Reduced complement-mediated immune complex solubilizing capacity and the presence of incompletely solubilized immune complexes in SLE sera.

Reduced complement-mediated solubilization (CMS) of pre-formed immune complexes (IC) was demonstrated in sera from 11 out of 12 SLE patients. The presence of incompletely solubilized endogeneous IC in SLE sera was indicated by the following findings: (1) When IC positive SLE sera with reduced CMS capacity were mixed with normal donor sera they inhibited the CMS of the latter sera. (2) Resuspended PEG (2.75%) precipitates obtained from SLE sera inhibited the CMS of normal donor sera. (3) Non-solubilized or incompletely complement solubilized IC in SLE sera give a strong response in the PEG-CC assay for IC. The IC activity of SLE sera was clearly reduced in this assay when the endogeneous IC were solubilized prior to testing. In contrast, sera of 14 rheumatoid arthritis (RA) patients exhibited normal CMS. IC which could be further solubilized by complement were not demonstrable although all RA sera were IC positive.     

Detection of circulating immune complexes by PEG precipitation combined with ELISA

An assay to detect circulating immune complexes is described and exemplified with serum from SLE and RA patients. The method is based on selective precipitation of immune complexes with PEG followed by a specific ELISA assay to detect the immunoglobulins present in the precipitate. The immunoglobulins of the precipitate were then monitored by their capacity to bind protein A and dDNA. With the PEG-protein A-ELISA procedure immune complexes were detected in 46% of the sera from SLE patients compared with 68% when using the PEG-DNA-ELISA.     

Quantitation and antigenic characterization of bound C3 of circulating immune complexes in systemic lupus erythematosus,rheumatoid arthritis,and primary biliary cirrhosis

In recent years defective function of the complement-mediated clearance of immune complexes (IC) has been reported in patients with immune complex disease. The defect has been found at different levels in the clearance system. An important event in this sequential system is the binding of C3-coated particles to C3 receptors on erythrocytes and phagocytes. This study focuses on immunochemical properties of IC-bound C3 that reflect the functional state of the molecule. Sera from patients with primary biliary cirrhosis (PBC), rhematoid arthritis (RA), and systemic lupus erythematosus (SLE) and from normal subjects were analyzed for their level of C3 precipitable in 2.7% (w/v) polyethylene glycol (PEG). The mean levels for the patient categories were significantly higher than that for the normal subjects. The immunochemical study revealed several differences among the different forms of PEG-precipitable C3. All forms expressed C3(D) antigens which are expressed by immune complex-associated and denatured forms but not by soluble physiological forms of C3. The expression of these antigens was proportionately lower for the complex-associated C3 of PBC compared to that of RA and SLE. Furthermore, employing monoclonal anti-C3(D) antibodies against the C3c and the C3d domain, distinct differences could be detected among all forms of PEG-precipitable C3. Sera from RA and SLE, in particular, contained PEG-precipitable C3 that exhibited distinctive immunochemical features with respect to these epitopes.     

Antibodies against Ku protein in sera from patients with autoimmune diseases

Immunoaffinity-purified Ku protein was used to screen sera from patients with systemic lupus erythematosus (SLE), scleroderma, myositis and Sjögren''s syndrome for anti-Ku antibodies in a quantitative immunoblot assay. Sixteen percent of the 159 studied sera were reactive with the Ku protein; significantly increased frequencies of anti-Ku antibodies were found in SLE (19%) and scleroderma (14%) sera. Patients with myositis and Sjögren''s syndrome showed similar frequencies. All positive sera had antibodies to the 86 kD subunit of Ku protein; only one serum did not react with 70 kD subunit. Frequencies of other autoantibodies were compared in anti-Ku positive and negative patients. Only anti-Sm antibodies, especially in the absence of anti-nRNP, appear to be associated with the presence of anti-Ku antibodies. A strong correlation between anti-Ku antibodies and the class II HLA antigen DQw1 (89% of the positive sera) was observed, suggesting participation of MHC genes in the mounting of the anti-Ku immune response.     

Cryoglobulinaemia and circulating immune complexes in tropical splenomegaly syndrome.

