Complement-mediated solubilization of immune complexes in systemic lupus erythematosus.

The capacity of complement-mediated solubilization of immune complexes (complex releasing activity: CRA) was studied in 63 sera from eight systemic lupus erythematosus (SLE) patients. CRA in sera of active SLE (35 +/- 17.%) was significantly lower than that of inactive SLE (64.1 +/- 24.1%, P less than 0.001). In addition, 20 of 23 sera collected during active diseases demonstrated CRA values less than 50% of the control pooled serum. On the other hand, CRA of 29 of 40 sera from inactive disease exceeded the 50% level. CRA in SLE sera correlated with complement component levels and in particular with the CH50. Serial determination of CRA and of levels of circulating immune complexes (CIC), C4 and C3 in two active patients indicated that the correlation between CRA and the complement components was positive, while that between CRA and CIC was negative. These studies provide evidence that CRA may be useful for following the activity of SLE and that CRA reflects the levels of the complement components of both classical and alternative pathways. The possibility that CIC may be solubilized and opsonized by complement and cleared by the reticuloendothelial system was discussed.     

Complement-mediated immune complex solubilization and precipitation inhibition in sera of patients with non-Hodgkin's lymphoma

The complement functions of 42 patients with non-Hodgkin's lymphoma have been examined. The patients were divided into groups according to the severity of their disease: 1st--patients with high-grade lymphomas, 2nd--with low-grade lymphomas and 3rd--with chronic lymphocytic leukaemia. The adopted methods were the measurements of complement-mediated immune complex solubilizing capacity (CMSC) and the complement-mediated immune complex precipitation inhibition capacity (IPIC). The CMSC and IPIC values were examined parallel with CH50, C3 complement levels and with levels of circulating immune complexes (CIC) in the sera of patients. The results indicated that the acquired deficiency of complement functions could be established by CMSC and IPIC measurements in the sera of patients with high-grade lymphomas. These defects were found to be milder in the group with low-grade lymphomas, and were not detectable in CLL. The changes of CH50 levels were found to be similar to that of IPIC values and the decrease in C3 levels was detectable in high-grade and low-grade lymphomas too. Elevated CIC levels were found in those cases in which both CMSC and IPIC were decreased.     

Complement-mediated solubilization of rat IgA immune precipitates

The complement-mediated solubilization (CMS) of immunoprecipitates (IP) consisting of DNP-rat serum albumin (RSA) and rat monoclonal anti-DNP antibodies of the IgA [both polymeric (p-) and monomeric (m-)] or IgG2b (sub)class was studied. In contrast to IgG2b IP, solubilization of IgA IP was only observed in an autologous system, with rat serum as the source of complement. IP prepared using m-IgA were solubilized faster than those prepared using p-IgA. Analysis of both affinity and avidity of the antibodies, indicated that this difference may be due to the lower avidity of the m-IgA antibodies as compared to p-IgA. Analysis of the solubilized IP revealed deposition of C3 and C4 on IgG2b, and only C3 on IgA IP. These results point toward a role of the alternative pathway in the solubilization of IgA IP. Size analysis of the solubilized IgA IP employing sucrose density gradient ultracentrifugation, indicated that these were heterogeneous, with a size generally larger than 19 S.     

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.     

DNA levels in circulating immune complexes decrease at severe SLE flares-correlation with complement component C1q.

The objective of this study was to investigate the relationship of DNA content in circulating immune complexes with disease course and activity in SLE. The DNA content in circulating immune complexes containing anti-DNA antibodies of IgG class was determined in serial samples from 28 patients with SLE by a quantitative immunochemical assay. The patients presented various active disease manifestations over 5-55 months. Disease activity (SLEDAI-score), drug treatment and ACR-criteria were recorded. Levels of anti-dsDNA, CRP, leukocytes, complement components C3, C4 and C1q were measured. Patients with severe flares and high SLEDAI scores had low Clq levels at onset of active disease manifestations. The patients with low C1q serum levels during flare (n=13) had significantly lower amounts of DNA in immune complexes than patients with normal Clq (P=0.001). Levels of DNA in immune complexes correlated with Clq at flares (r=0.62, P<0.0001) and correlated inversely with SLEDAI scores (r=-0.47, P=0.012). In conclusion, the low levels of DNA in circulating immune complexes found in severely ill SLE patients with concomitantly low serum concentrations of Clq prior to flares might be related to tissue deposition of immune complexes.     

