ANTIGENICITY OF POLY(I) AND ROS-POLY(I) AND THEIR RECOGNITION OF HUMAN ANTI-DNA AUTOANTIBODIES

The effect of hydroxyl radicals on polyinosinic acid [poly(I)] was studied. Strand breaks, base alteration and a decrease in absorbance at 248 nm (λmax) were observed upon ^?OH modification of poly(I). The broad antigen specificity of the induced anti-poly(I) and anti-ROS-poly(I) antibodies showed diverse antigen binding characteristics similar to those of SLE autoantibodies. Recognition of both poly(I) and ROS-poly(I) by human SLE anti-DNA autoantibodies was observed. The possible significance of these findings in the etiology of SLE has been discussed.     

Binding of SLE autoantibodies to native poly(I), ROS-poly(I) and native DNA: a comparative study.

Reactive oxygen species (ROS) are implicated in a variety of human diseases. The formation of pathogenic anti-DNA antibodies in systemic lupus erythematosus (SLE) has been extensively investigated. ROS-modified DNA has been found to be a better antigen for anti-DNA antibodies found in SLE sera. A comparative binding of SLE autoantibodies with native poly(I), ROS-poly(I) and nDNA has been studied. Affinity-purified SLE IgG exhibited a high degree of specificity towards the ROS-modified poly(I) in comparison to native DNA and native poly(I), reiterated visually by gel retardation assay. The data suggested that hydroxyl radical-modified nucleic acids like RNA and DNA might be agent for the induction of circulating SLE anti-DNA autoantibodies.     

Recent advances in understanding the clinical utility and underlying cause of antinucleosome (antichromatin) autoantibodies

The clinical utility of measuring antinucleosome autoantibodies (also known as antichromatin) in patients with systemic lupus erythematosus (SLE) has recently been evaluated by a number of different groups. Many studies found that antinucleosome autoantibodies were more prevalent than anti-DNA in SLE patients. In addition, antinucleosome autoantibodies were usually found to correlate with glomerulonephritis or disease activity better than anti-DNA in these patients. Antinucleosome autoantibodies are also found in patients with drug-induced lupus and Type I autoimmune hepatitis, but not usually in other diseases, thus showing good specificity for the above diseases. Several studies have shown that individuals with SLE have T cells reactive with nucleosomes and have increased levels of nucleosomes in their sera. The antinucleosome response in murine models of SLE is also T-cell-dependent and appears to be driven by self antigen. Nucleosome-antinucleosome immune complexes bind to glomeruli in vitro, and antinucleosome autoantibodies have been eluted from the kidneys of people and mice with glomerulonephritis. In one strain of mouse it was shown that antinucleosome autoantibodies were necessary, but not sufficient, to cause glomerulonephritis. These findings all show that antinucleosome autoantibodies are a sensitive and specific diagnostic marker for SLE and contribute to the pathology of glomerulonephritis.     

Association of neutropenia in systemic lupus erythematosus (SLE) with anti-Ro and binding of an immunologically cross-reactive neutrophil membrane antigen

SLE is associated with the production of autoantibodies to self-constituents. In particular, certain ribonucleoprotein particles are targeted. Despite the multitude of autoantibodies produced and the remarkable concentrations of these antibodies in the sera of SLE patients, there have been little data that the autoantibodies found in SLE are involved in the pathogenesis of disease or its manifestations. The present work demonstrates that anti-Ro (or SSA) is associated with granulocytopenia, binds the surface of granulocytes and fixes complement to this membrane surface. Binding is a property of anti-Ro Fab fragments and can be inhibited by 60-kD Ro. However, the antigen bound on the surface of granulocytes is a 64 000 mol. wt protein that is a novel autoantigen in SLE. As suggested by inhibition studies, sequence identity between 60-kD Ro and eight tandem repeats in the 64-kD antigen may be responsible for the observed serologic cross-reactivity. These data imply that anti-Ro antibodies that also bind the 64-kD protein mediate neutropenia in patients with SLE.     

Incidence of autoantibodies against type I and type II interferons in a cohort of systemic lupus erythematosus patients in Slovakia.

