Detection and characterization of circulating immune complexes by ultracentrifugation. Technical aspects

Most techniques for detection of immune complexes (IC) in biological fluids do not fulfil criteria essential for IC definition, i.e. identification of the antigen and antibody after dissociation and demonstration of their immunological binding. We have fractionated plasmas by sucrose density gradient ultracentrifugation at pH 7.4 and 2.8 and studied the effects of acid pH on isolated and plasma immunoglobulins, on antibody titers, on IC formed in vitro and on aggregated immunoglobulins. There was no effect on the distribution of isolated radiolabeled IgG, IgA or IgM or on IgG and IgA of fresh serum samples. Heavy IgG appeared at pH 2.8 in samples stored at ?20°C in excess of 1 month and also at pH 7.4 when stored in excess of 6 months. Heavy IgA was present in samples frozen for 1 week or more at both pHs. Anti-BSA and anti-DNA antibody titers and distribution were identical at both pHs. BSA : anti-BSA and DNA : anti-DNA IC formed in vitro were almost completely dissociated with recovery of antigen and antibody. IC obtained by fractionation at pH 7.4 could be refractionated at pH 2.8 with recovery of antibody and antigen. The distribution of aggregated IgG and IgA was unchanged at acid pH. This technique allows differentiation of complexed and aggregated Ig, identification of specific IC by comparing antibody titers at physiologic and acid pH, isolation of IC and the recovery of antigen and antibody from IC formed in vitro.     

Nature of circulating immune complexes in infective endocarditis.

Two percent polyethylene glycol (PEG) precipitation was found to be a useful method for detecting circulating immune complexes (CIC) and could be used diagnostically to implicate infective endocarditis. Complexes consisting of a least Clq, IgG, and IgA were typically detected in sera from patients with infective endocarditis. Serial studies showed that CIC detection and measurement could be used to monitor clinical progress. Successful clinical improvement was reflected by decreasing CIC levels and the disappearance of rheumatoid factor, and, where increasing amounts of CIC were found, this may indicate situations where treatment was insufficient or inappropriate. There was specific free antibody demonstrable in the serum of six out of nine patients against their own infecting organisms, but attempts to identify the specificity of the complexed antibody as being directed against these organisms or their extracellular products failed. We could not detect any radioactive precipitin arcs, indicative of the free antibody also being in the complex, between the F(ab')2 preparation from the complex and the electrophoresed bacterial antigens in a radio-immunoelectrophoresis system. Eleven out of 13 sera that contained Clq, IgG, and IgA in their complexes also contained rheumatoid factor. Immunisation against the patient's own persisting CIC may explain this phenomenon.     

Hepatic uptake of circulating IgG immune complexes.

IgG antibodies were found to increase the uptake of circulating dinintrophenylated human serum albumin (DNP-HSA) preparations by the nonparenchymal liver cells in rats. Highly DNP-conjugated HSA was taken up by the Kupffer cells both when given alone and when complexed by IgG. More lightly DNP-conjugated HSA was taken up mainly by the liver endothelial cells. Here, IgG promoted the antigen uptake both by the Kupffer cells and by the endothelial cells. Uptake of IgG immune complexes (IgG-ICs) by the sinusoidal endothelial cells of the liver is a new aspect on the function of these cells. Whether or not this phenomenon is Fc receptor-mediated is discussed. A heat-labile serum factor was found to direct the ICs to the Kupffer cells. This implies that serum complement and hepatic C3 receptors are essential for the physiological clearance of circulating immune complexes.     

Specific circulating immune complexes in amoebic liver abscess.

A micro enzyme-linked immunosorbent assay was used to detect the presence of amoebic antigen in polyethylene-glycol-precipitated circulating immune complexes (CIC). A cutoff dilution of greater than 1:160 for the precipitates was taken to be of clinical significance. Among the patients with amoebic liver abscess, 93% (14 of 15 confirmed cases) had indications of amoebic antigen in CIC. In 57% of the suspected cases of amoebic liver abscess, amoebic antigen could be detected in CIC. The specificity of the technique for the demonstration of specific amoebic antigen is indicated by the undetectable levels of amoebic antigen in CIC in serum samples from nine cases of nonamoebic hepatic diseases and 10 apparently healthy subjects. It is thus believed that the specific diagnosis of an individual case of amoebic liver abscess can be made by demonstrating specific amoebic antigen in CIC.     