Large amounts of cryoglobulins and soluble immune complexes were detected in the sera of thirteen patients with tropical spenomegaly syndrome (TSS). Complexes were detected by three different methods: radiobioassay, a modified rheumatoid factor-binding activity method and a modified Clq-binding assay. Protein precipitable by 4% polyethylene glycol (PEG) was also measured. The cryoglobulins contained IgM, IgG and in some cases, C3. It is likely that in TSS, marked immune complex formation is associated with hypergammaglobulinaemia and that continuous engulfment of these complexes by cells of the reticuloendothelial system (RES) is the cause of the hepatosplenomegaly.     

Comparison of some procedures detecting circulating immune complexes

Tests for circulating immune complexes were performed by means of 1) plain polyethylene glycol (PEG) precipitation (PEGprec), 2) immunoelectrophoresis of PEG precipitates (IEpp), 3) anti-antibody (AA) inhibition test with sera (AA-Is), and 4) AA inhibition test with PEG precipitates (AA-Ipp). The tests were performed with 156 pathological sera from patients with myasthenia gravis, syphilis, adenocarcinomas of the gastrointestinal tract, rheumatoid arthritis and systemic lupus erythematosus, and 51 normal sera from blood donors. PEGprec was positive with 76 sera, IEpp with 84 sera, AA-Is with 64 sera, and AA-Ipp with 74 sera. Comparison of results in all four tests showed high degree of correlation; all p values were below 0.005. The lower sensitivity of AA inhibition tests was due to the fact that these tests detect only complexes formed by IgG but not by IgM, whereas the remaining two tests detect complexes formed by antibodies of both these immunoglobulin classes. When sera of patients with rheumatoid arthritis and SLE were removed from the material studied, the four tests showed about equal sensitivity. PEGprec gave positive results with two normal sera and the remaining tests were negative with all these sera. It appears that the simultaneous application of PEGprec, IEpp, AA-Is and AA-Ipp will give sensitive and reliable procedure for detecting circulating immune complexes.     

Complement-mediated inhibition of immune precipitation in patients with immune complex diseases

The ability of human sera to prevent the precipitation of antigen-antibody complexes has been investigated. The early complement components including C3 are required for optimal prevention of immune precipitation, whereas the later components are not required. The sera of 36 of 75 patients with seropositive rheumatoid arthritis (RA), 14 of 32 with SLE and four of 17 with glomerulonephritis exhibited reduced capacities to prevent immune precipitation. In contrast sera from patients with seronegative RA, ankylosing spondylitis, psoriatic arthritis or degenerative joint disease were normal in this respect. In SLE and GN sera hypocomplementaemia was frequently associated but not always with failure to prevent immune precipitation, whereas only a small proportion of the patients with seropositive RA and reduced capacity to retain complexes in a soluble form were hypocomplementaemic. Thus the failure of sera to prevent the precipitation of antigen-antibody complexes is not always associated with hypocomplementaemia.     

Comparison of some procedures detecting circulating immune complexes

Tests for circulating immune complexes were performed by means of 1) plain polyethylene glycol (PEG) precipitation (PEGprec), 2) immunoelectrophoresis of PEG precipitates (IEpp), 3) anti-antibody (AA) inhibition test with sera (AA-Is), and 4) AA inhibition test with PEG precipitates (AA-Ipp). The tests were performed with 156 pathological sera from patients with myasthenia gravis, syphilis, adenocarcinomas of the gastrointestinal tract, rheumatoid arthritis and systemic lupus erythematosus, and 51 normal sera from blood donors. PEGprec was positive with 76 sera, IEpp with 84 sera, AA-Is with 64 sera, and AA-Ipp with 74 sera. Comparison of results in all four tests showed high degree of correlation; all p values were below 0.005. The lower sensitivity of AA inhibition tests was due to the fact that these tests detect only complexes formed by IgG but not by IgM, whereas the remaining two tests detect complexes formed by antibodies of both these immunoglobulin classes. When sera of patients with rheumatoid arthritis and SLE were removed from the material studied, the four tests showed about equal sensitivity. PEGprec gave positive results with two normal sera and the remaining tests were negative with all these sera. It appears that the simultaneous application of PEGprec, IEpp, AA-Is and AA-Ipp will give sensitive and reliable procedure for detecting circulating immune complexes.     

