closed-loop perfusion system. Each multiplexed chip contains six 3DvasPT models. We performed RNA-seq and compared the transcriptomic profile of proximal tubule epithelial cells (PTECs) and human glomerular endothelial cells (HGECs) seeded in our 3D vasPT-MCs and on 2D transwell controls with and without a gelatin-fibrin coating. Our results reveal that the transcriptional profile of PTECs is highly dependent on both the matrix and flow, while HGECs exhibit greater phenotypic plasticity and are affected by the matrix, PTECs, and flow. PTECs grown on non-coated Transwells display an enrichment of inflammatory markers, including TNF-a, IL-6, and CXCL6, resembling damaged tubules. However, this inflammatory response is not observed for 3D proximal tubules, which exhibit expression of kidney signature genes, including drug and solute transporters, akin to native tubular tissue. Likewise, the transcriptome of HGEC vessels resembled that of sc-RNAseq from glomerular endothelium when seeded on this matrix and subjected to flow. Our 3D vascularized tubule on chip model has utility for both renal physiology and pharmacology.
Subject(s)
Endothelial Cells , Kidney Tubules, Proximal , Humans , Kidney Tubules, Proximal/metabolism , Epithelium , Kidney , Epithelial Cells/metabolism , PhenotypeABSTRACT
Intranasal vaccination represents a promising approach for preventing disease caused by respiratory pathogens by eliciting a mucosal immune response in the respiratory tract that may act as an early barrier to infection and transmission. This study investigated immunogenicity and protective efficacy of intranasally administered messenger RNA (mRNA)-lipid nanoparticle (LNP) encapsulated vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Syrian golden hamsters. Intranasal mRNA-LNP vaccination systemically induced spike-specific binding [immunoglobulin G (IgG) and IgA] and neutralizing antibodies. Intranasally vaccinated hamsters also had decreased viral loads in the respiratory tract, reduced lung pathology, and prevented weight loss after SARS-CoV-2 challenge. Together, this study demonstrates successful immunogenicity and protection against respiratory viral infection by an intranasally administered mRNA-LNP vaccine.
Subject(s)
COVID-19 , Animals , Cricetinae , COVID-19/prevention & control , SARS-CoV-2 , Vaccination , Antibodies, Neutralizing , RNA, Messenger/geneticsABSTRACT
BACKGROUND: Neutrophil recruitment into glomerular tissues and reduced capillary wall integrity has been implicated in the development of vasculitic glomerulonephritis (VGN). This study investigated the stages and mechanisms through which neutrophil serine proteases (SPs), proteinase 3 (PR3) or elastase contribute to endothelial dysfunction. METHODS: Protease-induced damage to endothelium and adhesion molecule upregulation was measured by viability assays and ELISA. Neutrophil/platelet adhesion to human glomerular and umbilical vein endothelium was assessed using in vitro adhesion assays. RESULTS: PR3 and elastase (1 µg/mL, 2 h) significantly induced neutrophil adhesion to endothelial cells (EnC) whilst PR3 also enhanced platelet-EnC interactions. This neutrophil adhesion was associated with enhanced P-selectin expression and required CXCL8 receptor involvement, and could be inhibited by blocking the P-selectin ligand PSGL-1. SPs induced damage in a time- and dose-dependent fashion, decreasing cell monolayer integrity followed by cell membrane integrity, inducing caspase-3 activation and p21 cleavage. However, SPs caused significant EnC damage with increasing concentrations and prolonged exposures. CONCLUSION: Neutrophil SPs induce a pro-adhesive phenotype in glomerular endothelium primarily by inducing neutrophil and platelet adhesion that transits to dysfunction after high/prolonged exposures. Dysregulated release of these enzymes within glomeruli may contribute to injury during diseases such as VGN.
Subject(s)
Inflammation/enzymology , Kidney Glomerulus/enzymology , Kidney Glomerulus/immunology , Myeloblastin/physiology , Neutrophil Infiltration/physiology , Pancreatic Elastase/physiology , Urothelium/enzymology , Urothelium/immunology , HumansABSTRACT
A new vectored vaccine MVA-VLP-SUDV was generated against Sudan ebolavirus (SUDV) combining the advantages of the immunogenicity of a live attenuated vaccine vector (Modified Vaccinia Ankara, MVA) with the authentic conformation of virus-like particles (VLPs). The vaccine expresses minimal components to generate self-assembling VLPs in the vaccinee: the envelope glycoprotein GP and the matrix protein VP40. Guinea pigs vaccinated with one dose of MVA-VLP-SUDV generated SUDV-specific binding and neutralizing antibody responses as well as Fc-mediated protective effects. These responses were boosted by a second vaccine dose. All vaccinated animals which received either one or two vaccine doses were protected from death and disease symptoms following challenge with a lethal dose of SUDV. These data demonstrate single dose protection and potency of the MVA-VLP platform for use in emergency situations to contain outbreaks.
