Autoimmunity gene expression portrait: specific signature that intersects or differentiates between multiple sclerosis and systemic lupus erythematosus

Autoimmune diseases are either tissue-specific like multiple sclerosis (MS) or multisystemic like systemic lupus erythematosus (SLE), although clinically both exhibit common features. To gain insight into the properties of the genes involved in each disease we have investigated the gene expression signature of peripheral blood mononuclear cells (PBMC) in MS and SLE in comparison to healthy subjects. Total RNA was purified, hybridized to Genechip array and analysed in 36 subjects (13 relapsing-remitting MS patients, five SLE patients and 18 age-matched healthy subjects that served as controls). Additional blood samples from 15 relapsing-remitting MS patients, 8 SLE patients and 10 healthy subjects were used for confirmation of microarray gene expression findings by ELISA and RT-PCR. MS and SLE patients demonstrated a common gene expression autoimmune signature of 541 genes which differentiated them from healthy subjects. The autoimmune signature included genes that encode proteins involved in apoptosis, cell cycle, inflammation and regulation of matrix metalloproteinase pathways. Specifically, decreased TIMP1 gene expression in the autoimmunity signature suggests increased MMP activity in target tissues as a result of the lack of feedback mechanism. An additional different disease specific signature identified the gene expression pattern for MS (1031 genes), mainly associated with over-expression of adhesion molecules and down-expression of heat shock proteins; the SLE specific signature (1146 genes) mainly involved DNA damage/repair pathways that result in production of nuclear autoantibodies. These results provide insights into the genetic pathways underlying autoimmune diseases, and identify specific disease-associated signatures that may enable targetted disease-related specific therapies to be developed.     

Pure anti-dsDNA mAbs need chromatin structures to promote glomerular mesangial deposits in BALB/c mice

The glomerular targets for nephritogenic antibodies have been identified as membrane-associated chromatin fragments. The processes responsible for their deposition are poorly understood. To determine early events in antibody-mediated nephritis, we injected highly pure anti-dsDNA mAbs into BALB/c mice. Mice receiving one dose of anti-dsDNA mAbs were sacrificed 6 or 24 h later. No direct binding of mAbs to glomerular membranes or to the mesangial matrix was observed by immune electron microscopy. In contrast, repeated injections of the same antibodies over 4 weeks resulted in deposition of electron dense structures predominantly in the mesangial matrix. These structures contained mAbs and chromatin fragments as determined by co-localization immune electron microscopy. Biotinylated anti-dsDNA mAbs, injected into nephritic (NZB × NZW)F1 or MRL^lpr/lpr mice were detected in newly formed electron dense structures within glomerular capillary membranes. There were no correlation between mAb affinity for DNA, as determined by surface plasmon resonance analyses, and ability to bind chromatin fragments in vivo. No direct binding of mAbs to inherent membrane antigens was observed. Quantification of DNA in sera before and after one single injection of antibodies revealed increased DNA levels at 6 h after injection of anti-dsDNA mAb, and lower levels after 24 h. Repeated injections of anti-dsDNA caused an increase in circulating DNA. These results indicate that availability of chromatin fragments, presumable in circulation, is important for glomerular mesangial matrix deposition of anti-dsDNA antibody-containing immune complexes in context of lupus nephritis.     

Autoimmunity in focus: from mechanisms to treatment

The 5th Asian Congress on Autoimmunity took place in Suntec City, Singapore, on the 17–19 November 2011 under the presidency of Yehuda Shoenfeld (Chaim Sheba Medical Center, Ramat Gan, Israel). Senior investigators from a range of fields – including immunology, autoimmunity, rheumatology, neurology and hepatology – attended the conference. The scientific program placed an emphasis on the pathogenesis, genetic basis and mechanistic aspects of autoimmune diseases, as well as their clinical outcomes and treatment options. Particular focus was placed on systemic lupus erythematosus, rheumatoid arthritis, Type I diabetes, antiphospholipid syndrome and autoimmune hepatitis. Participants from over 50 countries attended the conference.     

