C3 Nephritic Factor Research Articles

Evidence that production of autoantibody to the alternative pathway C3 convertase is a normal physiologic event

The origin of autoantibody production was studied with the use of antibody to the alternative pathway C3 convertase (C3 nephritic factor (C3NeF), as a model. Pokeweed mitogen stimulation of peripheral mononuclear cells from newborn infants, normal adults, and patients with membranoproliferative glomerulonephritis indicated that the ability to make C3NeF is apparently present in everyone from the time of birth. In addition, C3NeF appeared to express a single or very limited idiotope (21/21 isolates). The data also suggest that the elaboration of C3NeF may approximate an antibody response after immunization. Thus the C3NeF fraction of the total IgG or IgM produced in culture by pokeweed mitogen-stimulated mononuclear cells from normal neonates and adults, as well as from patients, was in the range of the production of specific antibody. Further, both IgG and IgM C3NeF produced by cells from these normal individuals, including newborn infants, had an affinity for antigen (10(8) to 10(9) L/mol) that was also in the range of specific antibody. Most of the autoantibody molecules (5/7) from serum were IgG3; two B cell clones producing C3NeF were CD5-negative. These experiments indicate that unmutated germline genes are used in the production of C3NeF and that a limited spectrum of antiidiotypic antibodies regulate its production.

Autoantibody to complement neoantigens in membranoproliferative glomerulonephritis

With the exception of C3 nephritic factor, autoantibody formation has not been commonly associated with membranoproliferative nephritis (MPGN). We measured autoantibodies (nephritic factors) to the C3 convertases C3bBb (NFa) and C3bBbP (NFt), which result in fast and slow C3 activation, respectively, and to a neoantigen on C1q fixed to a solid phase (spC1q) in sera from 29 patients with MPGN type I, 26 with type II, and 28 with type III. Autoantibody formation was common in all MPGN types. An autoantibody to a C3 convertase neoantigen was identified in more than 75% of the hypocomplementemic MPGN sera tested. Anti-C3bBb (NFa) was present in 81% of patients with MPGN type II but was rarely found in either type I or type III. Anti-C3bBbP (NFt) was common in both MPGN I and III. Anti-spC1q was present in 74% of patients with type I and in 38% and 48% of types II and III MPGN, respectively. Patients with MPGN types I, II, and III had one and two serum autoantibodies detected significantly more frequently than did a group of healthy subjects. The presence of any one autoantibody was not specifically associated with the presence of any other autoantibody. The results indicate that multiple autoantibody formation is common in all MPGN types. MPGN II, and possibly MPGN I, tend to form more specific autoantibodies.

Familial Incidence of C3 Nephritic Factor, Partial Lipodystrophy and Membranoproliferative Glomerulonephritis

C3 nephritic factor is an IgG autoantibody that causes complement activation by stabilizing the alternative pathway C3 convertase. It is associated with partial lipodystrophy and membrano-proliferative glomerulonephritis. The occurrence of C3 nephritic factor, partial lipodystrophy and membranoproliferative glomerulonephritis, whether singly or in any combination, is usually sporadic. We describe the coexistence of all three of these conditions in members spanning two generations of a single family. This suggests that the pathogenesis of these conditions may be linked and that genetically determined factors may, in some circumstances, contribute to disease susceptibility.

Systemic lupus erythematosus in a patient with partial lipodystrophy

Systemic lupus erythematosus developed in a 35-year-old woman who had partial lipodystrophy since she was 7 years old. To our knowledge, this association has been reported only once. She also had hypocomplementemia, mesangiocapillary glomerulonephritis type II, and a serum assay with positive results for C3 nephritic factor. The association of partial lipodystrophy with other autoimmune disorders suggests an immunologic pathogenesis for this disease.

