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Many autoantibodies have been described in patients with systemic lupus erythematosus. However, besides antibodies to dsDNA, the majority of them has not been shown to help the clinician. It is interesting to note that the recently described autoantibodies to C1q strongly correlate with the occurrence or worsening of a preexisting lupus nephritis.
Previous studies have suggested that C1q reacts with DNA via both the globular region of C1q (GR) and the collagen-like region of C1q (CLR). In this study, the binding of dsDNA and ssDNA to GR and CLR was quantitated by a solid-phase assay. Both dsDNA and ssDNA bound to the GR and CLR of C1q in an ionic strength-dependent manner. Under physiologic salt concentrations, however, dsDNA and ssDNA bound preferentially to CLR and not to GR. The binding of dsDNA to C1q was not affected by heat inactivation of C1q or its exposure to pH 4.45, which abolished the binding of heat-aggregated human IgG (AHG) with C1q. The preincubation of the solid-phase C1q with AHG did not decrease the binding of dsDNA or ssDNA to the solid-phase C1q. These results indicate that the major sites for binding DNA to C1q are located in the CLR of C1q and are not overlapping with those for AHG or immune complexes.
An inhibitor of human C1q has been purified from serum and identified as a chondroitin 4-sulfate proteoglycan. A typical preparation contained 22% uronic acid, 20% hexosamine, 12% sulfate, and 9% protein. When chromatographed on Sepharose CL-2B, the proteoglycan was eluted as a broad peak with a mean Kav of 0.6, which indicates that it is polydisperse and has an average Mr = approximately 175,000 (range, 45,000-750,000). Unlike the major species of cartilage proteoglycans, the serum proteoglycan did not form a complex with hyaluronic acid. Additional evidence for the noncartilaginous origin of C1q inhibitor is that its glycosaminoglycan chains totally lack chondroitin 6-sulfate isomers. Furthermore, the glycosaminoglycan component of C1q inhibitor was eluted from Sepharose CL-6B with a Kav of 0.52, indicating that these polysaccharide chains are considerably larger than those of human articular cartilage proteoglycan. The interaction between the proteoglycan and C1q was clearly evident in 0.15 M NaCl, as demonstrated by a radial immunodiffusion technique. The interaction decreased with increasing ionic strength but was not entirely abolished even at 0.3 M NaCl. These findings suggest that the interaction between C1q and the C1q inhibitor may occur under physiological conditions and may be of importance in modulating C1q activity in vivo.
In order to characterize the immune complexes detected in patients with Sjogren's syndrome (SS) and with rheumatoid arthritis (RA), the sera of 19 patients with SS alone and 11 with SS plus RA were examined. Elevated quantities of circulating immune complexes (CIC) were detected in 67% by the C1q-binding assay (C1q-BA), 73% by the C1q-solid phase (C1q-SP) assay, 43% by the monoclonal rheumatoid factor solid phase assay (mRF-SP) and 33% by the mRF-inhibition assay (MRF-Inh). Elevated concentrations of IgM RF were detected in 83% and of IgG RF in 73% of the sera by radioimmunoassay. Strong correlations existed between RF of the IgM and IgG classes and both the C1q-BA and the C1q-SP. Three lines of evidence indicated that RF were important components of the immune complexes detected by these radioimmunoassays. These results indicated that in those patients with RA plus SS, as well as those with SS alone, both IgM and IgG RF made substantial contributions to immune complexes detected both by C1q-BA and C1q-SP.
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Binding sites for both the collagen-like and globular domains of C1q have been described on a variety of cell types. HUVEC were previously shown to express the 60- to 67-kDa receptor recognizing the collagen-like domain of C1q. This study demonstrates the presence of a distinct 28- to 33-kDa HUVEC protein (gC1qR) that interacts with the globular head domain of C1q. Polyclonal Abs raised against the Raji cell gC1qR partially inhibited HUVEC interaction with immobilized C1q and recognized a 28- to 33-kDa protein on Western blots. The Ab also reacted strongly with poly-L-lysine-immobilized, glutaraldehyde-fixed, intact HUVEC in ELISA assays. No significant difference in reactivity was noted if HUVEC were permeabilized with 0.2% Triton X-100. However, unfixed HUVEC grown on gelatin-coated microtiter wells to 80% confluence failed to express significant amounts of gC1qR Ag. Quantitation of HUVEC gC1qR by gel scanning suggested the presence of 5.7 +/- 3.8 x 10(6) molecules/cell (mean +/- SD; n = 4). A quantitative sandwich ELISA procedure, however, detected only 3.7 +/- 0.6 x 10(5) gC1qR molecules/cell (mean +/- SD; n = 4), consistent with previously described gC1qR multimerization. The capacity of endothelial cells to recognize both the collagen-like and globular domains of C1q via distinct binding sites may have implications for the role of C1q in vascular inflammatory and thrombotic lesions.