Circulating immune complexes, complement and complement component levels in childhood Hodgkin's disease.

Serum levels of circulating immune complexes (CIC) assayed by the Raji cell radioimmunoassay, total haemolytic complement (TCH50), Clq and C3 were correlated with clinical stage, histological type, age, sex and treatment of eighty-six children with Hodgkin's disease over a period of 4 years. Most significant findings were the changes of levels of CIC, TCH50, Clq and C3 during disease activity and following treatment. Significant perturbations were also seen in association with relapse. Levels of C and CIC were significantly elevated (P less than 0.001) at the time of diagnosis prior to splenectomy and/or any treatment. In the group before treatment, 81 percent of CIC levels were above 16 micrograms/ml with a maximum value of 1120 micrograms/ml. During treatment 33 percent were still above normal with a maximum of 320 micrograms/ml. Within 1 year after cessation of treatment, 37 percent also remained above normal levels with a maximum of 240 micrograms/ml. At relapse prior to treatment, 63 percent were again elevated with a maximum of 1280 micrograms/ml. The most significant difference on TCH50 levels relates to treatment periods. Sera of patients with active disease who are previously untreated show elevation of TCH50 levels (P less than 0.001) (average 127 CH50 mu/ml. During and after treatment eht TCH50 levels drop to 96 and 102 CH50 mu/ml, as compared to normal control of 100 CH50 mu/ml. In sera of patients at the first, second or third relapse, the combined TCH50 levels are significantly different from controls and across treatment periods (P less than 0.005).     

Studies on glomerular immune solubilization by complement in patients with IgA nephropathy

A study of the solubilization of glomerular immune deposits by serum or complement in patients with IgA nephropathy is described. Renal biopsy specimens were obtained from 15 patients with IgA nephropathy. These specimens were incubated with fresh and heated sera from healthy adults or with lyophilized complement components, i.e., C3 and C4, at 37 degrees C for one hour in plastic tubes. The sections were then stained with fluorescein isothiocyanate (FITC)-labelled anti-human IgA antisera and examined by fluorescence microscopy. Normal sera showed a marked capacity to solubilize the glomerular immune deposits characteristic of IgA nephropathy. The solubilization capacity was reduced after inactivation and absorption of sera with anti-human C3 antiserum. Lyophilized C3 or C4 did not show any ability to solubilize such deposits. It was concluded that the solubilization of glomerular immune deposits may require whole active (fresh) components of complement related to the alternative pathway.     

Circulating immune complexes and complement in bancroftian filariasis.

Sixty filarial cases, 30 endemic normal individuals and 10 non endemic subjects were investigated for the presence of Circulating Immune Complexes (CICs) and Complement Component C3. Using Polyethylene Glycol precipitation and Polyethylene Glycol precipitation-Complement Consumption methods, it was observed that CICs were raised significantly in chronic lymphatic filariasis and Tropical Pulmonary Eosinophilia (TPE) groups. The results observed by both the techniques for detection of CICs were comparable. Low levels of C3 were detected in chronic lymphatic filariasis cases by single radial immunodiffusion method, suggesting the utilization of complement by immune complexes.     

Circulating immune complexes and complement concentrations in patients with alcoholic liver disease.

A prospective evaluation of circulating immune complexes (CIC) and the activity of the complement system was undertaken in 53 alcoholic patients just before diagnostic liver biopsy. Circulating immune complexes were detected in 39% of patients with alcoholic steatosis (n = 26), 58% of patients with alcoholic hepatitis (n = 12), and 60% of patients with alcoholic cirrhosis (n = 15). No significant difference was found between the three group of patients. The activity of the complement system was within reference limits in the majority of patients and only slight differences were detected between the three groups. No significant differences were observed in liver biochemistry and complement concentrations in CIC-positive and CIC-negative patients. Detection of CIC in patients with alcoholic liver disease does not seem to be of any diagnostic value or play any pathogenic role. The high prevalence of CIC in these patients may be due to a depressing effect of ethanol on clearance of CIC or to increased immunological reactivity, or to both.     

Complement-mediated immune mechanisms in renal infection.

