Urs E. Nydegger

Circulating Immune Complexes in the Serum in Systemic Lupus Erythematosus and in Carriers of Hepatitis B Antigen QUANTITATION BY BINDING TO RADIOLABELED Clq

A sensitive and reproducible procedure for the detection of soluble immune complexes in sera from patients with various immunopathological disorders is reported. Radiolabeled C1q is reacted with sera containing immune complexes. Separation of free from complex bound [125I]C1q is achieved by selective precipitation with polyethylene glycol (PEG). The method is based on both the large molecular size and the C1q-binding property characterizing immune complexes. The minimal amount of aggregated immunoglobulins thus detected is about 10 μg and that of soluble human IgG-anti-IgG complexes is about 3 μg of complexed antibody. Some immune complexes formed in large antigen excess (Ag2Ab) can still be detected by this radiolabeled C1q binding assay. The specificity of the radiolabeled C1q binding test was documented by the inability of antigen-F(ab′)2 antibody complexes to lead to a precipitation of [125I]C1q in PEG. In a second step, this radiolabeled C1q binding assay was applied to an experimental model of immune complex disease and was shown to be efficient for the detection of in vivo formed immune complexes. Finally, the technique could be applied to the study of sera from patients with systemic lupus erythematosus (SLE) or to carriers of the hepatitis B antigen (HB-Ag). Significantly increased [125I]-C1q binding values were observed in 52 sera from SLE patients when compared to values obtained with healthy blood donors (P<0.001). Particularly high values were seen in active disease, a finding which was confirmed by follow-up studies performed with four SLE patients. No increased [125I]C1q binding was seen in 18 healthy carriers of the HB-Ag; whereas, sera from carriers with hepatitis appear to precipitate increased [125I]C1q percentages: 7/24 cases with acute transient and 4/7 cases with chronic persistent hepatitis were found to increasingly bind [125I]C1q. The results were also used for a correlative study of [125I]C1q binding to IgG levels in the sera but increased [125I]C1q binding could not be attributed to high serum IgG levels which are likely to account for gammaglobulin aggregates. These examples suggest the utility of the radiolabeled C1q binding assay for the evaluation of immune complex diseases in human pathology.

Circulating and intra-articular immune complexes in patients with rheumatoid arthritis. Correlation of 125I-Clq binding activity with clinical and biological features of the disease.

The correlation between the incidence and level of immune complexes in serum and synovial fluid and the various clinical and biological manifestations of rheumatoid arthritis has been studied. Immune complexes were quantitated using a sensitive radioimmunoassay, the 125I-Clq binding test, in unheated native sera and synovial fluids from 50 patients with seropositive (RA +) and 45 with seronegative (RA -) rheumatoid arthritis, 17 with other inflammatory arthritis, and 37 with degenerative and post-traumatic joint disease. The following observations were made: (a) when compared to the results from patients with degenerative and post-traumatic joint diseases, the 125I-Clq binding activity (Clq-BA) in synovial fluid was found to be increased (by more than 2 SD) in most of the patients with RA + (80%) and RA - (71%) and in 29% of patients with other inflammatory arthritis; the serum Clq-BA was also frequently increased in both RA + (76%) and RA - (49%) patients, but only exceptionally in patients with other inflammatory arthritis (6%); (b) a significant negative correlation existed between the Clq-BA and the immunochemical C4 level in synovial fluids from patients with RA + and RA -; (c) neither the serum nor the synovial fluid Clq-BA in rheumatoid arthritis significantly correlated with the erythrocyte sedimentation rate, the clinical stage of the disease, or the IgM rheumatoid factor titer; and (d) the serum Clq-BA in patients with rheumatoid arthritis and extra-articular disease manifestations (40 +/- 34% in those with RA +,32 +/- 29% in those with RA -) was significantly increased as compared to the serum Clq-BA in patients with joint disease alone (24 +/- 30% in those with RA +, 10 +/- 13% in those with RA -). Experimental studies were carried out in order to characterize the Clq binding material in rheumatoid arthritis. This material had properties similar to immune complexes: it sedimented in a high molecular weight range on sucrose density gradients (10-30S) and lost the ability to bind Clq after reduction and alkylation, or after acid dissociation at pH 3.8, or after passage through an anti-IgG immunoabsorbant. DNase did not affect the Clq BA. These results support the hypothesis that circulating as well as intra-articular immune complexes may play an important role in some pathogenetic aspects of rheumatoid arthritis. The 125I-Clq binding test may also be of some practical clinical value in detecting patients who have a higher risk of developing vasculitis.

Circulating Complement Breakdown Products in Patients with Rheumatoid Arthritis CORRELATION BETWEEN PLASMA C3d, CIRCULATING IMMUNE COMPLEXES, AND CLINICAL ACTIVITY

Quantitative determination of the small C3 breakdown product, C3d, was used to investigate complement activation in 45 plasma samples from 30 patients with rheumatoid arthritis (RA). The mean plasma C3e level in these samples (3.0 +/- 1.3 mg/100 ml) was significantly increased (P less than 0.001) as compared to patients with degenerative joint disease (0.9 +/- 0.4 mg/100 ml) and healthy blood donors (0.8 +/- 0.5 mg/100 ml). C3d levels were increased by more than s SD in 79% of RA samples. Plasma C3d levels were compared with C3d concentrations in synovial fluid. In most RA patients, the C3d levels were higher in synovial fluid than in plasma. A very significant correlation between plasma C3d levels and circulating immune complexes, as measured by determination of Clq binding activity (Clq BA), was observed (P less than 0.001). C3d levels were more elevated in RA patients with extra-articular disease manifestations (3.8 +/- 1.2 mg/100 ml) as compared to patients with joint disease alone (2.2 +/- 1.0 mg/100 ml). C3d levels and Clq BA were also significantly correlated (P less than 0.001) with the RA disease activity expressed by an index derived from sedimentation rate, joint score, and duration of morning stiffness. A close relationship between C3d levels, Clq BA, and the clinical activity further appeared during follow-up studies. The present observations suggest that a parallel but rather independent activation of the complement system may be induced by immune complexes in circulating blood and in the joint spaces during the course of rheumatoid arthritis.

