Autoantibody Profiling and Anti-Kinesin Reactivity in ANCA-Associated Vasculitis.

ANCA-associated vasculitides (AAV) are rare autoimmune diseases causing inflammation and damage to small blood vessels. New autoantibody biomarkers are needed to improve the diagnosis and treatment of AAV patients. In this study, we aimed to profile the autoantibody repertoire of AAV patients using in-house developed antigen arrays to identify previously unreported antibodies linked to the disease per se, clinical subgroups, or clinical activity. A total of 1743 protein fragments representing 1561 unique proteins were screened in 229 serum samples collected from 137 AAV patients at presentation, remission, and relapse. Additionally, serum samples from healthy individuals and patients with other type of vasculitis and autoimmune-inflammatory conditions were included to evaluate the specificity of the autoantibodies identified in AAV. Autoreactivity against members of the kinesin protein family were identified in AAV patients, healthy volunteers, and disease controls. Anti-KIF4A antibodies were significantly more prevalent in AAV. We also observed possible associations between anti-kinesin antibodies and clinically relevant features within AAV patients. Further verification studies will be needed to confirm these findings.

An adapted passive model of anti-MPO dependent crescentic glomerulonephritis reveals matrix dysregulation and is amenable to modulation by CXCR4 inhibition.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are severe inflammatory disorders that often involve focal necrotizing glomerulonephritis (FNGN) and consequent glomerular scarring, interstitial fibrosis, and chronic kidney disease. Robust murine models of scarring in FNGN that may help to further our understanding of deleterious processes are still lacking. Here, we present a murine model of severe FNGN based on combined administration of antibodies against the glomerular basement membrane (GBM) and myeloperoxidase (MPO), and bacterial lipopolysaccharides (LPS), that recapitulates acute injury and was adapted to investigate subsequent glomerular and interstitial scarring. Hematuria without involvement of other organs occurs consistently and rapidly, glomerular necrosis and crescent formation are evident at 12 days, and consequent glomerular and interstitial scarring at 29 days after initial treatment. Using mass-spectrometric proteome analysis, we provide a detailed overview of matrisomal and cellular changes in our model. We observed increased expression of the matrisome including collagens, fibronectin, tenascin-C, in accordance with human AAV as deduced from analysis of gene expression microarrays and tissue staining. Moreover, we observed tissue infiltration by neutrophils, macrophages, T cells and myofibroblasts upon injury. Experimental inhibition of CXCR4 using AMD3100 led to a sustained histological presence of fibrin extravasate, reduced chemokine expression and leukocyte activation, but did not markedly affect ECM composition. Altogether, we demonstrate an adapted FNGN model that enables the study of matrisomal changes both in disease and upon intervention, as exemplified via CXCR4 inhibition.

Isolation of Small Extracellular Vesicles from Human Sera.

Robust, well-characterized methods for purifying small extracellular vesicles (sEV) from blood are needed before their potential as disease biomarkers can be realized. Here, we compared isolation of sEV from serum by differential ultracentrifugation (DUC) and by exclusion chromatography using commercially available Exo-spin™ columns. We show that sEV can be purified by both methods but Exo-spin™ columns contain copious additional particles recorded by nanoparticle tracking analysis, invalidating its use for quantifying yields. DUC samples contained higher concentrations of exosome specific proteins CD9, CD63 and CD81 and electron microscopy confirmed that most particles in DUC preparations were sEV, whereas Exo-spin™ samples also contained copious co-purified plasma lipids. MACSPlex bead analysis identified multiple exosome surface proteins, with stronger signals in DUC samples, enabling detection of 21 of 37, compared to only 10 in Exo-spin™ samples. Nevertheless, the pattern of expression was consistent in both preparations, indicating that lipids do not interfere with bead-based technologies. Thus, both DUC and Exo-spin™ can be used to isolate sEV from human serum and what is most appropriate depends on the subsequent use of sEV. In summary, Exo-spin™ enables isolation of sEV from blood with vesicle populations similar to the ones recovered by DUC, but with lower concentrations.

Lymphangiogenesis in a mouse model of renal transplant rejection extends life span of the recipients.