Autoantibodies against interferon (IFN) can be found in patients with systemic lupus erythematosus (SLE). However, detailed information about the occurrence of type-specific antihuman IFN antibodies is not available. In this study, we investigated the incidence of autoantibodies specifically recognizing various type I IFNs (alpha1, alpha2, beta, omega) and type II IFN (gamma). Sera from 100 SLE patients were screened for the presence of IFN-binding antibodies by ELISA, using various types of recombinant IFNs as antigen. On the whole, autoantibodies against type I or type II or both IFNs were detected in 45% (45 of 100) of the serum samples investigated. More than half (56%) of the positive samples (25 of 45) contained antibodies specific only for type I IFNs, and 36% of positive sera (16 of 45) had autoantibodies only against type II IFN. Antibodies against both type I and type II IFNs were detected in 8% (4 of 45) of the positive samples. Among autoantibodies to type I IFNs, the most abundant were those against the type IFN-omega (15%) and the subtype IFN-alpha2 (11%). Autoantibodies binding subtype IFN-alpha1 and type IFN-beta were detected at a relatively lower incidence of about 3%-4%. The highest occurrence (20%) showed autoantibodies to the proinflammatory cytokine, IFN-gamma. We did not find any correlation between the production of autoantibodies against particular IFN species and an antibody response to other IFN species. We further observed that 84% (38 of 45) of the positive sera bound only one IFN species, and 13% (6 of 45) of positive samples contained antibodies against two IFN species of five different combinations (alpha1/beta, alpha1/omega, alpha2/omega, alpha2/gamma, omega/gamma). One sample uniquely showed reactivity with three IFN species (alpha2/omega/gamma). Our findings suggest that formation of autoantibodies could reflect humoral immune responses to increased spontaneous production of the respective IFN species in SLE patients.     

Autoantibodies directed against the amino-terminal domain I of human calpastatin (ACAST-DI Ab) in connective tissue diseases. High levels of ACAST-DI Ab are associated with vasculitis in lupus

To identify new autoantibody populations in patients with rheumatic diseases, a cDNA expression library was immunoscreened with a rheumatoid arthritis (RA) patient's serum which contains autoantibodies binding to uncharacterized polypeptides by Western-blotting. One clone encoding the amino-terminal region (Nt) [domain L and half of domain I] of human calpastatin was selected. Different fragments of the selected cDNA were prepared and the corresponding recombinant polypeptides were produced by in vitro translation and analysed by Western blotting. Most RA sera bound to recombinant amino-terminal region and domain I but not to domain L. This prompted us to use a recombinant polypeptide corresponding to the domain I of calpastatin as the antigen in a solid-phase ELISA to test sera from patients with various systemic rheumatic diseases and healthy controls.Anti-calpastatin domain I antibodies (ACAST-DI Ab), were detected by ELISA in RA, systemic lupus erythematosus (SLE), Sj?gren's syndrome and control sera at respective frequencies of 10, 9, 0 and 1%. These Ab did not have prognostic value in early RA; high levels were significantly associated with vasculitis in SLE. Antibodies reacting with the calpastatin amino-terminal region are produced during systemic rheumatic diseases and are predominantly directed against domain I. High levels of these Ab may constitute a marker of vasculitis in SLE.     

A common anti-DNA idiotype and other autoantibodies in sera of offspring of mothers with systemic lupus erythematosus.