Hepatitis B antigen and systemic lupus erythematosus

ZusammenfassungSeren von 22 Patienten mit Lupus erythematodes disseminatus (LED) wurden auf das Vorliegen des Hepatitis-B-Antigens (HBsAg) untersucht. Zu diesem Zwecke wurden 3 verschiedene Methoden eingesetzt: 1. Komplement-Fixationstest (KFT), 2. Überwanderungselektrophorese (UEP) und 3. Radio-Immunoassay.Seren von 8 Patienten zeigten positive Resultate im KFT. Dieselben Seren und solche von 28 weiteren LED Patienten enthielten kein HBsAg, wenn sie mit UEP und Radio-Immunoassay getestet wurden.Zusätzliche Studien dienten der Charakterisierung des für den positiven KFT verantwortlichen Faktors. Es zeigte sich, daß die LED Seren auch Komplement in Gegenwart von normalen Seren fixierten, wenn letztere mehrfach gefroren und aufgetaut oder auf 65°C erhitzt worden waren. Der Komplement-fixierende Faktor war absorbierbar mit aggregierten Gammaglobulinen, nicht aber mit HBsAg. Die Komplement fixierende Aktivität des Faktors wurde durch Behandlung der Seren mit 2-Merkapto-Aethanol abgeschwächt.Die falsch positiven Komplement-Fixationsteste mit LED Seren und anti HBsAg Antiseren können demzufolge auf das Vorliegen von Anti-Antikörpern mit Gammaglobulinspezifität zurÜckgeführt werden.SummarySera from 22 patients with systemic lupus erythematosus (SLE) were examined for the presence of hepatitis B antigen (HBsAg) by a complement fixation (CF) test, by an immunoelectrophoretic method (counterelectrophoresis-CEP), and by radioimmunoassay (RIA). The sera from 8 patients gave positive results using CF. However, the same sera and sera from 28 additional SLE patients, when tested with CEP and RIA, were not shown to contain HBsAg.Additional studies were carried out in order to characterize the factor responsible for the false positive CF of the 8 SLE sera. It was shown that the sera also fixed complement in presence of normal serum previously submitted to freezing and thawing or heating at 65°C. The complement fixing factor was readily absorbed by aggregated IgG but not by insolubilized HBsAg. Complement fixation was strongly diminished by 2-mercaptoethanol treatment of SLE serum. It thus appears that the false positive reactions for the presence of HBsAg obtained by CF are due to the occurrence of “anti-antibodies” reacting with aggregated IgG. There is no increased incidence of HBsAg in the serum of SLE patients.

RED CELL AUTOANTIBODIES

ABSTRACT Red blood cells (RBCs) are multifaceted targets for autoantibody recognition. A number of autoantibody-mediated autoimmune diseases will be associated to RBC lysis. Complement affects the susceptibility of RBC for antibody-mediated lysis, but in many instances, more particularly in experimental models of autoimmune haemolytic anaemia (AIHA), FcγR-mediated erythrophagocytosis apparently plays the major role. The possibilities for anti-RBC to recognize epitopes on RBC surfaces are abundant. Structural proteins, blood group proteins and carbohydrates are the major autoantigens. They are carried by a cell that lives 120 days and reach every corner of the lymphoid system. The prevailing specificity against which autoanti-RBCs react are the epitopes of the Rhesus system, but other blood group systems, such as MNS, Duffy, Kidd, or Kell, are also at stake. As detection techniques, the antiglobulin reaction is used in agglutination systems (direct antiglobulin test, DAT) or in cytofluorometric systems. Elution of antibodies from the RBC surface at low pH or high ionic strength allows for separation of the autoantibodies from their site of impact, and by simple centrifugation they may be separated and used for specificity studies with reference to RBCs. Therapy of AIHA, if acute and/or resistant to conventional drug therapy, is now feasible using extracorporeal immunoadsorptions. Clinically tolerated anticomplement agents such as C1 inhibitor, modified dextran sulfates, soluble complement receptor 1, or the complement scavengers i.v. immunoglobulins are part of ever-improving clinical study protocols. More recently, two genetic loci implicated in murine AIHA have been identified. Obviously, identification of the AIHA susceptibility genes is of paramount importance for the understanding of the mechanism of this disease, and is indeed a subject of extensive and active investigation.

Implications physio-pathologiques des complexes antigène-anticorps

Seventy years after the description of the Arthus phenomenon, the concept of immune complex mediated pathology is widely recognized in clinical pathophysiology. The availability of methods for the detection of soluble immune complexes opens new paths for clinical investigation in this field. This is demonstrated for systemic lupus erythematosus, viral hepatitis B and herpes zoster disease. New experimental approaches deal with the question of tissue localization of immune complex mediated diseases, e.g. through affinity of antigens for certain tissue structures.