Differential generation of chemiluminescence — detectable oxygen radicals by normal polymorphonuclear leukocytes challenged with sera from systemic lupus erythematosus and rheumatoid arthritis patients

Summary The generation of chemiluminescence (CL)-detectable oxygen radicals by normal human polymorphonuclear leukocytes (PMN) after challenging with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) sera is described. CL was measured in a luminol-dependent assay and referred to a standard obtained when preformed immune complexes (Ic) (human tetanus toxoid-antitoxoid Ic resuspended in normal pooled serum) were tested on PMN. Normal sera gave rise to CL activity by PMN between 0% and 50% of the standard Ic (mean±standard error of the mean (SEM): 20.7±4.8). Sera from SLE and RA patients induced strikingly different biological effects on PMN. SLE sera generally induced a high CL-detectable generation of oxygen metabolites which may be causally related to the intense tissue damage (vasculitis) frequently observed in this disease. In contrast to SLE, RA sera induced a CL-detectable respiratory burst by PMN that was included in the normal range. Thus, the biological effects of these sera in terms of stimulation of toxic oxygen radical generation by phagocytes are quite different. This generation of oxygen radicals might reflect a different clearance of circulating Ic by PMN in SLE and RA disease.Abbreviations CL Chemiluminescence - PMN Polymorphonuclear Leukocytes - SLE Systemic Lupus Erythematosus - RA Rheumatoid Arthritis - Ic immune complexes - TAT tetanus-antitetanus toxoid - TAT-S tetanus-antitetanus toxoid resuspended in pooled, non-inactivated human serum - SEM standard error for the mean - EHM Eagle-Hepes-Medium - PBS phosphate-buffered-saline - RF rheumatoid factor - SPA staphylococcal protein A - ANA antinuclear antibodies - KD kilodaltons - Ag antigen     

Autoantibodies from Rheumatoid Arthritis Patients Recognize Antigens on the Synoviocyte Surface

We have found autoantibodies in the sera from rheumatoid arthritis (RA) patients which recognize two cell surface antigens of approximately 70 kDa and 28 kDa from synoviocyte extracts as detected by immunoprecipitation analysis. These polypeptides were immuno-precipitated from extracts containing mainly macrophage-like synoviocytes (type A) but not from extracts of homogeneous fibroblast-like synoviocytes (type B). These autoantigens are not selectively expressed by RA synoviocytes, since both RA and non-rheumatoid synovia were reactive for RA sera. From the panel of different RA sera tested, 64% immunoprecipitated the 70 kDa band, and 27% recognized the 28 kDa polypeptide. These differences in the specificity of the sera seemed to be related to the clinical state of the donor. The sera from patients suffering from other autoimmune diseases such as autoimmune thyroiditis and systemic lupus erythematosus (SLE) do not appear to be reactive for these specificities, but sera from patients with Sjögren's syndrome, psoriatic arthritis, and Crohn's disease showed a weak cross-reactivity with the 70 kDa polypeptide. This autoreactivity against synovial cells in RA supports the idea that these cells participate in the initial immune response of the disease.     

Anti-fibronectin autoantibodies in systemic lupus erythematosus, rheumatoid polyarthritis, and various viral or bacterial infectious diseases

A micro-enzyme linked immunosorbent assay (ELISA) aimed at detecting anti-fibronectin (anti-Fn) antibodies has been developed and standardized. Fifty sera from systemic lupus erythematosus (SLE), 50 from rheumatoid arthritis (RA) patients, as well as 200 sera from patients with bacterial or viral infections were assayed for the presence of anti-Fn autoantibodies. The IgG fractions of three representative positive sera (1 SLE, 1 RA and 1 streptococcal endocarditis) were digested with pepsin and the resulting F(ab')2 fragment assayed in the test. The presence of the anti-Fn activity in these fragments as well as lack of correlation in individual sera between the level of anti-Fn (as determined by ELISA) and that of Ig or immune complexes, suggest that our anti-Fn autoantibodies are indeed detected in our assay. The meaning of these antibodies, which were also found with bacterial and viral infections is discussed within the frame of the fibronectin biological properties.     