Since the immune response in fetuses of mothers with systemic lupus erythematosus (SLE) is unknown, we investigated sera from six mothers and their paired offspring by enzyme-linked immunosorbent assay (ELISA) for the presence of a common anti-DNA idiotype (16/6 Id) and, as control, for the presence of an unrelated public idiotype of antibody to hepatitis B surface antigen (HBsAg). In addition, maternal as well as fetal sera were evaluated for the presence of antibodies to ssDNA, dsDNA, poly(I), poly (dT), RNA, cardiolipin, total histones and the presence of lupus anticoagulant. Clinically active SLE mothers showed in general increased IgG and, to a lesser extent, IgM autoantibody activity. Circulating lupus anticoagulant was detectable in clinically active mothers only. All offspring of clinically active SLE mothers showed increased IgG autoantibodies to a variety of antigens, while IgM antibodies were detected in only one fetus. In contrast, fetuses of clinically inactive mothers showed only minor IgG activity. Common anti-DNA-idiotype (16/6 Id) activity also correlated with disease activity in both maternal and fetal compartments. One clinically active mother was 16/6-negative; her offspring was, however, positive, indicating de novo production of the idiotype by the fetus. In contrast, a control anti-HBsAg idiotype was not detected in either maternal or fetal sera. It therefore appears that offspring of clinically active SLE mothers serologically reflect maternal disease activity. Furthermore, autoantibodies and common idiotype of autoantibodies can be found within the fetal compartment even in the absence of such antibodies in the maternal serum. Discrepancies between mothers and offspring in IgM-autoantibody levels and the presence of new idiotypes in fetuses are indicative of fetal de novo autoantibody production.     

Hydroxyl Radical Modification of Polyguanylic Acid: Role of Modified Guanine in Circulating SLE Anti‐DNA Autoantibodies

The hydroxyl radical generated by UV irradiation of hydrogen peroxide cause an extensive damage to guanine residues of ribohomopolymer, polyguanylic acid, poly (G) as investigated by spectrophotometric measurements, agarose gel electrophoresis, Sephadex G‐200 gel filtration and DEAE Sephadex A‐25 column chromatography. Native and ROS‐poly (G) were highly immunogenic inducing high titre antibodies in rabbits. The antibodies showed wide range of cross reactivity with various synthetic polynucleotides exhibiting B‐, A‐, and allied conformations. The diverse antigen binding characteristics of the induced antibodies resembles to those of naturally occurring lupus anti‐DNA autoantibodies. Sera from various SLE patients showed preferential binding to ROS‐poly (G) than native poly (G), indicating that oxidatively modified guanine residues are better recognised. The significance of these findings in the induction of SLE anti‐DNA autoantibodies by oxygen free radicals modified guanine residues in DNA has been discussed.     

Identification of autoantibody against poly (ADP-ribose) polymerase (PARP) fragment as a serological marker in systemic lupus erythematosus

OBJECTIVES: By utilizing serological analysis of a recombinant cDNA expression library (SEREX), we previously found that autoantibodies to poly (ADP-ribose) polymerase (PARP) are specifically present in the sera of patients with SLE. In this study, recombinant proteins of various domains of PARP were used to determine the PARP domain that is associated with SLE. METHODS: We produced four recombinant PARP proteins, which contained various PARP domains, and then carried out enzyme linked immunosorbent assay (ELISA) using these recombinant proteins to identify domains useful for SLE diagnosis. The recombinant proteins used in this analysis were; ADPNF (amino acids 1-234), ET-L2 (amino acids 339-680), ET-L3 (amino acids 681-1014), and ADPCF (amino acids 300-1014). RESULT: ELISA with ADPNF or ET-L2 showed low sensitivity in the sera of patients with SLE (14.3% and 17.0% respectively), whereas ELISA with ET-L3 or ADPCF showed high sensitivity in the sera of patients with SLE (34.0% and 49.1%, respectively). Autoantibodies to ADPCF were not found in the sera of patients with rheumatoid arthritis (0/30), systemic sclerosis (0/30) or healthy donors (0/54) and were rarely found in polymyositis/dermatomyositis (1/30) and Sjogren syndrome (1/14). Autoantibodies to ADPCF were closely associated with the presence of an oral ulcer in SLE (P=0.03, by the chi-square test). CONCLUSION: The high sensitivity and specificity shown by autoantibodies to ADPCF protein could be used as a valuable serologic maker for the diagnosis of SLE.     