Detection of circulating immune complexes in patients with glomerulonephritis.

143 patients were evaluated clinically on the basis of the renal biopsy. Three methods for detecting circulating immune complexes (CIC) were employed: complement consumption test, inhibition of erythrocyte antibody IgG-EA rosette forming test and optical density of 4% PEG precipitated sera. CIC were present in the sera of all patients with acute poststreptococcal glomerulonephritis (2 weeks after streptococcal infection of the throat). In a group of patients with chronic glomerulonephritis the highest values in positive results were observed in lupus nephritis, chronic proliferative glomerulonephritis and chronic submicroscopic glomerulonephritis. These results were compared with levels of total hemolytic complement, C3, C4 components and serum immunoglobulins (IgA, IgG, IgM).     

Detection of circulating immune complexes in alcoholic liver disease.

Sera of twenty-five patients with alcoholic liver disease and forty normal control sera were screened for circulating immune complexes by means of the anti-antibody neutralization test and by Raji-cell membrane immunofluorescence assay. IgG-containing immune complexes were detected in thirteen out of twenty-five patients with alcoholic liver diseases and in one out of forty normal individuals; in addition, IgA-containing complexes were demonstrated in seven out of thirteen sera positive for IgG complexes. The presence of immune complexes was restricted to alcoholic hepatitis and active cirrhosis, thus indicating a relationship with disease severity.     

Sequential determination of circulating immune complexes in experimental filariasis.

The role of endotoxin responsiveness in defense against gonococcal infection was studied in endotoxin-resistant (C3H/HeJ) and endotoxin-susceptible (C3H/HeN) mice by using a model of disseminated gonococcal infection (DGI) and a model of gonococcal survival in the female genital tract to determine the ability of the mice to eliminate gonococci. The 50% lethal dose in the DGI model was 10(9.6) for C3H/HeJ mice and 10(5.2) for C3H/HeN mice. Levels of bacteremia during infection indicated the C3H/HeJ mice cleared large numbers of gonococci from their peripheral blood by 24 h post-inoculation but that C3H/HeN mice did not. Additionally, the peritoneal leukocyte response after intraperitoneal inoculation of gonococci was greater in C3H/HeJ mice than in C3H/HeN mice, which suggested that the ability to mount an inflammatory response to endotoxin may be important in defense against DGI. Besides being different in susceptibility to DGI, C3H/HeJ mice were found to be more resistant then C3H/HeN mice to genital colonization by gonococci. The resistance of C3H/HeJ mice to genital colonization by gonococci appeared to be due to both the high numbers of polymorphonuclear leukocytes in the genital secretion and the predominance of inhibitory gram-negative genital flora in that mouse strain.     

Flow-cytometric detection of circulating immune complexes

In an effort to develop an assay which would rapidly detect and analyze circulating immune complexes, we have adapted the Raji cell radioimmunoassay (RIA) to a flow cytometric (FCM) analysis. The advantages of immune complex analysis by FCM are many. Foremost is the effectiveness and efficiency of the FCM method relative to the radioimmunoassay (RIA) method. The data demonstrated that FCM detection is three times more sensitive than RIA detection. Only populations of viable Raji cells bearing immune complexes are analyzed because parameters of the FCM analysis permitted the elimination (gating out) of dead cells. The determinations are rapid and the data are immediately available for several additional analyses. Because of the availability of many fluorescent monoclonal antibodies to complement components, viral antigens, light chains and immunoglobulin isotypes, it is possible to detect many components that might be present in the Raji cell bound complexes. Finally, the Raji cells can be characterized in different stages of their cell cycle to generate information about the state of the cells and the density of the receptors involved in binding the complexes.     

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.     