ABSTRACT
Dysregulated release of neutrophil azurophilic granules causes increased tissue damage and amplified inflammation during autoimmune disease. Antineutrophil cytoplasmic antibodies (ANCAs) are implicated in the pathogenesis of small vessel vasculitis and promote adhesion and exocytosis in neutrophils. ANCAs activate specific signal transduction pathways in neutrophils that have the potential to be modulated therapeutically to prevent neutrophil activation by ANCAs. We have investigated a role for diacylglycerol kinase (DGK) and its downstream product phosphatidic acid (PA) in ANCA-induced neutrophil exocytosis. Neutrophils incubated with the DGK inhibitor R59022, before treatment with ANCAs, exhibited a reduced capacity to release their azurophilic granules, demonstrated by a component release assay and flow cytometry. PA restored azurophilic granule release in DGK-inhibited neutrophils. Confocal microscopy revealed that R59022 did not inhibit translocation of granules, indicating a role for DGK during the process of granule fusion at the plasma membrane. In investigating possible mechanisms by which PA promotes neutrophil exocytosis, we demonstrated that exocytosis can only be restored in R59022-treated cells through simultaneous modulation of membrane fusion and increasing cytosolic calcium. PA and its associated pathways may represent viable drug targets to reduce tissue injury associated with ANCA-associated vasculitic diseases and other neutrophilic inflammatory disorders.
Subject(s)
Antibodies, Antineutrophil Cytoplasmic/immunology , Diacylglycerol Kinase/metabolism , Exocytosis/immunology , Neutrophils/cytology , Neutrophils/enzymology , Phosphatidic Acids/biosynthesis , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Actins/metabolism , Calcium/metabolism , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/metabolism , Diacylglycerol Kinase/antagonists & inhibitors , Enzyme Activation/drug effects , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin G/immunology , Intracellular Space/drug effects , Intracellular Space/metabolism , Ionomycin/pharmacology , Lysophosphatidylcholines/metabolism , Lysophosphatidylcholines/pharmacology , Membrane Fusion/drug effects , Neutrophils/immunology , Peroxidase/metabolism , Pyrimidinones/pharmacology , Tetraspanin 30/metabolism , Thiazoles/pharmacologyABSTRACT
The development of effective countermeasures against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent responsible for the COVID-19 pandemic, is a priority. We designed and produced ConVac, a replication-competent vesicular stomatitis virus (VSV) vaccine vector that expresses the S1 subunit of SARS-CoV-2 spike protein. We used golden Syrian hamsters as animal models of severe COVID-19 to test the efficacy of the ConVac vaccine. A single vaccine dose elicited high levels of SARS-CoV-2 specific binding and neutralizing antibodies; following intranasal challenge with SARS-CoV-2, animals were protected from weight loss and viral replication in the lungs. No enhanced pathology was observed in vaccinated animals upon challenge, but some inflammation was still detected. The data indicate rapid control of SARS-CoV-2 replication by the S1-based VSV-vectored SARS-CoV-2 ConVac vaccine.
