Neutrophil proteases degrade autoepitopes of NET-associated proteins.

Neutrophils can form neutrophil extracellular traps (NETs) to capture microbes and facilitate their clearance. NETs consist of decondensed chromatin decorated with anti-microbial proteins. Here, we describe the effect of neutrophil proteases on the protein content of NETs. We show that the neutrophil serine proteases degrade several neutrophil proteins associated with NETs. Interestingly, the anti-bacterial proteins associated with NETs, such as myeloperoxidase, calgranulin B and neutrophil elastase (NE), seem to be less susceptible to proteolytic degradation than other NET proteins, such as actin and MNDA. NETs have been proposed to play a role in autoimmune reactions. Our data demonstrate that a large number of the autoepitopes of NET proteins that are recognized by autoantibodies produced by systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients are also removed by the proteases. In conclusion, neutrophil serine proteases have a major impact on the NET proteome and the proteolytic changes of NET-associated proteins may counteract autoimmune reactions to NET components.

Circulating serum miR-223-3p and miR-16-5p as possible biomarkers of early rheumatoid arthritis.

Small non-coding RNAs have emerged as possible biomarkers for various diseases including autoimmune diseases. A number of studies have demonstrated that the expression of specific microRNAs (miRNAs) is dysregulated in rheumatoid arthritis (RA). So far, all studies on miRNAs in RA patients have been performed using either microarray or reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses. Compared to RT-qPCR and microarray analyses, next-generation sequencing (NGS) allows the genome-wide analysis of small RNAs and the differentiation between miRNAs that differ by a single nucleotide. The application of NGS to the analysis of small RNAs circulating in sera of RA patients has not been reported. This study provides a global overview of the circulating small RNAs in the sera of RA patients and healthy subjects and identifies differences between these groups using NGS. Several classes of small RNAs, including hY RNA-derived fragments, tRNA-derived fragments and miRNAs, were determined. Differentially expressed individual small RNAs were verified by RT-qPCR. The levels of two miRNAs, miR-223-3p and miR-16-5p, were significantly lower in the sera from early RA patients than in those from established RA patients and healthy controls. In contrast, the serum level of miR-16-5p was higher in patients with established RA than in healthy control samples. These miRNAs may not only serve as biomarkers, but may also shed more light on the pathophysiology of RA.

Cytosolic 5'-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis.

OBJECTIVES: Autoantibodies directed against cytosolic 5'-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5'-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis. MATERIALS AND METHODS: Data from various European inclusion body myositis registries were pooled. Anticytosolic 5'-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. RESULTS: Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5'-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients. INTERPRETATION: Differences were observed in clinical and histopathological features between anticytosolic 5'-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5'-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype.

From autoantibody research to standardized diagnostic assays in the management of human diseases - report of the 12th Dresden Symposium on Autoantibodies.

Testing for autoantibodies (AABs) is becoming more and more relevant, not only for diagnosing autoimmune diseases (AIDs) but also for the differentiation of defined AID subtypes with different clinical manifestations, course and prognosis as well as the very early diagnosis for adequate management in the context of personalized medicine. A major challenge to improve diagnostic accuracy is to harmonize or even standardize AAB analyses. This review presents the results of the 12th Dresden Symposium on Autoantibodies that focused on several aspects of improving autoimmune diagnostics. Topics that are addressed include the International Consensus on ANA Patterns (ICAP) and the International Autoantibody Standardization (IAS) initiatives, the optimization of diagnostic algorithms, the description and evaluation of novel disease-specific AABs as well as the development and introduction of novel assays into routine diagnostics. This review also highlights important developments of recent years, most notably the improvement in diagnosing and predicting the course of rheumatoid arthritis, systemic sclerosis, idiopathic inflammatory myopathies, and of autoimmune neurological, gastrointestinal and liver diseases; the potential diagnostic role of anti-DFS70 antibodies and tumor-associated AABs. Furthermore, some hot topics in autoimmunity regarding disease pathogenesis and management are described.

Autoimmune/inflammatory syndrome induced by adjuvants (ASIA) due to silicone implant incompatibility syndrome in three sisters.