The complement system is one of the important factors involved in the hyperacute rejection of xenografts. This report deals with the activation of the complement system in a clinical trial where pig kidneys were extracorporeally connected to two volunteer dialysis patients who were pretreated with plasmapheresis in order to substantially reduce anti-pig xenoantibodies. The clinical data of the perfusion experiments and the patients humoral immune response to pig xenoantigens have been reported in detail (Xenotransplantation 1996; 3:328-339, 340-353). Three consecutive daily plasmapheresis treatments of the patients reduced the plasma complement protein (C3, C4, and C5) concentrations to 8-27% of the baseline values. The perfusion of the pig kidney connected to patient 1 was terminated at 65 min due to graft rejection and this patient was not hemodynamically affected by the experiment. The second experiment was terminated at 15 min due to an anaphylactic like reaction of the patient. In patient 1 a slight reduction of plasma C3, C4, and C5 and an increase of C5a and SC5b-9 occurred, while C3a decreased during the perfusion. Patient 2 had an increase of all complement parameters, most prominent for C4d and SC5b-9, which occurred concomitant with the appearance of the anaphylactic like side effects. In general, plasma levels of PMN elastase, IL6 and IL8 increased in both patients during the perfusion. Immunohistochemical investigation of the kidney tissues revealed deposition of human complement factors C1q, C4c, and C3c in a congruent pattern with the vasculature of the kidney in patient 1. In kidney 2 only trace amounts of C1q and C3c were found. Both kidneys were negative for properdin. Therefore, in this experimental set up with extracorporeal connection of pig kidneys to the human circulation the human complement cascade is activated mainly through the classical pathway.
An immunohistological study was carried out on lungs and livers of pigs experimentally infected with two different African swine fever virus (ASFV) isolates. ASFV antigen, swine immunoglobulins (IgM and IgG) and (Clq) complement were demonstrated in both organs at different stages of infection. The ASFV antigen was mainly found in mononuclear phagocytic system (MPS) cells. Immunoglobulins and complement were observed in plasma, infected and non-infected phagocytic cells and cell debris. These findings suggest the presence, in acute infection, of immune complexes which may be involved in immunopathogenic mechanisms.
C1q, mannose-binding lectin (MBL), and pulmonary surfactant protein A (SPA) interact with human monocytes and macrophages, resulting in the enhancement of phagocytosis of suboptimally opsonized targets. mAbs that recognize a cell surface molecule of 126,000 Mr, designated C1qRP, have been shown to inhibit C1q- and MBL-mediated enhancement of phagocytosis. Similar inhibition of the SPA-mediated enhancement of phagocytosis by these mAbs now suggests that C1qRP is a common component of a receptor for these macromolecules. Ligation of human monocytes with immobilized R3, a IgM mAb recognizing C1qRP, also triggers enhanced phagocytic capacity of these cells in the absence of ligand, verifying the direct involvement of this polypeptide in the regulation of phagocytosis. While the cDNA for C1qRP encodes a 631 amino acid membrane protein, Chinese hamster ovary cells transfected with the cDNA of the C1qRP coding region express a surface glycoprotein with the identical 126,000 Mr in SDS-PAGE as the native C1qRP. Use of glycosylation inhibitors, cleavage of the mature C1qRP with specific glycosidases, and in vitro translation of C1qRP cDNA demonstrated that both posttranslational glycosylation and the nature of the amino acid sequence of the protein contribute to the difference between its predicted m.w. and its migration on SDS-PAGE. These results verify that the cDNA cloned codes for the mature C1qRP, that C1qRP contains a relatively high degree of O-linked glycoslyation, and that C1qRP cross-linked directly by monoclonal anti-C1qRP or engaged as a result of cell surface ligation of SPA, as well as C1q and MBL, enhances phagocytosis.