Epitope mapping with synthetic peptides of 52-kD SSA/Ro protein reveals heterogeneous antibody profiles in human autoimmune sera.

The reactivity of autoantibodies present in the sera of 489 patients with Sjögren's syndrome (SS), systemic lupus erythematosus (SLE) and other autoimmune diseases was investigated by ELISA using recombinant 52-kD SSA/Ro protein (rRo52) and 39 overlapping synthetic peptides representing the entire sequence of Ro52. We report that IgG antibodies reacting with rRo52 were present in the sera of a large number of patients with SS (67% of patients with primary SS and 46% of patients with SS associated with SLE), whereas they were less frequent (10-25%) in SLE, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA) and mixed connective tissue disease (MCTD), and absent in scleroderma. Among the 39 peptides tested, five were recognized by sera from 30-65% of patients with SS, namely peptides representing residues 2-11, 107-122, 107-126, 277-292 and 365-382. Patients with JCA had raised levels of IgG antibodies reacting with peptides 2-11 and 365-382, and 51% of patients with MCTD had raised levels of IgG antibodies reacting with peptide 365-382. None of the five peptides was recognized by more than 20% of sera from patients with SLE and RA. Interestingly, and of importance in the field of diagnostic tests based on peptides, the reactivity of antibodies to the Ro52 synthetic peptides varied greatly according to the origin of sera. Inhibition experiments using either patients' sera or antibodies induced in rabbits against Ro52 peptides showed that the four domains 2-11, 107-122, 277-292 and 365-382 are accessible on the surface of the Ro52 protein. These regions may thus be involved in the induction of specific antibodies in autoimmune patients.     

Polyethylene Glycol Precipitates of Serum Contain a Large Proportion of Uncomplexed Immunoglobulins and C3

High pressure liquid chromatography (HPLC) was used to fractionate redissolved polyethylene glycol (PEG) precipitates isolated from the sera of normal volunteers and from patients with IgA nephropathy (IgAN) and systemic lupus erythematosus (SLE), 2 diseases characterized by elevated levels of circulating immune complexes. The individual fractions were analyzed by solid phase ELISA for IgA, IgM, C3, IgG, and complexes of IgG-IgA and IgG-C3. Although PEG precipitates were enriched for high molecular weight IgA and IgG (presumably bound within CIC), significant amounts of IgM, unbound IgG and C3 were also present. The quantities of the PEG-precipitable proteins did not correlate with their serum concentrations. IgG-IgA and IgG-C3 complexes were found in all precipitates examined, but the levels of complexes were higher in both patient groups. These results indicate that PEG precipitates a considerable quantity of proteins not bound in immune complexes. There appeared to be greater protein precipitation from sera of the patient groups compared to the amount precipitated from the normal sera. These results suggest that an understanding of the mechanism of PEG precipitation may be important in defining abnormalities in IgAN, SLE and perhaps other diseases characterized by elevated levels of CIC. In addition, the possibility of undetected CIC in PEG precipitable material must be considered.     

Polyethylene Glycol Precipitates of Serum Contain a Large Proportion of Uncomplexed Immunoglobulins and C3

High pressure liquid chromatography (HPLC) was used to fractionate redissolved polyethylene glycol (PEG) precipitates isolated from the sera of normal volunteers and from patients with IgA nephropathy (IgAN) and systemic lupus erythematosus (SLE), 2 diseases characterized by elevated levels of circulating immune complexes. The individual fractions were analyzed by solid phase ELISA for IgA, IgM, C3, IgG, and complexes of IgG-IgA and IgG-C3. Although PEG precipitates were enriched for high molecular weight IgA and IgG (presumably bound within CIC), significant amounts of IgM, unbound IgG and C3 were also present. The quantities of the PEG-precipitable proteins did not correlate with their serum concentrations. IgG-IgA and IgG-C3 complexes were found in all precipitates examined, but the levels of complexes were higher in both patient groups. These results indicate that PEG precipitates a considerable quantity of proteins not bound in immune complexes. There appeared to be greater protein precipitation from sera of the patient groups compared to the amount precipitated from the normal sera. These results suggest that an understanding of the mechanism of PEG precipitation may be important in defining abnormalities in IgAN, SLE and perhaps other diseases characterized by elevated levels of CIC. In addition, the possibility of undetected CIC in PEG precipitable material must be considered.     