Three sisters who carried the BRCA-1 gene mutation had a preventive mastectomy and were reconstructed with silicone breast implants. After the reconstruction all three patients developed fatigue, arthralgia, myalgia and sleep disturbances within a period of four years. Because the complaints were thought to be related to the silicone breast implants, they were advised to have the implants replaced by non-silicone gel containing Monobloc Hydrogel breast implants. After this replacement operation, all complaints improved as evaluated 2.5 years later. Since the complaints developed during the presence of silicone implants and since the reversal was observed after replacement by hydrogel implants we postulate that our patients suffered from ASIA due to silicone implants, i.e. Silicone Implant Incompatibility Syndrome (SIIS). The generation of this syndrome in three sisters suggests that the susceptibility to the development of SIIS may be genetically determined.

Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes.

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.

Ribonucleoprotein complexes as autoantigens.

Many intracellular proteins and nucleic acids, that are involved in important biosynthetic pathways, are targeted by autoantibodies occurring spontaneously in the sera of patients with systemic autoimmune diseases. Frequently, the autoantigens are assembled into multicomponent complexes containing both nucleic acid(s) and proteins. Recently, progress has been made in the study of autoantigenic ribonucleoprotein complexes, the most important of which are spliceosomal ribonucleoproteins, nucleolar ribonucleoproteins, Ro/La ribonucleoproteins and complexes of aminoacyl-tRNA synthetase and tRNA. In addition to new structural and functional information, important results have been obtained on epitope spreading, as well as on a potential role for apoptosis during the development of an autoimmune response against these complexes.

Basic domains target protein subunits of the RNase MRP complex to the nucleolus independently of complex association.

The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both RNase MRP and RNase P accumulate in the nucleolus of eukaryotic cells. In this report we show that for three protein subunits of the RNase MRP complex (hPop1, hPop4, and Rpp38) basic domains are responsible for their nucleolar accumulation and that they are able to accumulate in the nucleolus independently of their association with the RNase MRP and RNase P complexes. We also show that certain mutants of hPop4 accumulate in the Cajal bodies, suggesting that hPop4 traverses through these bodies to the nucleolus. Furthermore, we characterized a deletion mutant of Rpp38 that preferentially associates with the RNase MRP complex, giving a first clue about the difference in protein composition of the human RNase MRP and RNase P complexes. On the basis of all available data on nucleolar localization sequences, we hypothesize that nucleolar accumulation of proteins containing basic domains proceeds by diffusion and retention rather than by an active transport process. The existence of nucleolar localization sequences is discussed.

Conserved features of Y RNAs: a comparison of experimentally derived secondary structures.

In this study, phylogenetically conserved structural features of the Ro RNP associated Y RNAs were investigated. The human, iguana, and frog Y3 and Y4 RNA sequences have been determined previously and the respective RNAs were subjected to enzymatic and chemical probing to obtain structural information. For all of the analyzed RNAs, the probing data were used to compose secondary structures, which partly deviate from previously predicted structures. Our results confirm the existence of two stem structures, which are also found at similar positions in hY1 and hY5 RNA. For the remaining parts of hY3 and hY4 RNA the secondary structures differ from those previously proposed based upon computer predictions. What might be more important is that certain parts of the RNAs appear to be flexible, i.e., to adopt several conformations. Another striking feature is that a characteristic pyrimidine-rich region, present in every Y RNA known, is single-stranded in all secondary structures. This may suggest that this region is readily available for base pairing inter-actions with other cellular nucleic acids, which might be important for the as yet unknown function of the RNAs.

Ro RNP associated Y RNAs are highly conserved among mammals.