ABSTRACT
Anti-proteinase 3 antibodies are implicated in the pathogenesis of small vessel vasculitis. These are primarily immunoglobulin G (IgG), with different subclasses predominating at different stages of disease. However, little is known of their respective roles in pathogenesis. We have previously shown that patient IgG4 was able to induce superoxide release from human neutrophils. To circumvent difficulties in separating the subclasses and additional differences in polyclonal patient antibodies we have generated monoclonal mouse/human IgG1 and IgG4 anti-proteinase 3 antibodies. Using these antibodies we have compared effects of IgG1 and IgG4 on human neutrophils in terms of superoxide release, cytokine production, degranulation and adhesion. Additionally we have investigated the interaction of the subclasses with Fc receptors expressed by the neutrophil. Chimeric antibodies were generated using human constant regions of each subclass and a variable region taken from a monoclonal antibody directed against proteinase 3. Superoxide release from neutrophils was measured by the reduction of ferricytochrome C, degranulation by the conversion of a synthetic colour substrate, cytokine release by interleukin-8 enzyme-linked immunosorbent assay, and adhesion by a flow-based adhesion assay. Fc receptor binding was assessed using blocking antibodies. The IgG4 anti-proteinase 3 was able to induce a dose-dependent release of superoxide, degranulation and adhesion. The antibody was not able to stimulate the secretion of interleukin-8. Fc receptors were essential for neutrophil stimulation and the constitutive Fc receptors were necessary for different stimulatory pathways. The IgG4 anti-proteinase 3 antibodies are able to stimulate neutrophils to undergo a pro-inflammatory response and may play a role in the pathogenesis of small vessel vasculitis.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Antibodies, Antineutrophil Cytoplasmic/immunology , Immunoglobulin G/immunology , Myeloblastin/immunology , Neutrophils/immunology , Cell Adhesion/immunology , Cell Degranulation/immunology , Cells, Cultured , Humans , Interleukin-8/biosynthesis , Neutrophil Activation/immunology , Receptors, IgG/metabolism , Recombinant Fusion Proteins/immunology , Respiratory Burst/immunologyABSTRACT
OBJECTIVE: To investigate whether single nucleotide polymorphisms (SNPs) within cytotoxic T-lymphocyte antigen-4 (CTLA-4) are associated with ANCA-associated small vessel vasculitis (SVV). METHODS: The CTLA-4 CT60 (exon 4), +49 (exon 1) and -318 (promoter region) genotypes were determined by PCR and restriction fragment length polymorphism (RFLP) in 222 white Caucasians of UK origin with SVV and 670 ethnically matched controls. RESULTS: The CTLA-4 exon 1 (+49) and 4 (CT60) polymorphisms are associated with SVV (+49: chi(2) = 10.965, P = 0.004; CT60: chi(2) = 12.017, P = 0.002). Both disease-susceptible and disease-protective haplotypes have been identified in this cohort, and their frequencies are similar in the subtypes of WG and microscopic polyangiitis. CONCLUSION: This study provides further evidence that CTLA-4, a susceptibility locus for a number of common autoimmune diseases, may also be involved in the development of ANCA-associated SVV.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/genetics , Antigens, CD/genetics , Polymorphism, Single Nucleotide , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , CTLA-4 Antigen , Case-Control Studies , Gene Frequency , Genetic Predisposition to Disease , Genotype , Haplotypes , HumansABSTRACT
Antineutrophil cytoplasm antibodies (ANCA) activate neutrophils to undergo a series of coordinated interactions, leading to transendothelial migration, eventually culminating in vascular destruction. The molecular events underlying neutrophil recruitment in ANCA-associated vasculitis need to be defined to enable effective therapeutic manipulation. A flow-based adhesion assay was used to investigate the role of beta2 integrins (CD11a/CD18 and CD11b/CD18) and chemokine receptors [CXC chemokine receptor (CXCR)1 and CXCR2] in neutrophil migration through the endothelium. Two endothelial models were used: a highly activated model stimulated with 100 U/ml tumor necrosis factor alpha (TNF-alpha) and a minimally activated model stimulated with 2 U/ml TNF-alpha and in which ANCA was present as a secondary neutrophil stimulus. CD11a/CD18, CD11b/CD18, and CXCR2 contributed to adhesion and transendothelial migration in both models. However, when the endothelium was minimally activated with TNF-alpha, CD11b/CD18 played an important role in stabilizing adhesion induced by ANCA immunoglobulin G (IgG). Analysis of beta2 integrins and chemokine receptors demonstrated that ANCA IgG had no effect on expression levels at the neutrophil surface but enabled an active conformational change of CD11b/CD18. Similar molecular mechanisms control neutrophil adhesion and migration through highly or minimally TNF-alpha-activated endothelium. However, the direct ANCA-mediated neutrophil stimulation is needed to drive migration through minimally activated endothelium, and CD11b/CD18 is recruited for greater stability of adhesion during this process and can undergo an activatory, conformational change in response to ANCA IgG.
Subject(s)
Antibodies, Antineutrophil Cytoplasmic/pharmacology , CD18 Antigens/metabolism , Cytokines/metabolism , Endothelium, Vascular/metabolism , Neutrophils/metabolism , Receptors, Interleukin-8A/metabolism , Receptors, Interleukin-8B/metabolism , Cell Adhesion , Cell Movement , Cells, Cultured , HumansABSTRACT
Small vessel vasculitis of the kidney is an inflammatory disorder that results in glomerulonephritis. Current treatment regimes have proved effective, with patient survival rates of 80% at five years. Of the 20% that die, a significant number of deaths are due to the side effects of therapies, and immunosuppression. A more tailored approach is required that targets the cells involved rather than generally restraining the immune system. This can only be achieved by increasing our knowledge of disease pathogenesis.