Renal allograft rejection can be prevented by immunological tolerance, which may be associated with de novo formed lymphatic vessels in the donor kidney after transplantation in man. A suitable mouse model of renal allograft rejection in which lymphangiogenesis can be deliberately induced in the graft is critical for elucidating the mechanisms responsible for the association between attenuated transplant rejection and abundance of lymphatic vessels. Here we describe the development of a novel mouse model of rapid renal transplant rejection in which transgenic induction of lymphangiogenesis in the immune-incompatible graft greatly extends its survival time. Thus, our novel approach may facilitate exploitation of lymphangiogenesis in the grafted organ.

Dendritic cells and routing cargo into exosomes.

Extracellular vesicles, released from cells, are important for intercellular communication. They are heterogeneous but fall into two broad categories based on origin and function: microvesicles formed by outward budding from the plasma membrane; and exosomes that originate as intraluminal vesicles in multivesicular endosomes that fuse with the plasma membrane to release them. Extracellular vesicles generally and exosomes in particular have powerful effects on specific immune responses, and recent advances highlight their potential therapeutic uses. Dendritic cells (DC) that have internalized antigen release exosomes that express MHC class II molecules loaded with antigenic peptides, co-stimulatory molecules and intact antigen. Depending on the setting, these stimulate CD4 T-cell proliferation either directly or only in the context of accessory antigen naïve DC. Here, we discuss the reasons for this; and review current knowledge about the loading of antigen, class II and other cargo into exosomes released by DC and other professional antigen-presenting cells in the context of advances in exosome biology more generally.

Surface LAMP-2 Is an Endocytic Receptor That Diverts Antigen Internalized by Human Dendritic Cells into Highly Immunogenic Exosomes.

The lysosome-associated membrane protein (LAMP) family includes the dendritic cell endocytic receptors DC-LAMP and CD68, as well as LAMP-1 and LAMP-2. In this study we identify LAMP-1 (CD107a) and LAMP-2 (CD107b) on the surface of human monocyte-derived dendritic cells (MoDC) and show only LAMP-2 is internalized after ligation by specific Abs, including H4B4, and traffics rapidly but transiently to the MHC class II loading compartment, as does Ag conjugated to H4B4. However, pulsing MoDC with conjugates of primary (keyhole limpet hemocyanin; KLH) and recall (Bet v 1) Ags (H4B4*KLH and H4B4*Bet v 1) induced significantly less CD4 cell proliferation than pulsing with native Ag or Ag conjugated to control mAb (ISO*KLH and ISO*Bet v 1). In H4B4*KLH-pulsed MoDC, the duration of KLH residence in MHC class II loading compartments was significantly reduced, as were surface HLA-DR and DR-bound KLH-derived peptides. Paradoxically, MoDC pulsed with H4B4*KLH, but not the other KLH preparations, induced robust proliferation of CD4 cells separated from them by a transwell membrane, indicating factors in the supernatant were responsible. Furthermore, extracellular vesicles from supernatants of H4B4*KLH-pulsed MoDC contained significantly more HLA-DR and KLH than those purified from control MoDC, and KLH was concentrated specifically in exosomes that were a uniquely effective source of Ag in standard T cell proliferation assays. In summary, we identify LAMP-2 as an endocytic receptor on human MoDC that routes cargo into unusual Ag processing pathways, which reduces surface expression of Ag-derived peptides while selectively enriching Ag within immunogenic exosomes. This novel pathway has implications for the initiation of immune responses both locally and at distant sites.

The Phenotypic Characterization of the Human Renal Mononuclear Phagocytes Reveal a Co-Ordinated Response to Injury.