Y RNAs are small cytoplasmic RNAs which are components of the Ro ribonucleoprotein complexes in higher eukaryotes. These complexes are frequently recognized by antibodies present in autoimmune sera. In this study we analysed the occurrence of Y RNAs in various mammalian and human cell lines and erythrocytes by means of hybridization with human Y RNA probes. Y RNAs homologous to their human counterparts, both in length and in sequence, were detected in all mammalian cells analysed. While hY1 and hY3 analogues were found in all cells, Y4 and Y5 RNA could not be detected in rodent cells. In addition, Y5 RNA was absent from bovine cells. Attempts to determine the sequence of rat Y RNAs by genomic cloning resulted in the isolation of a presumptive Y1 RNA pseudogene. Analysis of the hY RNA content of various human cell lines showed that all four human Y RNAs were present in all cell lines examined. However, the relative levels to which these RNAs were expressed showed marked differences.

Enhancement of DNA replication by transcription factors NFI and NFIII/Oct-1 depends critically on the positions of their binding sites in the adenovirus origin of replication.

The origin of DNA replication of many human adenoviruses is composed of a highly conserved core origin and an auxiliary region, containing the binding sites for NFI and NFIII/Oct-1. We examined enhancement of DNA replication in vitro by the purified functional DNA-binding domains of NFI (NFI-BD) and NFIII/Oct-1 (the POU domain), using origins in which the positions of the binding sites for these proteins were transposed. Insertion or deletion of two or three base pairs between the core origin and the NFI binding site resulted in a 3-5-fold decrease of stimulation, whereas larger insertions gradually reduced the stimulation further. Mutants in which the NFI binding site was separated approximately one or two helical turns from the core origin by AT-rich sequences could still be stimulated by NFI. In contrast, insertion of two or more base pairs between the NFI and NFIII/Oct-1 binding sites abolished stimulation by NFIII/Oct-1 almost completely. Furthermore, stimulation by this protein was lost when the Ad2 NFIII/Oct-1 binding site was transposed to a position closer to the core origin, destroying the NFI binding site. This shows that the position of the NFIII/Oct-1 binding site is essential for stimulation. Models to explain these position-dependent effects on stimulation are discussed.

Caspase-dependent cleavage of nucleic acids.

Autoimmune diseases are frequently characterized by the presence of autoantibodies directed against nucleic acid-protein complexes present in the nucleus of the cell. The mechanisms by which these autoantigenic molecules escape immunological tolerance are largely unknown, although a number of recent observations suggest that modified self-proteins generated during apoptosis may play an important role in the development of autoimmunity. It has been hypothesized that the recognition of these modified self-proteins by the immune system may promote autoantibody production. While apoptosis is specifically characterized by posttranslational modification of proteins, recent findings also show that nucleic acids are modified. This review summarizes the specific cleavages of some of these key nucleic acids, i.e. chromosomal DNA, ribosomal RNA and small structural RNAs (U1 snRNA, Y RNA), in apoptotic cells.

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule.

During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B'. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5 - 6 nucleotides including the 2,2, 7-trimethylguanosine (TMG) cap. The observations that U1 snRNA cleavage is very specific (no such modifications were detected for the other U snRNAs tested) and that U1 snRNA cleavage is markedly inhibited in the presence of caspase inhibitors, indicate that an apoptotically activated ribonuclease is responsible for the specific modification of U1 snRNA during apoptosis.

Caspase-mediated cleavage of the U snRNP-associated Sm-F protein during apoptosis.

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. The spliceosomal Sm proteins are recognized by the so-called anti-Sm autoantibodies, an antibody population found exclusively in patients suffering from systemic lupus erythematosus. We have studied the effects of apoptosis on the Sm proteins and demonstrate that one of the Sm proteins, the Sm-F protein, is proteolytically cleaved in apoptotic cells. Cleavage of the Sm-F protein generates a 9-kDa apoptotic fragment, which remains associated with the U snRNP complexes in apoptotic cells. Sm-F cleavage is dependent on caspase activation and the cleavage site has been located near the C-terminus, EEED(81) downward arrow G. Use of different caspase inhibitors suggests that besides caspase-8 other caspases are implicated in Sm-F cleavage. A C-terminally truncated mutant of the Sm-F protein, representing the modified form of the protein, is capable of forming an Sm E-F-G complex in vitro that is recognized by many anti-Sm patient sera.

The La (SS-B) autoantigen, a key protein in RNA biogenesis, is dephosphorylated and cleaved early during apoptosis.