Protein array autoantibody profiles for insights into systemic lupus erythematosus and incomplete lupus syndromes

The objective of this study was to investigate the prevalence and clinical significance of a spectrum of autoantibodies in systemic lupus erythematosus and incomplete lupus syndromes using a proteome microarray bearing 70 autoantigens. Microarrays containing candidate autoantigens or control proteins were printed on 16-section slides. These arrays were used to profile 93 serum samples from patients with systemic lupus erythematosus (SLE (n = 33), incomplete LE (ILE; n = 23), first-degree relatives (FDRs) of SLE patients (n = 20) and non-autoimmune controls (NC; n = 17). Data were analysed using the significance analysis of microarray (SAM) and clustering algorithms. Correlations with disease features were determined. Serum from ILE and SLE patients contained high levels of IgG autoantibodies to 50 autoantigens and IgM autoantibodies to 12 autoantigens. Elevated levels of at least one IgG autoantibody were detected in 26% of SLE and 19% of ILE samples; elevated IgM autoantibodies were present in 13% of SLE and 17% of ILE samples. IgG autoantibodies segregated into seven clusters including two specific for DNA and RNA autoantigens that were correlated with the number of lupus criteria. Three IgG autoantibody clusters specific for collagens, DNA and histones, were correlated with renal involvement. Of the four IgM autoantibody clusters, two were correlated negatively with the number of lupus criteria; none were correlated with renal disease. The IgG : IgM autoantibody ratios generally showed a stepwise increase in the groups following disease burden from NC to SLE. Insights derived from the expanded autoantibody profiling made possible with the antigen array suggest differences in autoreactivity in ILE and SLE. Determining whether the IgM aurotreactivity that predominates in ILE represents an early stage prior to IgG switching or is persistent and relatively protective will require further longitudinal studies.     

Specificities of anti-neutrophil autoantibodies in patients with rheumatoid arthritis (RA)

The objective of this study was to characterize antigens recognized by neutrophil-specific autoantibodies from patients with RA. Sera from 62 RA patients were screened by indirect immunofluorescence (IIF). Positive sera were further tested by ELISAs for antibodies against various granule proteins and by immunoblotting of electrophoretically separated cell, granule or nuclear extracts. Forty-two sera (68%) reacted with ethanol-fixed neutrophils. In the ELISAs 32% of the 28 medium to strongly IIF-positive sera were negative, while 43% were weakly positive for more than one antigen. Immunoblots of whole neutrophils showed IgG reactions at 25–35 kD, in the 55-kD region, at 80 kD, and at 110kD. Most sera reacted with more than one band. Except for the 55-kD antigen, none of the antigens appeared in lymphocytes. The most notable reactivity in subcellular fractions was with lactoferrin and with bands of 25–35 kD from nuclei. In conclusion, anti-neutrophil autoantibodies from RA patients recognize different antigens in the cytoplasm and in the nucleus. Lactoferrin is one of the common antigens recognized, but also unknown nuclear antigens of 25–35 kD mol. wt are involved.     

Human Anti-Ro Autoantibodies Bind Multiple Conformational Epitopes of 60-kD Ro Autoantigen

A gel filtration method was developed to estimate the number of conformational epitopes on the 60-kD Ro antigen. Anti-Ro Fab or Fab was incubated with native Ro antigen at different ratios and the Stokes radius molecular weight of complexes was estimated by gel filtration. Binding was saturated at 9 to 11 Fab molecules per bovine Ro molecule. Two additional Fab or Fab were bound if human Ro was used as the antigen. Isolated Ro antigen/anti-Ro Fab complexes were evaluated for the relative proportion of antigen to antibody at saturation of antigen with antibody and thus stoichiometry was determined. This provided data supporting there being between 7 and 11 binding sites, results similar to those with the gel filtration method. Experiments carried out with anti-Ro monoclonal antibodies showed one binding site per molecule of 60-kD Ro. Therefore, we have developed methods to count conformational epitopes on autoantigens and have applied it to the Ro/anti-Ro system. The data indicate that multiple conformational epitopes can be bound simultaneously by polyclonal anti-Ro sera from patients with systemic lupus erythematosus.     

Autoreactive B‐lymphocytes in SLE and RA patients: Isolation and characterisation using extractable nuclear and citrullinated antigens bound to immunobeads

Systemic lupus erythematosus and rheumatoid arthritis are autoimmune diseases characterised by B‐cell hyperactivation and production of autoantibodies (AutoAbs) against various self‐antigens, including extractable nuclear antigens and citrullinated peptides. Therefore, B lymphocytes and antibody‐secreting cells are considered relevant targets for therapies. However, isolation and characterisation of auto‐reactive specific B lymphocytes are limited, primarily due to technical issues. In this work, we purified extractable nuclear antigen‐specific and citrullinated peptide‐specific auto‐reactive B lymphocytes by magnetic selection with ENA‐ and citrullinated peptide‐bound immunobeads. We obtained blood auto‐reactive B lymphocytes from most patients. Their nature was primarily naïve B cells, some of them in an active status, with low levels of somatic hypermutations in the immunoglobulin heavy‐chain variable regions. Their presence correlated with serum levels of autoAb. Auto‐reactive B lymphocytes were able to differentiate into auto‐reactive antibody‐secreting cells under conditions of stimulation. In addition, based on the presence of circulating auto‐reactive B cells and/or antibody‐secreting cells, four different profiles were described in lupus patients. Thus, tracking auto‐reactive B cells and/or antibody‐secreting cells in patient blood could represent a biomarker for deciding whether to use therapies blocking either B cells, plasma cells or both, as well as a new tool for monitoring minimal residual autoimmune disease in patients.     