Subject(s)
Drug Delivery Systems , Immunosuppressive Agents/therapeutic use , Kidney Diseases/drug therapy , Vasculitis/drug therapy , Animals , Antibodies, Antineutrophil Cytoplasmic/metabolism , Apoptosis/drug effects , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Adhesion/drug effects , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Humans , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/pharmacology , Inflammation Mediators/metabolism , Kidney Diseases/immunology , Neutrophil Activation/drug effects , Neutrophil Infiltration/drug effects , Neutrophils/drug effects , Neutrophils/metabolism , Phagocytosis/drug effects , Signal Transduction/drug effects , Vasculitis/immunologyABSTRACT
We investigated the presence and potential role of N-glycosylation of the human (h) 5-hydroxytryptamine3 (5-HT3A) receptor subunit expressed in COS-7 cells. Incubation of cells with the N-glycosylation inhibitor, tunicamycin, reduced the molecular weight of the predominant immunoreactive h5-HT3A subunit species (from approximately 59 to 45 kDa) indicating that the h5-HT3A subunit is normally N-glycosylated. Site-directed mutagenesis studies individually substituting four identified N-terminal asparagines (N5, N81, N147, N163) demonstrated that each expressed mutant displayed a reduced molecular weight (by approximately 3 kDa) suggesting that each asparagine residue was subject to N-glycosylation. In addition, 5-HT3 receptor binding studies indicated that prevention of N-glycosylation, by individual amino acid substitution at each of the four asparagine residues, either prevented (N81, N147, N163) or greatly reduced (N5) the production of a 5-HT3 receptor binding site. Corresponding with the radioligand binding studies, immunocytochemical studies demonstrated that substitution of each asparagine either prevented (N81, N147, N163) or reduced considerably (N5) mutant protein expression within the cell membrane. The present study demonstrates an important role for N-glycosylation at multiple identified asparagine residues in the N-terminus of the h5-HT3A receptor subunit.
Subject(s)
Protein Subunits/metabolism , Receptors, Serotonin, 5-HT3/metabolism , Amino Acid Sequence , Animals , COS Cells , Glycosylation , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Processing, Post-Translational , Protein Subunits/genetics , Radioligand Assay , Receptors, Serotonin, 5-HT3/geneticsABSTRACT
Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulonephritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR2, TFLLR-NH(2) and SLIGKV-NH(2,) respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways.
Subject(s)
Endothelial Cells/cytology , Kidney Glomerulus/cytology , Myeloblastin/metabolism , Pancreatic Elastase/metabolism , Receptor, PAR-1/metabolism , Signal Transduction , von Willebrand Factor/metabolism , Calcium/metabolism , Endothelial Cells/metabolism , HEK293 Cells , Humans , ProteolysisABSTRACT
Patients with certain forms of systematic vasculitis, such as Wegener's granulomatosis, have circulating antineutrophil cytoplasmic antibodies (ANCA). These inappropriately stimulate circulating neutrophils adhere to and thereby obstruct small vessels. This, together with ANCA-induced degranulation and an oxidative burst, leads to local tissue damage. The signaling pathways that are activated by ANCA IgG are distinct from those that are involved in normal neutrophil activation. This study shows that diacylglycerol kinase is selectively activated by ANCA and that the generated phosphatidic acid is responsible for promoting neutrophil adhesion, in part through integrin activation. The data presented point to diacylglycerol kinase alpha as a novel but selective target for the development of drugs to treat this potentially fatal disorder.
Subject(s)
Antibodies, Antineutrophil Cytoplasmic/physiology , Diacylglycerol Kinase/physiology , Neutrophils/physiology , Phosphatidic Acids/biosynthesis , Calcium/metabolism , Catalysis , Cell Adhesion , Chromones/pharmacology , Humans , Immunoglobulin G/physiology , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/physiology , Phospholipase C gamma/physiology , Signal TransductionABSTRACT
BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) are associated with small-vessel vasculitis and have been implicated in its pathogenesis. The subclass distribution of ANCA IgG deviates from normal patterns, and it has been suggested that the IgG3 subclass may have pathogenic potential over the IgG1 subclass and may be more likely to be associated with active disease and renal involvement. OBJECTIVE: To deal with potential pathogenicity, chimeric antibodies were constructed of IgG1 and three subclasses with human IgG1 or three constant regions and a murine-derived variable region that binds an epitope within the ANCA antigen proteinase 3 (PR3) that is recognised by human autoantibodies. METHODS: The antibodies were characterised for binding to PR3, including affinity and avidity, before being used as tools to explore their ability to activate human neutrophils for superoxide release, cytokine release, degranulation and ability to induce neutrophil adhesion under flow. RESULTS: Both subclass antibodies elicited similar neutrophil responses for superoxide release, degranulation and interleukin (IL) 8 production, although quantitative responses showed that the IgG1 subclass favoured degranulation and the IgG3 subclass favoured IL8 production. Both antibodies were able to convert neutrophils from selectin-dependent rolling adhesion to integrin-dependent stationary adhesion in a flow assay. CONCLUSIONS: These findings indicate that humanised antibodies directed against a single epitope of PR3 can recapitulate the effects of polyclonal human ANCA, which recognises multiple PR3 epitopes. Further, PR3-ANCA of both IgG1 and IgG3 subclasses can activate neutrophils, although the more potent IL8 response by IgG3 PR3-ANCA may encourage further neutrophil recruitment and amplify injury.