Mammalian tissues contain networks of mononuclear phagocytes (MPh) that sense injury and orchestrate the response to it. In mice, this is affected by distinct populations of dendritic cells (DC), monocytes and macrophages and recent studies suggest the same is true for human skin and intestine but little is known about the kidney. Here we describe the analysis of MPh populations in five human kidneys and show they are highly heterogeneous and contain discrete populations of DC, monocytes and macrophages. These include: plasmacytoid DC (CD303+) and both types of conventional DC-cDC1 (CD141+ cells) and CD2 (CD1c+ cells); classical, non-classical and intermediate monocytes; and macrophages including a novel population of CD141+ macrophages clearly distinguishable from cDC1 cells. The relative size of the MPh populations differed between kidneys: the pDC population was bi-modally distributed being less than 2% of DC in two kidneys without severe injury and over 35% in the remaining three with low grade injury in the absence of morphological evidence of inflammation. There were profound differences in the other MPh populations in kidneys with high and low numbers of pDC. Thus, cDC1 cells were abundant (55 and 52.3%) when pDC were sparse and sparse (12.8-12.5%) when pDC were abundant, whereas the proportions of cDC2 cells and classical monocytes increased slightly in pDC high kidneys. We conclude that MPh are highly heterogeneous in human kidneys and that pDC infiltration indicative of low-grade injury does not occur in isolation but is part of a co-ordinated response affecting all renal DC, monocyte and macrophage populations.

A critical role for suppressor of cytokine signalling 3 in promoting M1 macrophage activation and function in vitro and in vivo.

Macrophages respond to their microenvironment and develop polarized functions critical for orchestrating appropriate inflammatory responses. Classical (M1) activation eliminates pathogens while alternative (M2) activation promotes regulation and repair. M1 macrophage activation is strongly associated with suppressor of cytokine signalling 3 (SOCS3) expression in vitro, but the functional consequences of this are unclear and the role of SOCS3 in M1-macrophage polarization in vivo remains controversial. To address these questions, we defined the characteristics and function of SOCS3-expressing macrophages in vivo and identified potential mechanisms of SOCS3 action. Macrophages infiltrating inflamed glomeruli in a model of acute nephritis show significant up-regulation of SOCS3 that co-localizes with the M1-activation marker, inducible nitric oxide synthase. Numbers of SOCS3(hi) -expressing, but not SOCS1(hi) -expressing, macrophages correlate strongly with the severity of renal injury, supporting their inflammatory role in vivo. Adoptive transfer of SOCS3-short interfering RNA-silenced macrophages into a peritonitis model demonstrated the importance of SOCS3 in driving production of pro-inflammatory IL-6 and nitric oxide, while curtailing expression of anti-inflammatory IL-10 and SOCS1. SOCS3-induced pro-inflammatory effects were due, at least in part, to its role in controlling activation and nuclear accumulation of nuclear factor-κB and activity of phosphatidylinositol 3-kinase. We show for the first time that SOCS3 also directs the functions of human monocyte-derived macrophages, including efficient M1-induced cytokine production (IL-1ß, IL-6, IL-23, IL-12), attenuated signal transducer and activator of transcription 3 activity and ability of antigen-loaded macrophages to drive T-cell responses. Hence, M1-associated SOCS3 was a positive regulator of pro-inflammatory responses in our rodent models and up-regulated SOCS3 is essential for effective M1-macrophage activation and function in human macrophages.

Autoantibodies to hLAMP-2 in ANCA-negative pauci-immune focal necrotizing GN.

Pauci-immune focal necrotizing GN (piFNGN) is usually associated with ANCAs that are thought to be pathogenic. However, 10%-15% of patients are ANCA negative and the cause of their injury is unknown. We previously reported a high frequency of autoantibodies to human lysosome-associated membrane protein-2 (hLAMP-2) in ANCA-associated piFNGN, and have now investigated whether the same is true in ANCA-negative patients. Of 11 patients, 8 (73%) had anti-hLAMP-2 antibodies detected by ELISA and confirmed by immunoblotting and indirect immunofluorescence. The autoantibodies from all 8 patients bound to native LAMP-2 purified from human glomeruli and recombinant hLAMP-2 expressed in ldlD cells, both with molecular masses of 110 kD. However, in contrast to anti-LAMP-2 antibodies from ANCA-positive patients, these antibodies from ANCA-negative patients failed to bind the more complexly glycosylated native neutrophil hLAMP-2 (190 kD). Treatment with the deglycosylating enzyme, endo-ß-galactosidase, reduced the mass of neutrophil hLAMP-2 to 110 kD and enabled autoantibody binding. Similarly, pretreating neutrophils with endo-ß-galactosidase or neuraminidase converted ANCA assay results from negative to positive. Finally, IgG from LAMP-2-positive ANCA-negative patients bound specifically to normal human kidney sections and to human glomerular endothelial cells in culture. In conclusion, in patients with ANCA-negative piFNGN, we have identified autoantibodies to hLAMP-2 that bind native glomerular but not neutrophil hLAMP-2, suggesting a role in pathogenesis.