The belief that the inactivation of complement by renal ammonia enhances the susceptibility of renal tissue to infection has been held for some years. This thesis has been investigated in the present experiments using cultures of renal tissue maintained in vitro under physiological conditions. The experiments have confirmed that exposure of normal serum to renal issue in culture does result in the rapid loss of complement activity, but that the inactivation was not due to renal ammonia. Furthermore, in quantitative experiments, the liver was found to have even greater anti-complementary activity than renal tissue. In experiments where the biological significance of this phenomenon was examined, it was shown that the bactericidal capacity of serum was maintained even after exposure to renal tissue. The results of these investigations suggest that the biological significance of the inactivation of complement by renal tissue in vitro has been over-emphasized and requires further studies in vivo.     

Reduced complement-mediated immune complex solubilization in leprosy patients.

The ability of sera from leprosy patients to solubilize immune precipitates in vitro through the complement system was studied. The solubilizing capacity of sera from patients who did not have any reactions during 2 years or more after starting chemotherapy was comparable with that of normal laboratory volunteers. On the other hand, sera from borderline tuberculoid and lepromatous leprosy patients in reaction had markedly decreased levels of solubilization. Their total and the alternative pathway haemolytic levels did not show a corresponding decrease. Although the circulating immune complexes and serum C3d of these patients came down after the subsidence of reaction, their solubilization remained consistently low during a 3 month follow-up period.     

Pre-operative levels of serum immunoglobulins, circulating immune complexes and complement proteins in patients with different types of neoplasms

In the sera of 30 neoplasm patients without metastases, the average IgG level was higher than in the control group (CG) (18.16+/-5.10 vs. 12.62+/-2.14 g/l or 12.22+/-2.14 after excluding an outier). Average concentrations of circulating immune complexes (CIC), IgM, complement 1 inhibitor (Cli), C3c and C4 did not statistically differ between the groups. Dividing the patients' group into: breast or ovary cancer (BC), melanoma (M), digestive tract cancer (DT) and other neoplasms (ON) subgroups revealed that the IgG increase did not apply to the BC group. Relatively decreased CIC concentrations in the BC and DT group and an increased Cli in the DT group were found. Several diversities detected in the humoral immunity indices' distributions and correlations suggest activation of different mechanisms depending on the neoplasm types.     

Circulating immune complexes and complement in rheumatoid arthritis

Rheumatoid arthritis is characterized immunologically by the detection of rheumatoid factor (RF) and circulating immune complexes (CIC). The pathogenesis role played by these CIC has been discussed a long time. A part of this theoretical question, it could be of interest to know if these technics could help the clinician in the diagnosis or in the follow up of the patients with RA.     

Anti-leprosy drugs inhibit the complement-mediated solubilization of pre-formed immune complexes in vitro.

Incubation of pre-formed immune complexes (IC) (125I-HSA--anti-HSA) with normal human serum resulted in solubilization of IC. When various anti-leprosy drugs were added to human sera, solubilization of IC was fairly explicit with clofazimine, whereas this effect was marginal with dapsone. Rifampicin hardly displayed this effect. Aspirin, chloroquine, and prednisolone, the drugs used in addition to multi-drug therapy to control reactions in leprosy, were in a position to inhibit the solubilization of 125I HSA--anti-HSA by normal serum only at a very high dose. From the current data of the inhibition of solubilization of pre-formed IC along with our earlier observations on the modulation of complement-mediated haemolysis by these drugs, it may be possible to postulate that clofazimine as well as chloroquine affect early complement components. This may in turn be responsible for preventing the deposition of C3 complement onto IC.     

Reduction of the complement activation capacity of soluble IgG aggregates and immune complexes by IgM-rheumatoid factor.

The influence of IgM-rheumatoid factor (IgM-RF) on the activation of isolated C1 by soluble IgG aggregates (AIgG) and immune complexes was studied. IgM preparations obtained from the sera of four patients with seropositive rheumatoid arthritis markedly reduced the C1 activation capacity of AIgG, especially when large aggregates were tested. The results of parallel experiments with radiolabelled AIgG indicated that this inhibitory effect of IgM-RF was accompanied by a very large increase of the aggregate size. A comparable IgM preparation isolated from pooled normal human serum influenced neither the size nor the C1 activation capacity of AIgG. The inhibitory effect of IgM-RF on C1 activation was also demonstrated for soluble tetanus-anti-tetanus immune complexes. Thus, in spite of the established C activation ability of IgM-RF and the fact that, in general, larger IgG aggregates and immune complexes activate C1 more efficiently, cross-linking and size enlargement of soluble IgG complexes and aggregates by IgM-RF lead to a decrease of the C1 activation capacity. As a consequence, IgM-RF may reduce plasma complement activation by soluble IgG complexes in the circulation of patients with seropositive rheumatic diseases.     