Subject(s)
Antibodies, Antineutrophil Cytoplasmic/immunology , Immunoglobulin G/immunology , Myeloblastin/immunology , Neutrophils/immunology , Animals , Antibody Affinity/immunology , Cell Adhesion/immunology , Cells, Cultured , Cricetinae , Cytokines/immunology , Dose-Response Relationship, Immunologic , Epitopes/immunology , Humans , Immunoglobulin G/biosynthesis , Mice , Pancreatic Elastase/immunology , Receptors, IgG/immunology , Signal Transduction/immunology , Superoxides/immunologyABSTRACT
Antineutrophil cytoplasm antibodies (ANCA) are implicated in the pathogenesis of systemic vasculitis. ANCA are directed against antigens expressed on the surface of cytokine-primed neutrophils. It was shown previously that whole IgG ANCA and its fraction antigen binding [F(ab')(2)] fragment can activate the GTPase p21(ras). This study shows a functional involvement of this molecule in the ANCA activation of neutrophils by inhibiting the production of superoxide with farnesylthiosalicylic acid. Using the ras activation assay, farnesylthiosalicylic acid inhibits p21(ras) binding to its substrate at comparable concentrations to those seen for superoxide inhibition. It is also shown that activation of p21(ras) by ANCA is transient, peaking at 5 to 10 min and returning to baseline by 30 min. The use of ras isoform-specific antibodies in Western blots established, for the first time, that Harvey-ras is not present in human neutrophils, but both Kirsten-ras (K-ras) and Neuronal-ras are. Stimulation with ANCA is able to differentially activate K-ras without effects on neuronal-ras. The activation of p21(ras) by ANCA and its F(ab')(2) is prevented by inhibition of both Src kinases and phosphatidylinositol-3-kinase, indicating a cooperative role for both molecules in the G protein pathway activated by ANCA F(ab')(2) upstream of p21(ras). It is concluded that ANCA selectively activates K-ras during induction of a respiratory burst via pathways involving multiple upstream kinases.
Subject(s)
Antibodies, Antineutrophil Cytoplasmic/immunology , Farnesol/analogs & derivatives , Neutrophils/immunology , Proto-Oncogene Proteins p21(ras)/immunology , Proto-Oncogene Proteins p21(ras)/metabolism , Antibodies, Antineutrophil Cytoplasmic/pharmacology , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Farnesol/pharmacology , Humans , Immunoglobulin G/immunology , Immunoglobulin G/pharmacology , Isomerism , Morpholines/pharmacology , Neutrophils/drug effects , Proto-Oncogene Proteins p21(ras)/chemistry , Respiratory Burst/immunology , Salicylates/pharmacology , Superoxides/metabolismABSTRACT
ANCA (anti-neutrophil cytoplasm antibody)-associated small vessel vasculitis is an inflammatory condition associated with the production of autoantibodies to neutrophil cytoplasmic components. The disorder results in destruction of the microvasculature, infiltration of neutrophils into tissues, which is followed later by mononuclear cells, leading to injury and the formation of granulomatous lesions. Initiators for the disease are undetermined but a pro-inflammatory environment is required. Other influencing factors may include environmental triggers, genetic propensity or infectious agents. The primary cellular event in the condition involves the neutrophils, which are likely to be responsible for the majority of tissue injury. Binding of the autoantibody to neutrophils initiates cell activation via a complex intracellular signalling cascade, culminating in the release of pro-inflammatory mediators, proteolytic enzymes and reactive oxygen species. Adhesion of neutrophils to endothelial cells is observed in vitro and more investigations in this area may explain the focussing of the disease to certain vessels/tissues. Current treatment regimens have substantial toxicity. Although newer developments are an improvement there is still a pressing need for more targeted therapies, which could be provided by extrapolating information emerging from basic scientific research.