Evidence for circulating immune complexes in sarcoidosis

Immune complexes were detected by the platelet aggregation technique in sera of six out of twenty-six patients with sarcoidosis. Five of these patients had acute bilateral hilar lymphoma syndrome, four of them with concomitant erythema nodosum. The size of the immune complexes was 19S or larger.     

IgA-containing circulating immune complexes in gluten-sensitive enteropathy

Since mucosal immune response involving IgA may be particularly important in the pathogenesis of gluten-sensitive enteropathy (GSE), we examined the sera of 22 patients with GSE for IgA-containing circulating immune complexes using a sensitive radioimmunoassay, the Raji cell assay for IgA-containing circulating immune complexes. The Raji cell assay for IgG-containing circulating immune complexes and the ¹²⁵I-C1q-binding assay were also used to measure IgG- or IgM-containing circulating immune complexes in these patients. Ten of 22 (45%) patients had IgA-containing circulating immune complexes, while 11 of 22 (50%) had IgG- or IgM-containing circulating immune complexes. Thirteen of 22 (59%) patients had circulating immune complexes detected by at least one of the assays used. Neither the presence nor level of immune complexes correlated with disease activity in any of the patients studied. Five patients, whose disease was well controlled on a gluten-free diet, were studied serially during dietary challenge with gluten. It was found that IgA-containing circulating immune complexes did not develop or increase in amount in the serum of these patients despite the induction of gastrointestinal symptoms. In addition, no significant change in IgG- or IgM- containing circulating immune complexes occurred in any of the challenged patients. No significant abnormalities of serum complement levels (C3, C4, factor B) were detected in any of the patients including those challenged with gluten. Sucrose density-gradient ultracentrifugation studies revealed that the IgA-containing circulating immune complexes had sedimentation characteristics between 9S and 13S. The presence of circulating immune complexes in only 59% of patients with GSE, their lack of correlation with disease activity, and their failure to change during dietary gluten challenge suggests that circulating immune complexes do not play a primary role in the pathogenesis of GSE.     

HIV antigen detection in circulating immune complexes

Various methods were evaluated for their effectiveness in releasing HIV antigen (Ag) from artificial immune complexes (IC) and from IC present in serum from HIV antibody (Ab) positive subjects. The most effective methods for recovering HIV Ag from IC were those which included a denaturation step to prevent reassociation of Ag with Ab. IC precipitation in 2.5% polyethylene glycol followed by acid treatment with 1 M glycine.HCl (pH 2) for 10 min at 70 degrees C in the presence of 0.05% SDS gave very satisfactory results. With this method, IC were detected in sera from HIV antibody positive Caucasian subjects at all stages of infection. After HIV IC dissociation, HIV Ag was detected in a significant number (8/17 or 47%) of asymptomatic subjects. IC were most prevalent during the late stages of infection. A substantial increase in HIV Ag positivity was also observed in 20 Senegalese HIV Ab positive sera. After HIV IC dissociation HIV antigen detection increased from 2/20 to 12/20. The relevance of IC detection is discussed.     

The detection of circulating immune complexes in renal transplant patients.

The Raji cell assay and radiolabelled Clq binding method were used to detect circulating immune complexes in the sera of renal transplant patients. Complexes were found in seven of twelve patients using the Raji cell assay; only one serum sample was positive by the Clq method. In five patients the complexes were detected prior to the clinical diagnosis of rejection and in those in whom treatment reversed the rejection the complexes rapidly disappeared. The presence of complexes correlated with a vascular type of rejection characterised by fibrin deposits in the glomeruli in the absence of immunoglobulin or C3 deposits. In two patients, in whom anti red cell antibodies were present, irreversible rejection occurred without the presence of detectable complexes in the sera.     

Demonstration of two disease specific antigens in circulating immune complexes.

A method is described to assess antigenic cross-reactivity between soluble immune complexes precipitated from sera with polyethylene glycol. The precipitated complex from one serum was dissociated in acid and used to coat a plastic cup. Radioiodinated complexes from another serum were dissociated in the cup, neutralized and allowed to reassociate overnight. The binding of the labelled complex was used to measure the cross-reactivity between the complexes. Using this technique, complexes from a group of patients with haematuria and hypertension have been found to share an antigen, and a different antigen was found in patients with bullous pemphigoid. The participation of rheumatoid factors in the cross-reactions is unlikely, and no cross-reactivity of either group was found with sera from patients with rheumatoid arthritis.     