Erythrocyte complement receptor type 1 (CR1) expression and circulating immune complex (CIC) levels in hydralazine-induced SLE.

Family studies were carried out to look at CR1 expression in 24 hydralazine-induced SLE patients (Hz Reactors), who had been off the drug for at least 1 year and were clinically well at the time of the study. Mean expression of CR1 was reduced by 27% in the group of hypertensives who had developed Hz-induced SLE compared with a group of 35 normal individuals. CR1 expression was also slightly reduced in the relatives of the Hz Reactors compared to the normal group. Using a solid-phase Clq binding assay, CIC levels were found to be elevated in the plasma of the Hz reactors and an inverse relationship was found between CR1 levels and CIC levels in this patient group. Both CR1 levels and CIC levels in Hz Reactors and normal individuals were constant over the 36 weeks studied. This study suggests that there is an association between an inability to deal efficiently with CIC and susceptibility to developing Hz-induced SLE.     

Lack of complement activation by human IgA immune complexes.

Complement activation may play an important role in renal injury associated with glomerular deposition of IgA immune complexes. The ability of naturally occurring human IgA immune complexes (IgA-IC) and covalently cross-linked human IgA oligomers (X-IgA) to activate complement were examined in vitro and in vivo. Large-sized IgA-IC were isolated from a patient's serum by affinity purification (Jacalin-Sepharose) and gel chromatography. Stable X-IgA were prepared by chemical cross-linking with a heterobifunctional reagent, N-succinimdyl 3-(2-pyridyl-dithio) propionate (SPDP). Treatment of fresh normal human serum with large amounts of either IgA-IC or X-IgA failed to activate C3. The C3 consumption was measured immunochemically by the decrease of the B antigen on the native C3 and by the generation of iC3b. Addition of these complexes to serum did not result in cleavage of factor B. Administration of human IgA-IC or X-IgA to mice, killed after 6 h, resulted in glomerular deposition of IgA. Despite the presence of intense glomerular IgA deposits no C3 was detected. Collectively, these findings suggest that neither soluble nor renal localized human IgA complexes activate complement.     

Family studies of erythrocyte complement receptor type 1 levels: reduced levels in patients with SLE are acquired, not inherited.

It has been claimed that patients with systemic lupus erythematosus (SLE) have an inherited deficiency of erythrocyte complement receptor type 1 (CR1, with ligand binding specificity for C3b, iC3b and C4b). CR1 functions as the only cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg. The activity of this receptor on red cells may be an important mechanism for handling immune complexes which have bound C3b or iC3b. Radioligand binding studies were performed using a monoclonal antibody to CR1, E11, to enumerate these receptors accurately. The results confirmed that patients with SLE have a reduced number of CR1 molecules per red cell, but showed no reduction in CR1 levels amongst their consanguineous relatives. Study of 13 normal families suggested the presence of heritable factors controlling the numbers of erythrocyte CR1 molecules; in particular there was a correlation between mean parental CR1 numbers and CR1 numbers in their children. However, amongst 17 families of 19 patients with SLE, four families were identified in which genotypically 'high CR1' SLE patients had persistently low phenotypes. This is not compatible with the hypothesis that the reduction in erythrocyte CR1 numbers in these patients is inherited.     

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.     

Dissociation of primary antigen-antibody bonds is essential for complement mediated solubilization of immune precipitates.

The role of dissociation of primary antigen-antibody bonds in the solubilization of immune complexes (IC) has been investigated using photo-affinity crosslinked IC comprising NAP15-BSA and murine monoclonal anti-DNP antibodies. Non-covalently linked IC were solubilized rapidly when incubated with normal human serum (NHS), whereas covalently-linked IC were solubilized poorly or not at all. The rate and extent of complement activation produced by incubating covalently-linked and non-covalently linked IC with NHS was similar as assessed by the production of the C1s:C1-inhibitor, C3:properdin and C5b-9 complexes and the anaphylatoxins C4a and C3a. Thus, the inability of serum to solubilize photo-affinity crosslinked IC must be due to failure of dissociation of primary antigen-antibody bonds.