Detection of a circulating form of vascular cell adhesion molecule-1: raised levels in rheumatoid arthritis and systemic lupus erythematosus.

We have developed a panel of MoAbs against four separate but overlapping epitopes on endothelial cell (EC) vascular cell adhesion molecule-1 (VCAM-1). Two of the MoAbs (1G11 and 1E5) inhibited T cell adhesion to tumour necrosis factor (TNF)-activated EC, whilst two MoAbs (1.4C3 and 6D9) did not. Using these MoAbs we have identified a circulating form of VCAM-1 (cVCAM-1) which has identical epitope distribution to the EC form, and which is able to support the adhesion of the human lymphoblastoid cell line Jurkat J6 by a VLA-4- and VCAM-1-dependent mechanism when immobilized from plasma. cVCAM-1 isolated by immunoaffinity and size-exclusion chromatographies was shown by SDS-PAGE to have an apparent mol. wt of 85-90 kD. Levels of cVCAM-1 were significantly raised (P < 0.001) in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) compared with normal individuals. It is possible that cVCAM-1 may be a useful plasma marker for the diagnosis and management of patients with inflammatory diseases. Furthermore, detection of elevated cVCAM-1 levels may act as a guide to the importance of VCAM-1-dependent cell adhesion in different pathological settings.     

Increased serum levels of soluble interleukin-2 receptor in patients with systemic lupus erythematosus and rheumatoid arthritis

In this study we investigated the serum levels of a released soluble form of the interleukin-2 receptor (sIL-2R) in 42 patients with rheumatoid arthritis and in 12 cases of systemic lupus erythematosus. Data were evaluated in relationship to the clinical phase and compared with those observed in normal controls (N=56) and in osteoarthritis (N = 7). Increased levels were observed in both rheumatoid arthritis (mean ± SE, 604±49 U/ml) and systemic lupus erythematosus (1438±481 U/ml). These values were significantly higher than in control (256±15 U/ml;P<0.001) and in osteoarthritis (298±33 U/ml;P<0.001) groups. In addition, the highest values were associated with the active phases of both rheumatoid arthritis (active vs inactive, 771±78 vs 451±39 U/ml;P<0.001) and systemic lupus erythematosus (active vs inactive, 2108±489 vs 499±75 U/ml;P<0.001). Our findings suggest that the detection of sIL-2R in rheumatoid arthritis and in systemic lupus erythematosus may represent a good marker of disease activity, which indirectly indicates the ongoing activation and/or proliferation of immunoreactive cells which are involved in the pathogenetic events of these autoimmune conditions.     

Autoimmune,rheumatic, chronic inflammatory diseases: Neutrophil extracellular traps on parade

Abstract

Rheumatic diseases are a group of inflammatory conditions that affect joints and connective tissues and are often accompanied by pain and restriction of motility. In many of these diseases, autoantibodies develop that react with molecules/structures commonly found hidden in neutrophils. Neutrophil extracellular trap (NET) formation and release is considered a defense mechanism against pathogens or endogenous danger signals and it has been associated with initial inflammatory responses. NETs are also endowed with an important resolution potential based on its intrinsic enzymatic activity, but in the case they are not timely removed from the crime scene they might modulate subsequent immune responses and contribute to the pathogenesis of chronic inflammatory diseases. In this review, we will summarize the actual knowledge about the multifaceted roles of NETs in the etiology and pathogenesis of rheumatic autoimmune diseases.     

Molecular cloning of metaphase chromosome protein 1 (MCP1), a novel human autoantigen that associates with condensed chromosomes during mitosis

Systemic lupus erythematosus autoantibodies were used to identify and to characterize new human chromosome-associated proteins. Previous immunolocalization studies in human and murine tissue culture cells showed that some of these monoclonal antibodies recognize nuclear antigens that associate with condensed chromosomes during mitosis. One antibody was selected for screening a human HeLa S3 cDNA expression library, and cDNAs that code for an antigen of 31--33 kDa were isolated. Immunological, biochemical and cell fractionation data indicate that the 31- to 33-kDa antigen corresponds to the chromosome-associated protein recognized by the original monoclonal antibody. Sequence analysis shows that we isolated a novel human gene. Immunolocalization to human tissue culture cells shows that during interphase the antigen is dispersed in the nucleus and that during mitosis it associates exclusively with condensed chromosomes. A similar pattern of localization was also observed in mouse fibroblasts, suggesting that the antigen is conserved among different species. Finally, we show that part of the antigen remains bound to the scaffold/matrix component, even after high salt extraction.     