In the past few years, a role for apoptotic processes in the development of autoimmune diseases has been suggested. An increasing number of cellular proteins, which are modified during apoptosis, has been described, and many of these proteins have been identified as autoantigens. We have studied the effects of apoptosis on the La protein in more detail and for the first time demonstrate that this autoantigen is rapidly dephosphorylated after the induction of apoptosis. Dephosphorylation of the La protein was observed after induction of apoptosis by several initiators and in various cell types. Furthermore, we demonstrate that at least a subset of the La protein is proteolytically cleaved in vivo, generating a 45 kDa fragment. Dephosphorylation as well as cleavage of La is inhibited by ZnSO4 as well as by several tetrapeptide caspase inhibitors, indicating that these processes require the activation of caspases. Dephosphorylation of La is inhibited by low concentrations of okadaic acid, suggesting that a PP2A-like phosphatase is involved. Generation of the 45 kDa fragment is consistent with proteolytic cleavage at amino acids 371 and/or 374. The possible significance of the apoptotic changes in the La protein for autoantibody production is discussed.

Ro ribonucleoprotein assembly in vitro. Identification of RNA-protein and protein-protein interactions.

The human Y RNAs, small RNAs with an unknown function, are complexed with at least three proteins: the 60,000 M(r) Ro protein (Ro60), the 52,000 M(r) Ro protein (Ro52) and the La protein (La). In this study we examined the intermolecular interactions between the components of these so-called Ro ribonucleoprotein (Ro RNP) complexes. Incubation of 32P-labelled hY1 RNA in HeLa S100 extract allows the reconstitution of Ro RNP complexes, which were analysed by immunoprecipitation with monospecific antisera. By immunodepletion of HeLa S100 extracts for either Ro60, Ro52 or La, followed by supplementation with recombinant Ro60 or La, it was demonstrated that both Ro60 and La bind to hY1 RNA directly without being influenced by one of the other proteins. However, binding of Ro52 to hY1 RNA required the presence of Ro60, which strongly suggests that the association of Ro52 with Ro RNPs is mediated by protein-protein interactions between Ro60 and Ro52.

Intracellular localization and nucleocytoplasmic transport of Ro RNP components.

The Ro and La autoantigens, which were originally identified by sera from autoimmune patients, consist of RNA-protein complexes (RNPs), in which the protein components carry most of the autoantigenic determinants. In human cells, Ro RNPs consist of one of four small RNA molecules, termed hY1, hY3, hY4 and hY5 [35], associated with several proteins, Ro60, Ro52 and La [8, 35, 106] and possibly other, yet unidentified polypeptides. Controversial data have been published on the association of the protein calreticulin with Ro RNPs [53, 58, 75, 78], but recently in vitro evidence has been obtained that non-phosphorylated calreticulin is able to interact with hY RNAs directly [17]. The physiological significance of this interaction remains to be established. In addition to its association with hY RNAs, La is (in most cases transiently) associated with all other RNA polymerase III transcripts. The hY RNAs are the only known cellular RNAs stably bound by La. While the Ro RNPs were found to reside mainly in the cytoplasm, the other La RNPs are mainly nuclear. In this review recent progress made on the intracellular localization and transport of Ro RNP components and of assembled Ro RNPs will be discussed.

Anti-cyclic citrullinated peptide positivity in non-rheumatoid arthritis disease samples: citrulline-dependent or not?

