Detection of antibodies to the antigens involving differentiation of myeloid cells in sera from patients with systemic lupus erythematosus

The sera from 66 patients with systemic lupus erythematosus (SLE) were examined by the immunoblotting method to detect antibodies to the antigens on the cultured myeloid cell lines, fresh monocytes and granulocytes, and were compared with the sera from 26 healthy subjects sex- and roughly age-matched to the patients. The sera from SLE patients demonstrated antibodies to many antigens on myeloid cells at high frequencies, compared with healthy subjects. A high reactivity with similar patterns was demonstrated with K562, KG-1 and HL60 cells, while reactivity to U937, monocytes and granulocytes was rather low. In particular, the sera from SLE patients were found to contain the antibody to the antigens with Mr of 60K on K562, KG-1, and HL60 cells, which are known to express a good amount of c-myc products. However, the sera from healthy subjects demonstrated hardly any antibody to the 60K antigen on HL60 cells. After an incubation of HL60 cells with TPA or vitamin D3 to induce their monocytic differentiation, the SLE sera became able to detect the 55K antigen on the differentiated HL60 cells, while the 60K antigen turned to undetectable or only faintly detected. These findings suggested that the 60K antigen on HL60 cells may be related to a gene product involving cell growth or differentiation, such as c-myc protein. Actually, polyclonal antibody to myc-specific peptide could identify the 60K antigen as one of the cellular products of HL60.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-mannose binding lectin antibodies in sera of Japanese patients with systemic lupus erythematosus

Mannose-binding lectin (MBL) is a key element in innate immunity with functions and structure similar to that of complement C1q. It has been reported that MBL deficiency is associated with occurrence of systemic lupus erythematosus (SLE). We hypothesized that anti-MBL antibodies, if present, would affect the occurrence or disease course of SLE, by reduction of serum MBL levels, interference of MBL functions, or binding to MBL deposited on various tissues. To address this hypothesis, we measured the concentration of anti-MBL antibodies in sera of 111 Japanese SLE patients and 113 healthy volunteers by enzyme immunoassay. The titres of anti-MBL antibodies in SLE patients were significantly higher than those in healthy controls. When the mean + 2 standard deviations of controls was set as the cut off point, individuals with titres of anti-MBL antibodies above this level were significantly more frequent in SLE patients (9 patients) than in controls (2 persons). One SLE patient had an extremely high titre of this antibody. No associations of titres of anti-MBL antibodies and (i) genotypes of MBL gene, (ii) concentrations of serum MBL, or (iii) disease characteristics of SLE, were apparent. Thus, we have confirmed that anti-MBL antibodies are indeed present in sera of some patients with SLE, but the significance of these autoantibodies in the pathogenesis of SLE remains unclear.     

Antinucleosome antibodies and decreased deoxyribonuclease activity in sera of patients with systemic lupus erythematosus

Nucleosomes are the dominant autoantigens in patients with systemic lupus erythematosus (SLE), and immune complexes involving nucleosomes are the major cause of tissue damage. The activity of DNase I, the enzyme responsible for nucleosome degradation, has been found to be decreased in patients with SLE. However, it is not known whether DNase activity is a clinically useful parameter. The aim of our study was to assess DNase activity in a prospective study of 113 patients with SLE in relation to disease activity and organ involvement. We included two control groups: 9 patients with undifferentiated connective tissue disease (UCTD) and 14 healthy individuals. DNase activity was found to be lower in patients with SLE (63.75%+/-12.1%) than in the controls (81.3%+/-9.25%) (P<0.001). DNase activity in patients with UCTD (64.9%+/-18.2%; P=0.854) did not differ from that in patients with SLE. Patients with SLE had higher antinucleosome antibody titers (356.3+/-851) than the controls (1.44+/-2.77; P<0.01) or UCTD patients (39.9+/-57.7; P<0.01). In addition, samples positive for antinucleosome antibodies displayed low levels of DNase activity. Within the SLE group, the presence of renal disease had no impact on DNase activity or antinucleosome antibody titers. Also, the SLE disease activity index showed no correlation with DNase activity. In a longitudinal study of six SLE patients, DNase activity did not follow disease activity or autoantibody titers. Our results confirm that serum DNase activity is decreased in patients with SLE, but we conclude that it is not a clinically useful parameter for the prediction of flare-ups of disease or renal involvement.     

Lymphocytotoxic antibodies in patients with systemic lupus erythematosus

Lymphocytotoxic activity could be demonstrated in the sera of patients with systemic lupus erythematosus. The number of positive reactions varied with temperature of incubation. Lymphocytotoxicity was present in 88%, 49% and 11% of sera tested at 15°C, 22°C and 37°C respectively. At an incubation temperature of 22°C, the presence of the lymphocytotoxic antibody in the sera could be correlated with significantly higher titres of anti-nuclear factor, anti-single-stranded DNA and the histological appearance of active diffuse glomerulonephritis in renal biopsies.     