Hydroxyl radical modification of polyguanylic acid: role of modified guanine in circulating SLE anti-DNA autoantibodies

The hydroxyl radical generated by UV irradiation of hydrogen peroxide cause an extensive damage to guanine residues of ribohomopolymer, polyguanylic acid, poly (G) as investigated by spectrophotometric measurements, agarose gel electrophoresis, Sephadex G-200 gel filtration and DEAE Sephadex A-25 column chromatography. Native and ROS-poly (G) were highly immunogenic inducing high titre antibodies in rabbits. The antibodies showed wide range of cross reactivity with various synthetic polynucleotides exhibiting B-, A-, and allied conformations. The diverse antigen binding characteristics of the induced antibodies resembles to those of naturally occurring lupus anti-DNA autoantibodies. Sera from various SLE patients showed preferential binding to ROS-poly (G) than native poly (G), indicating that oxidatively modified guanine residues are better recognised. The significance of these findings in the induction of SLE anti-DNA autoantibodies by oxygen free radicals modified guanine residues in DNA has been discussed.     

Autoantibodies to topoisomerase I (Scl-70): analysis by gel diffusion, immunoblot, and enzyme-linked immunosorbent assay

Anti-topoisomerase I autoantibodies (anti-topo I, anti-Scl-70) are associated with proximal scleroderma and are of prognostic significance in patients with Raynaud's disease. To establish a highly sensitive and specific system for the detection of anti-topo I, we have investigated sera from 409 patients and controls by Ouchterlony gel diffusion, Western immunoblot on chromosome proteins, and solid-phase enzyme-linked immunosorbent assay (ELISA) with purified topoisomerase I as antigen. The ELISA was more sensitive than the gel diffusion technique and was more specific than the Western immunoblot, while the immunoblot may identify additional autoantibodies.     

Expression of an anti‐RNA autoantibody in a mouse model of SLE increases neutrophil and monocyte numbers as well as IFN‐I expression

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of antinucleic acid autoantibodies, high levels of circulating type I interferon (IFN‐I), and an IFN‐I‐dependent elevated expression of activating FcγR. Increases in neutrophils and monocytes are often observed in clinical SLE, but how these contribute to autoantibody and IFN‐I production is poorly understood. Here, we analyzed SLE pathogenesis in 564Igi mice, an SLE‐model strain carrying gene‐targeted heavy and light chain antibody genes encoding an anti‐RNA autoantibody in a C57BL/6 background. Similar to human SLE patients, 564Igi mice produce anti‐RNA autoantibodies and expanded neutrophil and monocyte populations. These myeloid cells produced IFN‐I and exhibit increased FcγRIV expression induced via an IFN‐I autocrine loop. A direct effect of IFN‐I on 564Igi BM B cells and neutrophils was supported by their upregulation of “IFN‐I signature genes”. In addition, 564Igi developing B cells showed upregulated TLR7 resulting in IgG2a/2b class switch recombination and autoantibody production. Our results indicate that the production of anti‐RNA autoantibody is sufficient to induce an increase of BM, blood, and spleen IFN‐I‐producing neutrophils, and suggest a mechanism by which autoantibody and IFN‐I contribute to SLE by activating B lymphocytes, neutrophils, and monocyte effector cells in vivo.     

Extractable nuclear antigen (ENA) autoantibodies in SLE: an immunogenetic relationship with HLA, C4 and Bf alleles.

Sera from 36 subjects with systemic lupus erythematosus (SLE) were examined for extractable nuclear antigen (ENA) autoantibodies by immunoassay with the ribonucleoprotein (RNP) subset being determined by immunodiffusion. The prevalence of the genetic markers of HLA, the fourth complement component (C4) and properdin factor (Bf), which are all coded for within the major histocompatibility complex on chromosome 6 were analysed in relation to various parameters of these autoantibodies. The following associations were observed: The lowest ENA antibody titres of the RNP negative group were associated with HLA A9 (P less than 0.05), while the lowest RNA-ase sensitive ENA (RSE) subset antibody levels were associated with HLA Dr 1 (P less than 0.05). For the complement markers, C4 AQo was associated with the lowest affinity ENA antibodies (P less than 0.05), while the BF F allele and Fs phenotype had lower RSE antibody levels than did the S allele (P less than 0.05) and the SS phenotype (P less than 0.05) respectively. This study demonstrated diverse association between various MHC markers and ENA antibody parameters, indicating that there are distinctive immunogenetic influences over ENA autoantibodies in SLE.     