Serum and organ-associated anti-hemoglobin humoral autoreactivity: association with anti-Sm responses and inflammation

The release of hemoglobin (Hb) occurs in some infectious and autoimmune diseases characterized by inflammation. As levels of haptoglobin (Hp) fall, free Hb can cause pathology. Humoral autoreactivity to human Hb was demonstrated in the sera of systemic lupus erythematosus (SLE), leishmania and malaria patients. Serum anti-murine Hb antibody levels in lupus-prone mice also exhibited an age-dependent increase, with progressive organ sequestration; significant isotypic correlation was observed with anti-dsDNA antibodies. A suggestive link between anti-Hb and anti-Sm responses was observed: Human lupus sera expressing anti-Sm antibody reactivity preferentially contained heightened levels of anti-Hb autoantibodies, and immunization of lupus-prone mice with Sm led to enhanced anti-murine Hb reactivity. Human and murine anti-Hb monoclonal antibodies were generated, some of which were preferentially reactive toward disease-associated methemoglobin. Epitope-mapping studies revealed evidence of intra-molecular cross-reactivity. One such autoantibody synergized with Hb to enhance the secretion of pro-inflammatory cytokines while eliciting the increased production of monocyte migratory signals from endothelial cells. Preferential usage of specific variable region gene segments was not observed, although somatic mutations were documented. These studies reveal that, while the etiology, specificity and sequences of anti-Hb autoreactive antibodies can vary, they occur quite frequently and can have inflammatory consequences.     

血清IV型胶原和层粘连蛋白测定在结缔组织病中的意义

目的 探讨血清Ⅳ型胶原（ＩＶ．Ｃ）和层粘连蛋白（ＬＮ）在类风湿关节炎（ＰＡ）、系统性红斑狼疮（ＳＬＥ）和其它结缔组织病（ＣＴＤ）患者中的意义。方法 采用放射免疫法（ＰＩＡ）对３６例ＲＡ、３４例ＳＬＥ和１７例其它ＣＴＤ患者以及６３例正常人的血清ＩＶ．Ｃ和ＬＮ含量进行了测量。结果 各组患者血清ＩＶ．Ｃ和ＬＮ含量均较对照组明显升高（Ｐ〈０．０１～０．０００１）,且ＳＬＥ组血清ＬＮ水平明显提高ＲＡ组（Ｐ〈     

Loading of nuclear autoantigens prototypically recognized by systemic lupus erythematosus sera into late apoptotic vesicles requires intact microtubules and myosin light chain kinase activity

Most cases of systemic lupus erythematosus (SLE) are characterized by an impaired clearance of apoptotic cells in various tissues. Non-cleared apoptotic waste is considered an immunogen driving the autoimmune response in patients with SLE. During the execution of apoptosis, membrane blebs are formed and filled with cellular components. Here, we evaluate the cytoskeletal pathway(s) responsible for the loading of SLE prototypic nuclear autoantigens into the apoptotic cell-derived membranous vesicles (ACMV) generated during late phases of apoptosis. HeLa cells expressing a fusion protein of histone H2B with green fluorescent protein (GFP) were irradiated with ultraviolet (UV)-B to induce apoptosis. The appearance and trafficking of chromatin-derived material was monitored by fluorescence microscopy. Specific inhibitors of cytoskeletal pathways were employed to identify the motile elements involved in translocation and trafficking of the nuclear components. We observed that immediately after their appearance the ACMV did not contain histone H2B^GFP; in this phase the fluorescence was contained in the nuclear remnants and the cytoplasm. Within consecutive minutes the ACMV were loaded with chromatin-derived material, whereas the loading of simultaneously created ACMV with histone H2B^GFP was not uniform. Some ACMV were preferentially filled and, consequently, showed a remarkably higher histone H2B^GFP accumulation. Inhibitors of the cytoskeleton revealed that functional microtubules and myosin light chain kinase are required for nuclear shrinkage and loading of nuclear material into the ACMV, respectively.     