The immune system and kidney disease: basic concepts and clinical implications.

The kidneys are frequently targeted by pathogenic immune responses against renal autoantigens or by local manifestations of systemic autoimmunity. Recent studies in rodent models and humans have uncovered several underlying mechanisms that can be used to explain the previously enigmatic immunopathology of many kidney diseases. These mechanisms include kidney-specific damage-associated molecular patterns that cause sterile inflammation, the crosstalk between renal dendritic cells and T cells, the development of kidney-targeting autoantibodies and molecular mimicry with microbial pathogens. Conversely, kidney failure affects general immunity, causing intestinal barrier dysfunction, systemic inflammation and immunodeficiency that contribute to the morbidity and mortality of patients with kidney disease. In this Review, we summarize the recent findings regarding the interactions between the kidneys and the immune system.

What is the evidence for antibodies to LAMP-2 in the pathogenesis of ANCA associated small vessel vasculitis?

PURPOSE OF REVIEW: This review critically analyses the data implicating antibodies to lysosome associated membrane protein-2 (hLAMP-2) in ANCA-associated vasculitis (AAV). It addresses recent controversies over prevalence of anti-hLAMP-2 antibodies as well as their potential for diagnosis and monitoring disease activity. RECENT FINDINGS: Anti-hLAMP-2 antibodies were first described in the 1990s and have become the focus of intense clinical interest in the past 4 years. This followed the demonstration of their very high prevalence in untreated patients presenting with AAV but absence when patients were in remission. The data also demonstrated molecular mimicry between hLAMP-2 and the bacterial protein FimH. The same group later confirmed the original findings and showed the anti-hLAMP-2 autoantibodies have different kinetics to those recognising myeloperoxidase and proteinase-3 and are less likely to be detectable when the disease is in remission. By contrast, a different group reported a lower prevalence of anti-hLAMP-2 antibodies in AAV and questioned their relevance to pathogenesis. Critical analysis of these studies suggests that the differences are largely attributable to selection criteria of the AAV patients studied and the assays used. SUMMARY: Anti-hLAMP-2 antibodies are frequently found in AAV but attempts to define their consequences have been frustrated by lack of generally available assays for them.

Characterisation of tumour-infiltrating macrophages: impact on response and survival in patients receiving primary chemotherapy for breast cancer.

The role of the tumour microenvironment and complex cellular interactions has attracted interest in responses to primary chemotherapy. Of particular interest are tumour-infiltrating T cells and tumour-infiltrating macrophages (TIMs). We evaluated TIMs and their key activation markers in patients with breast cancer undergoing primary chemotherapy related to response and survival. One hundred and ninety nine patients with large or locally advanced breast cancers received primary chemotherapy. Clinical data, histopathological responses to chemotherapy and survival were examined related to infiltrating cells in tumour microenvironments: cluster of differentiation (CD)3 (pan T cell); CD4 (helper T cells); CD8 (cytotoxic T cells); CD25 (activated T cells); CD68, suppressor of cytokine signalling (SOCS)1, SOCS3 (macrophages); and CD11c and CD205 (dendritic). In tumours demonstrating better responses to chemotherapy, there were significantly fewer CD4(+) T-helper cells than a poorer response (p < 0.05). There were increased numbers of SOCS3 expressing macrophages (pro-inflammatory) in tumours with complete pathological responses compared with no response to chemotherapy (p < 0.05). There was no association between SOCS1 expressing macrophages (anti-inflammatory) and tumour response. Multivariate analysis revealed that factors indicating better survival were receiving anthracycline plus docetaxel (ExpB = 1.166; p = 0.006), better pathological chemotherapy response (ExpB = 0.309; p = 0.009) and a low macrophage SOCS1 expression (ExpB = 13.465; p = 0.044). This study highlights the heterogeneity of TIMs and provides further insight into complex interactions within tumours. The results emphasise the importance of characterising activation status of infiltrating macrophages and provides proof of principle for using macrophage SOCS protein expression as a survival predictor. The apparent impact of macrophage subsets on overall survival underlines the therapeutic potential of manipulating macrophage activation in cancer.