Elevation of the levels of SmB, B' and D proteins but not that of the La (SS-B) antigen during HSV infections

Three Sm proteins, B, B' and D, which are highly immunoreactive in systemic lupus erythematosus (SLE), increase in abundance in Vero cells infected with laboratory strains and clinical isolates of herpes simplex virus (HSV) types 1 and 2. The autoimmune antigen La (SS-B) does not accumulate in identical infections. The significance of the Sm antigen accumulation is discussed in terms of its possible role in HSV infection and in connection with the origin of autoantibodies in SLE.     

Distinction between natural and pathological autoantibodies by immunoblotting and densitometric subtraction: liver-kidney microsomal antibody (LKM) positive sera identify multiple antigens in human liver tissue.

Using one-dimensional and two-dimensional immunoblotting techniques the reactions of sera from 14 patients with liver-kidney microsomal (LKM) antibody positive chronic active hepatitis (CAH) with human liver microsomal preparations was compared with the reaction of sera from 12 healthy persons and from five patients with systemic lupus erythematosus (SLE). All sera displayed a multiplicity of reactions. This demonstrates the presence of many autoantibodies in normal human sera. It could be shown that all sera react with the cytoskeletal antigens cytokeratin, actin and actomyosin. These reactions were more marked in the autoimmune sera, i.e. LKM-positive CAH and SLE. Densitometric subtraction was found to be a reliable technique to distinguish the natural antigen/autoantibody reactions from pathological, disease-characteristic autoantibodies. It was shown that the pathological LKM autoantibodies recognize non species-specific microsomal proteins at 50 kD of pI 7.5-8.0 at high titres, which are only very weakly recognized by normal or SLE sera. We recommend sensitive immunoblotting techniques and densitometric subtraction as the currently most accurate method to distinguish natural from pathological autoantibodies.     

Amelioration of experimental systemic lupus erythematosus (SLE) by retrovirus infection

Experimental SLE can be induced in susceptible 129/J mice by immunization with a human anti-DNA antibody bearing a common idiotype designated 16/6 Id. Immunized mice develop autoantibodies, leukopenia, proteinuria, and immune complex deposits in renal glomeruli. Case reports have described clinical improvement in SLE in individuals becoming infected with HIV-1. Because 129/J mice are susceptible to experimental SLE and to infection with the BM5def murine leukemia virus (MuLV) mixture but do not develop the lymphoproliferative/immunodeficiency disorder known as murine AIDS (MAIDS), we superimposed this infection on immunization with the 16/6 Id. Multiple effects were observed. First, we noted an amelioration in the course of experimental SLE. Second, both in experimental SLE and in BM5def MuLV infection, immunoreactivity to HIV-1 gp120 was demonstrated, although gp120 is not present in the BM5def MuLV viruses. Third, production of autoantibodies characteristically found in SLE, e.g., anti-DNA, anti-RNP, and anti-SSA, was seen in BM5def MuLV-infected mice, demonstrating that an immune response as a consequence of infection had occurred despite the absence of MAIDS induction. We conclude that (1) retrovirus inoculation may ameliorate the course of experimental SLE; and (2) retrovirus inoculation, even in the absence of MAIDS induction, induces an immunologic response which promotes the production of potentially pathogenic autoantibodies.     

HLA-A,B,C and DR antigens, GLO I and Bf marker profiles in 75 Cape Coloured patients with systemic lupus erythematosus (SLE)

HLA-A,B,C and DR antigens were tested in 75 Cape Coloured systemic lupus erythematosus (SLE) patients, and the GLO I and Bf markers in 51. The patients with HLA-DR2 had a relative risk significantly greater than one (p=0.0005). Twenty-two (29%) patients had only one detectable DR antigen. Of these, 11 (50%) were found to have DR2 only. The HLA-DR7 antigen was associated with severe disease (p less than 0.02). Bf and GLO I markers were not associated with SLE.     

The significance of antibodies to poly(adenosine diphosphate-ribose) in systemic lupus erythematosus.