Lack of a strong association of CTLA-4 exon 1 polymorphism with the susceptibility to rheumatoid arthritis and systemic lupus erythematosus in Japanese: an association study using a novel variation screening method

CTLA-4 is considered to be one of the attractive candidates for the susceptibility genes to rheumatic diseases. In the present study, the association of CTLA-4 polymorphism with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) was examined in the Japanese population using the case-control association analysis. Polymerase chain reaction-preferential homoduplex formation assay (PCR-PHFA) was applied for the screening of genetic variations and for the genotyping of a large number of samples. A greater proportion of Japanese patients with RA (44%) and SLE (44%) compared with healthy individuals (37%) had exon 1 49 G/G genotype, but the difference did not reach statistical significance. However, when the patients with RA and healthy individuals were stratified according to HLA-DRB1 alleles, a weakly significant increase of the positivity of CTLA-4 49G allele was observed in HLA-DRB1*0405-positive patients (87%) compared with DRB1*0405-positive healthy individuals (71%) (P = 0.014, odds ratio = 2.77). These results indicate that CTLA-4 exon 1 polymorphism does not contribute greatly to the susceptibility to RA and SLE, at least in Japanese, although the presence of CTLA4 49G allele could be a minor predisposing factor for RA in HLA-DRB1*0405-positive individuals. In addition, PCR-PHFA was shown to be useful for a mass screening of gene variations.     

Juvenile onset systemic lupus erythematosus thyroid dysfunction: A subgroup with mild disease?

The aim of this study was to evaluate the frequency of thyroid dysfunction and thyroid antibodies in patients with juvenile onset Systemic Lupus Erythematosus (JOSLE) and its association with clinical and immunological features. Seventy-seven patients with JOSLE, 64 females, median age 19 years, were consecutively enrolled from March to December 2007. Clinical data related to thyroid dysfunction and lupus were obtained by chart review and patient interview. Serum levels of TSH, free T4, anti-thyroglobulin (TgAb), anti-thyroperoxidase (TPOAb), TRAb and lupus related autoantibodies were analyzed by standard techniques. Nine patients were diagnosed as hypothyroidism and 4 hyperthyroidism. 28% JOSLE patients had moderate/high titer of thyroid antibodies: 23% TgAb, 2.6% TPOAb and 3.9% TRAb. JOSLE patients with positive thyroid autoantibodies had higher frequency of anti-U1RNP antibodies than patients without these antibodies (40.9 vs. 14.5%, OR:0.25, CI:0.08–0.76, p = 0.017). Furthermore, renal/neurological/hematological involvement was less frequently observed in patients with hypothyroidism (55.6 vs. 87.5%, OR:0.18, CI:0.04–0.81, p = 0.035) and with thyroid antibodies (68.4 vs. 90.9%, OR:0.22, CI:0.06–0.82, p = 0.027) than in patients without these alterations. No association with PTPN22 polymorphism was found. In conclusion, JOSLE patients have high prevalence of subclinical hypothyroidism. The novel association of anti-thyroid antibodies with anti-U1RNP antibodies in JOSLE seems to identify a subgroup of patients with less life-threatening organ involvement.     

Gene expression profiles of systemic lupus erythematosus and rheumatoid arthritis

Gene expression profiling using microarray technology is being employed to define specific molecular mediators and pathways involved in immunobiology, to understand the intricate interplay of genes participating in the pathogenesis, and to develop biomarkers of disease activity in both systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). This review summarizes the latest information on the pathogenesis of SLE and RA and describes the utilization of microarray technology in these systemic autoimmune diseases.     

Analysis of a functional BTNL2 polymorphism in type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus

The aim of this study was to test whether the functional variant rs2076530 of the BTNL2 gene confers susceptibility to the autoimmune diseases type 1 diabetes (T1D), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). Our study populations consisted of 326 patients with T1D and 351 healthy subjects, 808 patients with RA and 1137 healthy controls, and 372 patients with SLE and 280 healthy controls. Genotyping of the BTNL2 gene rs2076530 polymorphism was performed by real-time polymerase chain reaction technology, using the TaqMan 5′-allele discrimination assay. We observed statistically significant differences in the distribution of BTNL2rs2076530 alleles between patients with T1D, RA, and SLE and healthy controls (p = 0.0035, 0.000003, and 0.00002, respectively), but in two divergent ways: the G allele was associated with T1D and RA, and the A allele was associated with SLE. However, the polymorphism exhibited strong linkage disequilibrium with HLA DQB1–DRB1 haplotypes previously identified as predisposing to the diseases. When the BTNL2 polymorphism was tested conditional on HLA DQB1–DRB1haplotypes, the BTNL2 effect was no longer significant in all three study populations. The BTNL2 rs2076530 polymorphism is associated with T1D, RA, and SLE because of its strong linkage disequalibrium with predisposing HLA DQB1–DRB1 haplotypes in Caucasian populations.