Genetically distinct subsets within ANCA-associated vasculitis.

BACKGROUND: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a severe condition encompassing two major syndromes: granulomatosis with polyangiitis (formerly known as Wegener's granulomatosis) and microscopic polyangiitis. Its cause is unknown, and there is debate about whether it is a single disease entity and what role ANCA plays in its pathogenesis. We investigated its genetic basis. METHODS: A genomewide association study was performed in a discovery cohort of 1233 U.K. patients with ANCA-associated vasculitis and 5884 controls and was replicated in 1454 Northern European case patients and 1666 controls. Quality control, population stratification, and statistical analyses were performed according to standard criteria. RESULTS: We found both major-histocompatibility-complex (MHC) and non-MHC associations with ANCA-associated vasculitis and also that granulomatosis with polyangiitis and microscopic polyangiitis were genetically distinct. The strongest genetic associations were with the antigenic specificity of ANCA, not with the clinical syndrome. Anti-proteinase 3 ANCA was associated with HLA-DP and the genes encoding α(1)-antitrypsin (SERPINA1) and proteinase 3 (PRTN3) (P=6.2×10(-89), P=5.6×10(-12,) and P=2.6×10(-7), respectively). Anti-myeloperoxidase ANCA was associated with HLA-DQ (P=2.1×10(-8)). CONCLUSIONS: This study confirms that the pathogenesis of ANCA-associated vasculitis has a genetic component, shows genetic distinctions between granulomatosis with polyangiitis and microscopic polyangiitis that are associated with ANCA specificity, and suggests that the response against the autoantigen proteinase 3 is a central pathogenic feature of proteinase 3 ANCA-associated vasculitis. These data provide preliminary support for the concept that proteinase 3 ANCA-associated vasculitis and myeloperoxidase ANCA-associated vasculitis are distinct autoimmune syndromes. (Funded by the British Heart Foundation and others.).

High prevalence of autoantibodies to hLAMP-2 in anti-neutrophil cytoplasmic antibody-associated vasculitis.

The involvement of autoantibodies to human lysosome-associated membrane protein-2 (hLAMP-2) in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is controversial because of the absence of confirmatory data subsequent to the initial reports of their high prevalence in this disease. We characterized three assays for anti-hLAMP-2 antibodies: ELISA and Western blotting assays using unglycosylated recombinant hLAMP-2 expressed in Escherichia coli, and an indirect immunofluorescence assay using stably transfected ldlD cells that expressed glycosylated full-length hLAMP-2 on the plasma membrane. The assays detected autoantibodies to hLAMP-2 in human sera reproducibly and with comparable sensitivity and the assays gave the same results in 80.5% of the test panel of 40 selected positive and negative sera. In untreated patients at presentation, the frequencies of autoantibodies to LAMP-2 were 89%, 91%, and 80%, respectively, among three groups of patients with ANCA-associated vasculitis from Vienna, Austria (n=19); Groningen, the Netherlands (n=50) and Cambridge, United Kingdom (n=53). Prevalence of LAMP-2 autoantibodies was similar in both those with myeloperoxidase-ANCA and proteinase 3-ANCA. Furthermore, we detected LAMP-2 autoantibodies in two ANCA-negative patients. LAMP-2 autoantibodies rapidly became undetectable after the initiation of immunosuppressive treatment and frequently became detectable again during clinical relapse. We conclude that when robust assays are used, circulating autoantibodies to hLAMP-2 can be detected in most European patients with ANCA-associated vasculitis. Large-scale prospective studies are now needed to determine whether they are pathogenic or merely an epiphenomenon.

The renal mononuclear phagocytic system.