Epstein-Barr virus and molecular mimicry in systemic lupus erythematosus

Systemic lupus erythematosus (SLE or lupus) is a complex disease with a multifactoral etiology, with genetic, hormonal, and environmental influences. Molecular mimicry as a result of viral infection may contribute to the development of lupus. The pattern of autoantibody development in lupus is consistent with initiation through molecular mimicry, as the initial autoantigenic epitopes that have been observed are limited and cross-reactive with viral proteins. Autoantibody specificity may then later diversify to other autoantigens through B-cell epitope spreading. Epstein-Barr virus (EBV) is an excellent candidate to be involved in molecular mimicry in lupus. EBV infection has been associated with lupus through serological and DNA studies. Infection with EBV results in the production of the viral protein Epstein-Barr virus nuclear antigen-1 (EBNA-1), antibodies against which cross-react with lupus-associated autoantigens, including Ro, Sm B/B', and Sm D1, in lupus patients. The immune response against EBV, and EBNA-1 in particular, differs among lupus patients and healthy controls, with controls maintaining a limited humoral response and failing to produce long-standing cross-reactive antibodies. We hypothesize that the humoral immune response to EBNA-1 in susceptible individuals leads to the generation of cross-reactive antibodies. Through the process of epitope spreading, these cross-reactive antibodies target additional, non-cross reactive autoepitopes, spread to additional autoantigens, and become pathogenic, leading eventually to clinical lupus. This paper reviews some of the current literature supporting roles for EBV exposure and epitope spreading in SLE.     

Enhancement of Epstein-Barr virus antibody production in systemic lupus erythematosus patients.

Two groups of 22 patients suffering from either systemic lupus erythematosus (SLE) or infectious mononucleosis (IM) were checked for Epstein-Barr virus capsid antigen antibody (EB-VCA) production. The average significant antibody levels as well as the frequency of their occurrence were clearly higher in SLE than in IM patients.     

Immunological characterization of small nuclear ribonucleoproteins reactive with sera of patients with systemic lupus erythematosus

We have previously reported the purification of Sm and RNP antigens from goat liver and identified two polypeptides of molecular weights 70 and 80–90 kd as RNP specific and of 14 and 30 kd as Sm specific. In this communication the effect of ribonuclease and trypsin on Sm and RNP antigens was studied at the polypeptide level. We found that the RNP antigenic determinant polypeptides of 70 and 80–90 kd are lost as a result of such treatment, whereas there is no effect on the Sm-specific 14- and 30-kd polypeptides. The role of RNA in the antigenicity of Sm and RNP was studied by dissociation and reconstitution studies. The antigens were fractionated into protein and RNA and the individual fractions were tested for Sm and RNP activity by counterimmunoelectrophoresis (CIE) and enzyme-linked immunosorbent assay (ELISA). The RNA fraction did not react alone with anti-Sm and anti-RNP sera with either of the assays. Conversely when the protein fraction was tested by CIE, only Sm antigenicity was detectable. In the ELISA both Sm and RNP activities were demonstrated in the protein fraction. These results show that the presence of RNA is important in the immunoprecipitation reactions involving only RNP antigen, whereas Sm activity is independent of RNA. In addition, when the reaction is carried out by an assay involving primary antigen-antibody reaction (e.g., ELISA), RNP antibodies react with protein fractions alone, without the presence of RNA. We also report the glycoprotein nature of Sm-specific polypeptides. The antigen was found to react specifically with concanavalin A (Con A), indicating the presence of glycosyl and/or mannosyl residues. The observed glycoprotein nature of the Sm-specific polypeptides possibly explains their remarkable stability, unlike RNP-specific polypeptides, which are susceptible to proteolytic attack.     

Detecting Epstein-Barr virus DNA from peripheral blood mononuclear cells in adult patients with systemic lupus erythematosus in Taiwan

Epstein-Barr virus (EBV) has been found by many serology studies to be associated with systemic lupus erythematosus (SLE). However, the results of DNA studies have been conflicting. Therefore, instead of antibody to EBV, we studied the association between EBV DNA and SLE. In this case-control study in Taiwan, we enrolled 87 SLE patients and 174 age- and sex-matched controls. Peripheral blood mononuclear cells of SLE patients and matched controls were tested for EBV DNA by polymerase chain reaction (PCR) and Southern blot. Of the 87 SLE patients, 71 (81.6%) were found to be positive for EBV DNA, while 85 (48.9%) of the 174 controls (odds ratio 4.64, 95% confidence interval 2.50–8.62, P<0.0001) were positive. While the EBV DNA-positive rate did not decline with age in SLE patients (P>0.05), it did decline with age in controls (P<0.05). Furthermore, based on a real-time quantitative PCR study, we have found a significant difference between EBV viral load in SLE and controls (P=0.008). Therefore, in our molecular study of DNA level, we found evidence for the association of EBV infection and SLE, suggesting that EBV contributes, if not to the development of SLE, then to disease perpetuation.     