A rapid and reproducible method for the analysis of immune complexes using affinity chromatography and Western blotting

A new procedure which couples different analytical techniques in a format permitting the rapid analysis of immune complex components is described. Complexes obtained from sera by polyethylene glycol (PEG) precipitation were resuspended and then added, using a batch method, to antibody coupled to Sepharose beads. Antibody directed against either human C1q or human C3c were used in the present study. Bound immune complexes were washed and then eluted from the Sepharose by sodium dodecyl sulphate (SDS) treatment and simultaneously reduced with dithiothreitol. Individual components were separated by SDS gradient polyacrylamide gel electrophoresis and then transferred to nitrocellulose by Western blotting. Individual strips of nitrocellulose were investigated using specific antisera and a radiolabelled probe. Immune complexes (IC) isolated from the sera of 7 rheumatoid arthritis (RA) patients were analysed using this method and the results obtained for both affinity adsorbents compared.     

Evidence for a disease specific antigen in circulating immune complexes in ankylosing spondylitis.

The presence of circulating immune complexes (CIC) has been documented in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and ankylosing spondylitis (AS) by various investigators. It has been suggested that these may be used as probes to identify antigens playing a role in these pathological processes. Using a solid-phase cross-reaction assay to establish if these complexes reacted with each other in specific disease groups, it was found that polyethylene glycol (PEG) precipitates of six AS patients cross reacted in 29 of 36 tests, but reacted with SLE and RA PEG precipitates in only two of 24 tests in each case. SLE PEG precipitates cross-reacted in four of 14 tests and reacted with none of the six AS and four RA precipitates. Similarly, RA PEG precipitates did not cross-react (none of 16 tests), nor did they react with AS (none of 24 or SLE precipitates (none of 16). Similar results were observed when IgG, obtained after acid dissociation on sucrose density gradients of CIC isolated by PEG precipitation and staphylococcal protein A chromatography, was used as the solid phase component. F (ab')2 fragments with similar antibody specificity were obtained by pepsin digestion of isolated CIC from three of six AS patients. These bound radiolabelled AS PEG precipitates (2.02-2.40%) significantly more than SLE (0.22-0.28%) or RA (0.29-0.35%) precipitates. These studies demonstrate the feasibility of obtaining F (ab')2 fragments with antibody activity from isolated CIC and the presence of a disease specific antibody specificity in AS CIC. The nature of the antigen involved remains to be elucidated. Such a cross-reactive antibody specificity was not found in RA nor SLE CIC.     

A fluorimetric assay for human antibodies to all the histones

We describe a fluorescence immunoassay for anti-histone antibodies in human sera. Histones are bound to immobilised tyrosine-glutamic acid copolymer on a polystyrene cuvette. With mixed histones as antigen normal sera showed low levels of antibody binding. Much higher values were obtained with some sera from rheumatoid arthritis (RA) patients positive for antinuclear antibodies, and from patients with vasculitic RA, systemic lupus erythematosus (SLE) and drug induced LE. Antibodies to all 5 individual histones were elevated in SLE and vasculitic RA patients. Preliminary results suggest that differences in response patterns may be disease related.     