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A case of hereditary angioedema secondary to C1 esterase inhibitor deficiency associated with lupus-like nephritis is reported. The patient was initially treated with both corticosteroids and danazol and subsequently had Guillain-Barré syndrome together with appearance of circulating immune complexes and an increase in total complement and C1q, C3, C4, B, and C1 esterase inhibitor levels. Guillain-Barré syndrome might be secondary to danazol therapy since this drug could increase both circulating immune complex production and complement synthesis, thereby providing additional substrate for the underlying immune complex disease. Normalization of complement might therefore be hazardous in lupus with underlying complement deficiency states.
ZnSO4 promotes the binding of C1q to immune complexes over the same concentration range (10(-5)-10(-4) M) that it inhibits binding of C1 to cell-bound immunoglobulin [Biochem. Biophys. Res. Commun. (1981) 103, 856-862]. At higher concentrations (10(-3)-2 X 10(-2) M) ZnSO4 inhibited the binding of C1q to immune complexes, [Ki = (6 +/- 2) X 10(-3) M]. This inhibition could be correlated with a ZnSO4-induced change in the tryptophan fluorescence of C1q [delta F 25%, Kd = (9.9 +/- 1.0) X 10(-3) M].
Experiments were undertaken to determine if porcine C1q could replace human C1q in the solid-phase immunoassay of human immune complexes (ICs). Porcine C1q was obtained by a two-cycle precipitation method involving dialysis against chelating agents in low ionic strength buffer. C1q was adsorbed to polystyrene beads and in vivo- or in vitro-formed ICs binding to the solid-phase C1q were detected with 125I-labeled or horseradish peroxidase-conjugated anti-human gamma antibodies. Unfractionated, heat-aggregated human gamma globulin (delta IgG) could be detected at 20 ng/ml when diluted in buffer only. The detection threshold changed to 40-80 ng delta IgG/ml when the assay was run with buffer containing normal human serum diluted 1: 1000 (the serum dilution used for detecting natural ICs). Analysis of systemic lupus erythematosus sera revealed that 60% contained highly significant levels of ICs (binding greater than or equal to 3 S.D. above the mean of controls). Comparison with platelet aggregation test results revealed a highly significant correlation between the two methods (P less than 0.0001), even though each assay detected ICs in several serum specimens negative in the other test. These results demonstrate that porcine C1q can functionally replace human C1q in the solid-phase immunoassay of human ICs. Since porcine blood is normally a waste product of the meat-processing industry, it is an obvious source of easily isolated C1q for use in such an assay.
Several parameters of the 125I-C1q-binding test were investigated: ionic strength, pH, concentration of EDTA, influence of serum C1q and the possibility of interference by polyanions. Lowering the ionic strength of the borate buffer resulted in increased precipitation of 125I-C1q in normal human serum. This increase was dependent on the presence of serum proteins, probably immunoglobulins. When the concentration of the EDTA was decreased, increased precipitation of 125I-C1q in normal human serum was also observed. This was prevented by adding NaCl to the EDTA solution. However at very low concentrations of EDTA (too low to chelate all calcium ions in the serum), increased precipitation of 125I-C1q in normal human serum was observed even in the presence of added NaCl. Addition of purified C1q to sera from patients with very low C1q levels had varying effects on the results of the C1q-binding test: (a) it decreased the C1q-binding activity of some sera, probably by competition with 125I-C1q for binding sites on the immune complexes; (b) it increased the C1q-binding activity of other sera, probably by enhancing the precipitation of immune complexes as a consequence of the cross-linking effect of C1q; or (c) it had no influence, possibly due to the opposite effects of (a) and (b). The addition of dextran sulphate resulted in a dose-dependent increase in the 125I-C1q-binding activity of normal human serum. This effect was dependent on the interaction of dextran sulphate with either C1q or low-density lipoproteins and was prevented by addition of polybrene to the assay. However, addition of polybrene to sera with a high C1q-binding activity scarcely influenced binding activity.
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Circulating immune complexes (CIC) containing HBsAg and HBeAg were identified in sera of 5 out of 6 children with hepatitis B mediated membranous glomerulonephritis. CIC were precipitated from sera by 3.5% PEG, washed and subsequently analysed after acid dissociation and trypsin digestion. HBsAg, anti-HBs and albumin; HBeAg, anti-HBe and anti-HBc were recovered from the isolated complexes and these findings are discussed. Analysis of 3.5% PEG mediated precipitate of human serum proteins showed the relatively high content of IgG classical pathway complement components: C1q, C4 and C3.
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