Detection of immunoprecipitated systemic lupus erythematosus antigens by immunoblot analysis

When antigens are isolated from staphylococcal protein A immunoprecipitation pellets for analysis by SDS polyacrylamide gel electrophoresis and immunoblotting, severe background problems, due to the presence of antisera and bacterial proteins, can result. We describe a procedure for the analysis of immunoprecipitated systemic lupus erythematosus antigens (e.g., La, Ro, and Sm) which significantly reduces this background while retaining sensitivity with respect to antigen detection. We have adapted a method previously described (MacSween, J.M. and Eastwood, S.L. Methods Enzymol. 1981; 73:459-471) in which lithium diiodosalicylate is used to separate the immunoprecipitated antigen from a covalent antibody-staphylococcal protein A complex. In addition, a modified series of immunoblot incubations was employed, in which antigenic proteins were identified by incubating blots with the antiserum used for the original immunoprecipitation (e.g., La) followed by protein A-biotin and avidin-alkaline phosphatase. Overall, the procedure is straight-forward and may be applicable to other immunoblot systems.     

Spontaneous production of antibodies to deoxyribonucleic acids in patients with systemic lupus erythematosus

In vitro spontaneous anti-DNA antibody production in patients with systemic lupus erythematosus (SLE) was examined. SLE lymphocytes produced IgG and IgM anti-DNA antibody from the third culture day, and reached a plateau on the seventh culture day. This anti-DNA antibody activity in 7-day culture supernatant was abolished by pretreatment of the lymphocytes with cycloheximide, suggesting de novo immunoglobulin synthesis was required for this spontaneous anti-DNA antibody production. Lymphocytes from patients with rheumatoid arthritis (RA) and other collagen diseases including progressive systemic sclerosis, polymyositis/dermatomyositis, and polyarteritis nodosa did not produce IgG and IgM anti-DNA antibody spontaneously, but SLE lymphocytes produced substantial amounts of IgG and IgM anti-DNA antibody spontaneously. Furthermore, active SLE produced a larger amount of IgG anti-DNA antibody than inactive SLE. We observed a significant negative correlation between the number of Ia+ T cells and IgG, but not IgM, anti-DNA antibody production. Furthermore, spontaneous IgG anti-DNA antibody production was elevated after pretreatment of SLE T cells with anti-Ia and complement, suggesting that Ia+ T cells in SLE bring about suppression of autologous B cells producing IgG anti-DNA antibody.     

Antigranulocyte antibodies in patients with systemic lupus erythematosus (SLE)

Antigranulocyte antibodies were studied in patients with systemic lupus erythematosus. The frequency and type of the antibodies were identified with ELISA (Enzyme-Linked Immunosorbent Assay) and MGCT (microgranulocytotoxicity test). To check antibody specificity, a LCT (lymphocytotoxicity test) was used parallel with the above technique. The ELISA proved to be suitable for determining antigranulocyte antibodies. There was a correlation between the serum level of IgG-type antigranulocyte antibodies and granulocytopenia, but the IgM-type antigranulocyte antibodies failed to show a similar correlation. A good parallelism was found between MGCT--a test to be used to determine IgM-type antibodies--and the IgM-type antigranulocyte antibodies determined by ELISA. Of 25 SLE sera, 13 were found positive for antigranulocyte antibodies. LCT was used to examine the presence of HLA antibodies in these sera and 39% of the sera positive for antigranulocyte antibodies appeared to be granulocyte-specific while 61% reacted in the LCT, too.     

Autoantibodies from patients with systemic lupus erythematosus bind a shared sequence of SmD and Epstein-Barr virus-encoded nuclear antigen EBNA I

SmD is one of the small nuclear ribonucleoproteins frequently targeted by autoantibodies in systemic lupus erythematosus. We isolated and characterized the antibodies present in lupus sera that are specific for the C-terminal region of SmD (sequence 95–119). This region is highly homologous to sequence 35-58 of the EBNA I antigen, one of the nuclear antigens induced by infection with Epstein-Barr virus. Antibodies affinity purified over a peptide 95–119 column were able to recognize this sequence in the context of the whole SmD molecule, as they reacted with blotted recombinant SmD. Anti-SmD 95-119 antibodies bound also the EBNA I 35–58 peptide and detected the EBNA I molecule in a total cell extract from Epstein-Barr virus-infected lines. A population of anti-SmD antibodies is, therefore, able to bind an epitope shared by the autoantigen and the viral antigen EBNA I. To investigate the involvement of this shared epitope in the generation of anti-SmD antibodies, we immunized mice with the EBNA I 35-58 peptide. Sera from immunized animals displayed the same pattern of reactivity of spontaneously produced anti-SmD antibodies. They reacted in fact with the EBNA peptide as well as with SmD 95-119 and recombinant SmD. These data suggest that molecular mimicry may play a role in the induction of anti-SmD autoantibodies.