Human complement receptor 1 (CR1, CD35, C3b/C4b receptor) is a membrane glycoprotein found on erythrocytes and most white blood cells. Reduced CR1 expression has been reported in patients with systemic lupus erythematosus (SLE) (1, 2). The origin of soluble CR1 (sCR1) might be direct secretion or proteolytic cleavage at the cell surface. Microvesiculation does occur, suggesting that vesiculation can also be the reason for CR1 loss (3). Elevated serum levels of CR1 have been observed in patients with different diseases but not in those with SLE (4). The aim of our study was to define whether a part of sCR1 is bound to the complement component opsonized circulating immune complexes (CICs) in sera from patients with SLE.
A heterogeneous population of 61 patients with SLE and 30 healthy donors was investigated. Patients fulfilled at least 4 criteria for a diagnosis of SLE as defined by the American College of Rheumatology (5) but were chosen irrespective of their disease duration, disease activity, and the therapy used. Three groups of patients were formed, based on the level of renal involvement. Patients in group I (60.6%) had urinary abnormalities classified as World Health Organization (WHO) class I, 19.7% of patients (group II) were designated as WHO class II and III, and 19.7% of the patients (group III) had diffuse proliferative glomerulonephritis and were designated as WHO class IV. Informed consent was obtained from both patients and healthy controls.
In order to quantitate serum sCR1 CICs, a novel enzyme-linked immunosorbent assay (ELISA) was developed in which anti-CR1 mouse monoclonal antibody HB8592 (a generous gift from R. P. Taylor) as capture antibody and mouse IgG1 as control were fixed on flat-bottomed microtiter plates (Greiner, Frickenhausen, Germany). Serum samples obtained from patients and controls were added (1:40 dilution) to phosphate buffered saline, and the plates were incubated for 1.5 hours at 37°C and washed. The IgG components of CICs were recognized by an anti-human IgG conjugated with horseradish peroxidase (Dako, Glostrup, Denmark). The color was developed by o-phenylenediamine–H2O2, and absorbances were measured with an ELISA reader (Labsystems Multiscan MS, Helsinki, Finland) at 492 nm. The mean background binding of mouse IgG1 was subtracted from the optical density measured for the samples of patients, and the results were expressed as nanograms per milliliter of equivalent human IgG on the basis of an IgG calibration curve. The level of sCR1 in the same sera was determined by a capture ELISA (4). The immune complex levels in the same sera were determined with a commercial enzyme immunoassay (C1q-IC; Hycor Biomedical, Penicuik, Scotland, UK), according to the manufacturer's instructions.
The analytic properties of the novel assay were evaluated, and the intraassay and interassay coefficients of variation were 9.3% and 10.6%, respectively. The mean ± SD of the sCR1 CIC concentration was significantly higher (P < 0.0001) in sera from 61 patients with SLE than that in sera from 30 controls (Figure 1). The average value of sCR1 CIC in group II (n = 12) and group III (n = 12) was also significantly increased (P = 0.001) compared with group I (n = 37), indicating that sCR1 CIC levels are elevated in groups having severe histologic lesions. The sCR1 CIC and C1q immune complex levels in patients, as determined by these ELISAs, were compared by Pearson's regression analysis. A strong correlation (r = 0.844, P < 0.0001) was observed between the C1q–immune complex and sCR1 CIC concentrations in sera from patients with SLE. The levels of sCR1 found in sera from patients with SLE were not different from those observed in healthy individuals (mean ± SD 191.1 ± 139.0 versus 160.1 ± 67.2 ng IgG equivalent/ml), supporting previous findings.
CICs are heterogeneous particles consisting of antigen, antibodies (immunoglobulins), and complement components, including C3b/C4b. Enhanced renal deposition of CICs and renal damages are involved in the pathomechanism of immune complex diseases, including SLE. Erythrocyte CR1 has an important role in the clearance of C3b/C4b opsonized immune complexes from the circulation (1). CR1 is mainly involved in binding and transporting them to the liver and spleen macrophages, where they are subsequently bound and ingested (6–8). The reduced levels of erythrocyte CR1 detected in SLE patients were acquired rather than inherited and correlate with the renal involvement of the patients (2, 5). One reason for these deficiencies could be that CR1 was lost progressively even from normal erythrocytes during in vivo aging, while in contrast stimulation of erythropoiesis (9) and plasmapheresis (10) increases the amount of CR1 expressed on erythrocytes. Anti-CR1 autoantibodies recognize mostly CR1 epitope(s) not present on native molecules, suggesting that they were not directly involved in the loss of CR1 as sCR1–anti-CR1 CICs (11).
In conclusion, we observed that an important part of cleaved CR1 appeared as sCR1 CICs in the circulation of patients with SLE, especially those with severe renal lesions, and their levels correlate with the concentration of serum immune complexes. These results strongly suggest that sCR1 CIC has some role in the clearance of CICs and in the pathomechanism of SLE; moreover, it should also be considered in other diseases, such as infections, in the future.