The renal mononuclear phagocytic system, conventionally composed of macrophages (Mø) and dendritic cells (DCs), plays a central role in health and disease of the kidney. Overlapping definitions of renal DCs and Mø, stemming from historically separate research tracks and the lack of experimental tools to specifically study the roles of these cells in vivo, have generated confusion and controversy, however, regarding their immunologic function in the kidney. This brief review provides an appraisal of the current state of knowledge of the renal mononuclear phagocytic system interpreted from the perspective of immunologic function. Physical characteristics, ontogeny, and known functions of the main subsets of renal mononuclear phagocytes as they relate to homeostasis, surveillance against injury and infection, and immune-mediated inflammatory injury and repair within the kidney are described. Gaps and inconsistencies in current knowledge are used to create a roadmap of key questions to be answered in future research.

Suppressor of cytokine signaling (SOCS)1 is a key determinant of differential macrophage activation and function.

Macrophages become activated by their environment and develop polarized functions: classically activated (M1) macrophages eliminate pathogens but can cause tissue injury, whereas alternatively activated (M2) macrophages promote healing and repair. Mechanisms directing polarized activation, especially in vivo, are not understood completely, and here, we examined the role of SOCS proteins. M2 macrophages activated in vitro or elicited by implanting mice i.p. with the parasitic nematode Brugia malayi display a selective and IL-4-dependent up-regulation of SOCS1 but not SOCS3. Using siRNA-targeted knockdown in BMDM, we reveal that the enhanced SOCS1 is crucial for IL-4-induced M2 characteristics, including a high arginase I:iNOS activity ratio, suppression of T cell proliferation, attenuated responses to IFN-γ/LPS, and curtailed SOCS3 expression. Importantly, SOCS1 was essential in sustaining the enhanced PI3K activity that drives M2 activation, defining a new regulatory mechanism by which SOCS1 controls M2 polarization. By contrast, for M1 macrophages, SOCS1 was not only an important regulator of proinflammatory mediators (IL-6, IL-12, MHC class II, NO), but critically, for M1, we show that SOCS1 also restricted IL-10 secretion and arginase I activity, which otherwise would limit the efficiency of M1 macrophage proinflammatory responses. Together, our results uncover SOCS1, not only as a feedback inhibitor of inflammation but also as a critical molecular switch that tunes key signaling pathways to effectively program different sides of the macrophage balance.

Risk HLA-DQA1 and PLA(2)R1 alleles in idiopathic membranous nephropathy.

BACKGROUND: Idiopathic membranous nephropathy is a major cause of the nephrotic syndrome in adults, but its etiologic basis is not fully understood. We investigated the genetic basis of biopsy-proven cases of idiopathic membranous nephropathy in a white population. METHODS: We performed independent genomewide association studies of single-nucleotide polymorphisms (SNPs) in patients with idiopathic membranous nephropathy from three populations of white ancestry (75 French, 146 Dutch, and 335 British patients). The patients were compared with racially matched control subjects; population stratification and quality controls were carried out according to standard criteria. Associations were calculated by means of a chi-square basic allele test; the threshold for significance was adjusted for multiple comparisons (with the Bonferroni method). RESULTS: In a joint analysis of data from the 556 patients studied (398 men), we identified significant alleles at two genomic loci associated with idiopathic membranous nephropathy. Chromosome 2q24 contains the gene encoding M-type phospholipase A(2) receptor (PLA(2)R1) (SNP rs4664308, P=8.6×10(-29)), previously shown to be the target of an autoimmune response. Chromosome 6p21 contains the gene encoding HLA complex class II HLA-DQ alpha chain 1 (HLA-DQA1) (SNP rs2187668, P=8.0×10(-93)). The association with HLA-DQA1 was significant in all three populations (P=1.8×10(-9), P=5.6×10(-27), and P=5.2×10(-36) in the French, Dutch, and British groups, respectively). The odds ratio for idiopathic membranous nephropathy with homozygosity for both risk alleles was 78.5 (95% confidence interval, 34.6 to 178.2). CONCLUSIONS: An HLA-DQA1 allele on chromosome 6p21 is most closely associated with idiopathic membranous nephropathy in persons of white ancestry. This allele may facilitate an autoimmune response against targets such as variants of PLA2R1. Our findings suggest a basis for understanding this disease and illuminate how adaptive immunity is regulated by HLA.