Isolation and characterization of circulating immune complexes in cystic fibrosis.

A methodology for the isolation and immunologic characterization of IgG-containing circulating immune complexes (IgG-CIC) as detected by the 125IClq-binding assay (ClqBA) is described. We applied this methodology to sera from patients with cystic fibrosis (CF), both positive and negative for IgG-CIC. We used latex-fixation-positive rheumatoid arthritis sera and normal human sera as positive and negative controls, respectively. All IgG-CIC-positive serum samples from CF patients were found to contain antibodies against Pseudomonas aeruginosa in the isolated complexes. Some patients also had antibodies in CIC specific for Staphylococcus aureus and Candida albicans. CIC specificity corresponded to respiratory tract colonization for each patient.     

Significance of circulating immune complexes in pulmonary tuberculosis

In the present study we have tried to demonstrate circulating immune complexes (CIC) in sera from patients with pulmonary tuberculosis (TB) by three techniques; latex agglutination; 3.5% PEG precipitation and determination of optical density at 280 nm and RIA of CIC using bovine spermatozoa. About 40 normal control sera and 100 TB patients sera were investigated for the presence of CIC. Seventeen per cent cases of pulmonary TB were positive by latex agglutination while none of the control was positive. Levels of CIC as detected by PEG precipitation and RIA were significantly elevated in patients as compared to normal controls. While IgG, IgA and IgM were elevated in the CIC of patients, IgM immunoglobulins were detected only in patients and not in controls. Detection of CIC may at times be useful in diagnosis, prognosis and therapeutic monitoring of disease processes, but it is the characterization of immune complexes (IC) and identification of the specific components of these complexes which holds the greatest potential for better understanding of disease mechanisms. CIC were precipitated using 3.5% PEG from sera of patients suffering from TB. The specific anti-TB antibody component of complex was determined using S. aureus protein A as a solid phase, Anti-BCG antibody and 125I-labelled TB antigen. The specific TB antigen component of the IC was dissociated thermally from TB antibody and assayed by a radioimmunoassay technique developed in our laboratory. Patients were classified into two groups. Those those sputum was positive for Mycobacterium tuberculosis by smear and/or culture and those whose sputum was negative. The TB antigen concentrations of CIC was higher 19.1 +/- 2.3 ng/ml (mean +s.e.) in sputum positive cases, and 9.9 +/- 1.9 ng/ml in sputum negative cases as compared to 2.2 +/- 0.3 ng/ml in controls. Patient groups were significantly different from controls as well as from each other (P less than 0.001). Anti-TB antibody ratios were 11.7 +/- 1.48, 5.1 +/- 1.5 and 0.6 +/- 0.1 in sputum positive, sputum negative and controls. The significance of differences between the groups was P less than 0.001. The effect of treatment administered over a period of 12 weeks or more was evaluated. It was observed that in patients with persistent demonstration of M. tuberculosis in the sputum, the TB antigen and TB antibody levels of CIC were consistently high. In patients who responded to anti-tubercular drugs the TB antigen levels decreased progressively while TB antibody levels remained high.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of circulating immune complexes in heart disease

Circulating immune complexes (CIC) were characterized for the content in IgG, IgA, IgM, C3 and C4 in patients with heart disease. The levels of IgG, IgM and C4 in PEG-precipitates of patients' sera were significantly higher than those found in controls, and the precipitation profile was similar to that of rheumatoid arthritis patients. Differences were observed in the composition of CIC: IgM was highest in association with myocarditis, and C3 predominated in cases of valvular disease. Complex-bound C4 was significantly higher in patients with myocardial infarction which developed pericarditis either early or late in the evolution of the disorder. Antimyocardial antibodies could be detected in sera and in corresponding PEG-precipitates. The bulk of the data suggests that CIC might play a pathogenetic role in various heart diseases.