BACKGROUND: Antibodies directed against citrullinated proteins (eg anti-cyclic citrullinated peptide (CCP)) have excellent diagnostic and good prognostic potential for rheumatoid arthritis. Type 1 autoimmune hepatitis (AIH-1) is a chronic liver disease characterised by a variety of serum autoantibodies. Recently, in a large group of patients with AIH-1 without clear rheumatoid arthritis overlap, a relatively high percentage (9%) of anti-CCP2 positivity was scored. OBJECTIVES: To characterise the citrulline-dependence of the observed anti-CCP2 positivity in AIH-1 sera as well as in other groups of patients without rheumatoid arthritis (mainly rheumatic diseases). METHODS: Serum samples of 57 patients with AIH-1 and 66 patients without rheumatoid arthritis, most of them reported as anti-CCP positive, were tested for citrulline-specific reactivity with a second generation anti-CCP kit, with the citrullinated and the corresponding non-citrullinated (arginine-containing) antigen. A subset of AIH-1 sera was also tested with a CCP1 ELISA (and arginine control). RESULTS: The anti-CCP2 reactivity of most non-rheumatoid arthritis rheumatic diseases samples (87-93%) was citrulline-specific, whereas a relatively high percentage of AIH-1 samples (42-50%) turned out to be reactive in a citrulline-independent manner. The use of citrullinated and non-citrullinated CCP1 peptides confirmed a high occurrence of citrulline-independent reactivity in AIH-1 samples. CONCLUSIONS: In rheumatoid arthritis and most non-rheumatoid arthritis rheumatologic disease sera, anti-CCP positivity is citrulline-dependent. However in some patients, particularly patients with AIH-1, citrulline-independent reactivity in the anti-CCP2 test can occur. A positive CCP test in a non-rheumatic disease (eg liver disease) should therefore be interpreted with care, and preferably followed by a control ELISA with a non-citrullinated antigen.

Fine specificity of the anti-citrullinated protein antibody response is influenced by the shared epitope alleles.

OBJECTIVE: In classic studies on the genetic background of antibody production, the major histocompatibility complex (MHC) has been shown to act as the most prominent immune response gene that controls the magnitude and the specificity of antibody production. The strongest genetic risk factor for rheumatoid arthritis (RA), the human MHC HLA-DRB1 shared epitope (SE) alleles, predisposes for antibodies against citrullinated proteins (ACPAs). ACPA levels are higher in SE-positive patients with RA than in SE-negative patients with RA. The aim of the present study was to determine whether SE influences not only the magnitude but also the specificity of the ACPA response. METHODS: In 2 cohorts of anti-citrullinated peptide 2-positive patients with RA, one from a study of recent-onset arthritis (n = 206) and the other from a treatment strategy study (n = 141), serum antibodies against a citrullinated peptide derived from vimentin (cVim) and antibodies against a citrullinated fibrinogen peptide (cFibr) were determined by enzyme-linked immunosorbent assay. HLA-DRB1 genotyping was performed. RESULTS: In the first cohort, SE alleles were significantly associated with the presence of antibodies against cVim (odds ratio [OR] 4.95, 95% confidence interval [95% CI] 1.87-15.3) and were not significantly associated with the presence of antibodies against cFibr (OR 1.71, 95% CI 0.70-4.14). These results were replicated in the second cohort (OR 5.05, 95% CI 1.92-13.6 and OR 1.19, 95% CI 0.30-3.97, respectively). CONCLUSION: In 2 cohorts of ACPA-positive patients with RA, SE alleles predisposed for the development of antibodies against cVim but not for the development of antibodies against cFibr. These data indicate that SE alleles act as "classic" immune response genes in the ACPA response, because they influence both the magnitude and the specificity of this RA-specific antibody response.

Epitope specificity determines the ability of anti-Ro52 autoantibodies to precipitate Ro ribonucleoprotein particles.

Ro ribonucleoprotein particles (Ro RNPs) are evolutionarily conserved cytoplasmic complexes of unknown function. They are composed of several proteins and a small, RNA polymerase III-transcribed Ro or Y RNA. Abs directed against the protein moiety of Ro RNPs are often found in sera of patients suffering from certain autoimmune disorders. The association of one of the Ro proteins, a protein of 52 kDa (Ro52), with Ro RNPs is still questionable. In this study, we have used anti-Ro52 Abs isolated from autoimmune sera to locate the antigenic determinants of Ro52 and to analyze the correlation between regions of Ro52 recognized by these Abs and their ability to immunoprecipitate Ro RNPs. The results indicate that the autoimmune response against Ro52 is heterogeneous and that the exclusive recognition of certain epitopes does not result in immunoprecipitation of Ro RNPs.