Poly(adenosine diphosphate-ribose) and ds-DNA binding activity have been measured in thirty-nine systemic lupus erythematosus (SLE) sera, nineteen rheumatoid arthritis sera, fourteen sera from non-SLE rheumatic and non-rheumatic diseases and in ten normal sera. Antibodies to poly(ADP-ribose) were found only in the SLE and in three SLE-like rheumatic diseases. Anti-DNA antibodies, on the other hand, were found not only in the SLE and SLE-like diseases, but also in rheumatoid arthritis and chronic active hepatitis. Estimation of poly(ADP-ribose) binding was, therefore, more specific for, and more discriminatory of SLE from other diseases, than the estimation of ds-DNA binding. The results indicate that the estimation of poly(ADP-ribose) binding in serum may be more useful in the diagnosis of SLE than the presently employed estimation of DNA binding using the Amersham kit. DNA-anti-DNA immune complexes are detected in some of the SLE sera after deoxyribonuclease I digestion, confirming earlier reports of the existence of circulating DNA-anti-DNA complexes in SLE patients. Snake venom phosphodiesterase treatment of some of the SLE sera also resulted in increased poly(ADP-ribose) binding activity, suggesting the existence of poly(ADP-ribose)-anti-poly(ADP-ribose) immune complexes in the circulation of SLE patients. This observation raises the possiblity that poly(ADP-ribose) immune complexes may play some part in the pathogenesis of some cases of SLE.     

Antiribonuclease H2 antibodies are an immune biomarker for systemic lupus erythematosus

We previously reported that autoantibodies against the proliferating cell nuclear antigen protein (PCNA)-binding protein chromatin assembly factor-1 (CAF-1) are specifically found in patients with systemic lupus erythematosus (SLE). PCNA and its complex constituents elicit autoimmune responses in patients with SLE, suggesting that autoantibody diversification likely occurs owing to epitope spreading. Therefore, we sought to clarify whether patients with SLE exhibit an autoimmune response to Ribonuclease H2 (RNase H2), another PCNA-binding protein that regulates cell division. As results, RNase H2 autoantibodies were detected in the sera of 33.9% (19/56) of SLE patients, which was significantly higher than that observed in sera from other patients with systemic autoimmune diseases (polymyositis/dermatomyositis, systemic sclerosis, Sjogren’s syndrome, mixed connective tissue disease and rheumatoid arthritis) and healthy controls. Regression analysis also showed that serum anti-RNase H2 levels were strongly correlated to that of CAF-1 in SLE patients. Our data support the use of RNase H2 autoantibodies as a serum biomarker for SLE diagnosis. Moreover, the strong correlation observed between RNase H2 and CAF-1 suggests that intermolecular epitope spreading may play a critical role in autoantibody production and diversification in SLE.     

Over-expression of TATA binding protein (TBP) and p53 and autoantibodies to these antigens are features of systemic sclerosis, systemic lupus erythematosus and overlap syndromes

The aim of this study was to determine the expression levels of p53 and TATA binding protein (TBP) and the presence of autoantibodies to these antigens in Asian Indian patients with systemic sclerosis (SSc), overlap syndromes (OS) and systemic lupus erythematosus (SLE). Fifty patients with SSc, 20 with OS, including mixed connective tissue diseases (MCTD), 20 with SLE, 10 disease controls (DC) and 25 controls (C) were studied. The over-expression of p53 and TBP antigen was determined quantitatively by sandwich enzyme-linked immunosorbent assay (ELISA), varies between four- and sevenfold higher in patients with SSc, OS and SLE, in comparison to DC and C. The expressed protein antigens were not present as free antigens but as immune-complexes. Autoantibodies to p53 were detected by ELISA in 78% subjects with SSc, 100% with OS and 80% with SLE. Autoantibodies to TBP were observed in 28% patients with SSc, 25% with OS and 15% with SLE. In comparison to healthy controls, the titre of antibodies to p53 was significantly higher in patients with SSc (P = 0.00001) than the patients with OS (P = 0.00279) and SLE (P = 0.00289), whereas the titre of antibodies to TBP was higher in patients with OS (P = 0.00185) than the SLE (P = 0.00673) and the SSc (P = 0.00986) patients. Autoantibodies to p53 and TBP were detected in all these patients and the levels of these two autoantibodies showed weak negative correlation with each other. We propose that the over-expression of these antigens might be due to hyperactive regulatory regions in the p53 and TBP gene.