Elevated levels of soluble CD40 ligand (sCD40L) in serum of patients with systemic autoimmune diseases

The CD40–CD40L costimulatory pathway is involved in the evolution of many autoimmune diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and Sjögren's syndrome (SS). Increased levels of sCD40L in the serum have been associated with disease activity in SLE. The aim of this study was to investigate the role of sCD40L in the development of lupus nephritis and examine its possible association with cryoglobulinemia in Sjögren's syndrome. We used a 2-site sandwich ELISA to measure the levels of sCD40L in sera, from 64 patients with SLE, RA and SS and 17 healthy blood donors. Biological specimens from the affected tissues such as urine from patients with lupus nephritis and saliva from patients with SS were also tested. In this regard, paired sera and first morning urine samples from 6 SLE patients (3 with active lupus nephritis and 3 with inactive lupus nephritis) were tested with the sCD40L ELISA protocol as well as paired sera and salivary samples from 5 patients with SS and cryoglobulinemia, 5 patients with SS and anti-Ro or anti-La autoantibodies and 5 age-matched healthy control donors. We also examined possible correlations of sCD40L levels with several laboratory and clinical parameters in SS and SLE. We found that sera from SLE and SS patients had significantly higher levels of sCD40L compared to sera from healthy control donors. No sCD40L was detected, in urine samples of patients with either active or inactive nephritis and in salivary samples from SS patients or normal subjects. Soluble CD40L is elevated in sera of SS and SLE patients but further investigation is needed to determine its possible role in SLE nephritis and Sjögren's syndrome.     

Immune deposits in normal skin of patients with systemic lupus erythematosus: relationship to the serum capacity to solubilize immune complexes

Immunofluorescent deposits at the dermal-epidermal junction (DEJ) of the skin (lupus band test or LBT) were evaluated in 134 patients with various connective tissue diseases. LBT was found positive in 23 of 32 (71.8%) patients with systemic lupus erythematosus (SLE), in 3 of 53 (5.6%) patients with rheumatoid arthritis (RA), and in 1 of 5 cases of mixed connective tissue disease (MCTD). No deposits were found in patients with systemic sclerosis and with vasculitis. In patients with SLE a positive LBT showed a direct correlation with serum Clq binding activity (ClqBA) and with hypocomplementemia. The mean ClqBA was 13.49 +/- 12.85 and 2.38 +/- 2.27% in SLE patients with positive and negative band test, respectively (P less than 0.005). Likewise depressed mean serum levels of C3 and C4 were detected in patients with skin deposits (P less than 0.005). Sera from SLE patients showed an overall decreased capacity to solubilize preformed immune complexes when compared to normal sera. Furthermore 10 band-positive patients were less able to solubilize immune complexes than sera from LBT-negative lupus patients (45 +/- 16 and 62 +/- 11%, respectively; P less than 0.01). Also the capacity to inhibit the precipitation of immune complexes was decreased in SLE patients with negative LBT (P less than 0.05). In conclusion our data suggest that in SLE patients a decreased complement-mediated solubilization of immune complexes is involved in the persistence of high levels of circulating immune aggregates and, considered its correlation with positive LBT, may be responsible for the deposits of immunoglobulins at the dermal-epidermal junction of the skin.     

IgG antibodies from patients with primary Sjögren''s syndrome and systemic lupus erythematosus recognize different epitopes in 60-kD SSA/Ro protein.

Five synthetic peptides corresponding to the N-, the C- and a central domain in 60-kD SSA/Ro protein were prepared and tested with sera from 112 patients with systemic lupus erythematosus (SLE), 55 with primary Sjögren's syndrome (pSS) and 29 with rheumatoid arthritis. Among these five fragments, one representing residues 21-41, was recognized by antibodies in 57% of pSS patients. Interestingly, this peptide was recognized by only a few (≤7%) of SLE sera, while 63% of pSS sera and 46% of SLE sera tested in parallel possessed antibodies reacting in ELISA with purified 60-kD SSA protein. The ELISA results were compared with the pattern of reactivity obtained in immunodiffusion and immunoblotting. The results indicate that the sensitivity of ELISA using peptide 21-41 and pSS sera was in the same range as immunoblotting and higher than immunodiffusion. Thus the peptide 21-41 proved useful for the detection of anti-SSA antibodies in the sera of patients with pSS. Furthermore, a positive ELISA using peptide 21–41 could be of potential use to discriminate pSS with systemic features from SLE. The fact that peptide 21–41 is recognized by antibodies in pSS but only by very few SLE sera implies that different mechanisms are involved in the anti-SSA immune response in these two autoimmune diseases.