Quantitation of C4 nephritic factor by an enzyme-linked immunosorbent assay

We have developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of C4 nephritic factor (C4NeF). Incubation of the C4NeF-positive serum from patient M.I. with normal human serum (NHS) in the presence of human aggregated IgG (AHG) resulted in the formation of stable C4-C2 complex. No complex was formed in EDTA or under the condition free of AHG. The reaction mixture was filtered through an ACA 22 column, from which the C4-C2 complex was eluted at the first protein peak. When IgG purified from M.I. serum was incubated with NHS and AHG, C4-C2 complex also increased in proportion to dose of the purified M.I. IgG. These results show that C4NeF in M.I. serum stabilizes C4b2a convertase of the classical complement pathway, and is quantified by the ELISA. C4NeF activity was measured, using the ELISA method, in patients with various glomerular diseases, and found elevated in three of 24 patients with membranoproliferative glomerulonephritis (MPGN) type I and slightly but distinctly positive in seven of 24. No C4NeF was detected in two C3 nephritic factor-positive patients with MPGN type II and six with active systemic lupus erythematosus. The new method was more simple and quantitative than C4b2a stabilization assay for C4NeF.

Production of C3 nephritic factor by cultured lymphocytes derived from glomerulonephritic patients

C3 nephritic factor (C3 NeF) has been found mainly in the sera of patients with membranoproliferative glomerulonephritis (MPGN) and partial lipodystrophy (PLD). We examined whether peripheral blood mononuclear cells (PBMC) from patients with PLD and MPGN could produce C3 NeF. We investigated the in vitro immunoglobulin synthesis of PBMC with mitogen. We further studied whether or not C3 NeF was included in the IgG of the culture supernatants by the C3bBb stabilizing activity and agglutination assay. The IgG of PLD patient was able to agglutinate only EAC4bBb cells and none of the other intermediate cells. We thus demonstrated that C3 NeF could be produced in vitro by PBMC derived from the patient with PLD. However, as regard case of C3 NeF weakly positive and C3 NeF negative patients, C3 NeF couldn't be produced in vitro by PBMC derived from the patients.

Persistent Low C3 Levels Associated with Meningococcal Meningitis and Membranoproliferative Glomerulonephritis

Recurrent meningococcal meningitis is usually related to terminal complement factor deficiencies (C5-C8); however it is not frequent with isolated primary C3 deficiency. Similarly, membranoproliferative glomerulonephritis has been described in association with primary C3 deficiencies and the presence of C3 nephritic factor. We present a case of an 18-year-old woman with relapsing meningococcal meningitis in whom membranoproliferative glomerulonephritis and persistent low serum C3 levels were found. A detailed immunological study was performed, but no other abnormalities in the complement components were found. C3 Nef was also negative. Moreover, the familiar complement studies showed an asymptomatic C4 deficiency in her mother and borderline C3 levels in her sister. The presence of persistent low serum C3 levels in the absence of other immunological abnormalities suggests that this is the cause of the relapsing meningococcal infections and the glomerular disease of this patient. We suggest that a complement deficiency, including isolated C3 deficiency, should be ruled out in all cases of relapsing meningitis. Further, the possibility of glomerular disease should be carefully considered in these patients.

Mesangiocapillary glomerulonephritis type II (dense-deposit disease): clinical features of progressive disease.

Twenty-seven patients presenting to the Royal Melbourne Hospital between 1968 and 1988 with mesangiocapillary glomerulonephritis type II with intramembranous dense deposits (dense-deposit disease, DDD) are analyzed. Patients were divided into two groups on the basis of whether renal function deteriorated (14 patients) or remained stable (13 patients). At presentation or during the course of the disease, heavy proteinuria, macroscopic hematuria, and high quantitative urinary red cell or white cell counts characterized patients with progressive disease. Patients with crescents on their initial renal biopsy or with large numbers of polymorphs in glomerular capillaries corresponding with sterile pyuria were more likely to have deterioration of renal function. The average time from onset of symptoms to development of end-stage renal disease was over 16 years. The patient's clinical course could not be anticipated by serum complement profiles, the presence of C3 nephritic factor, or partial lipodystrophy. Pregnancy did not affect the course of the disease. Six patients underwent renal transplantation and the disease recurred on renal biopsy in four. However, only two individuals lost renal allografts due to recurrent DDD.

Interaction between C3 nephritic factor and erythrocyte membranes. Presence of nephritic factor in patients' erythrocytes.

The presence of IgG has been observed by fluorescence microscopy in the erythrocytes of 5 patients with NEF antibody. IgG subclass distribution of the IgG bound was analyzed by fluorescence-activated cell sorter. NEF activity was also determined in the eluted antibodies from the erythrocyte membrane. Three of five patients had NEF antibody bound to the erythrocyte membrane. IgG subclass distribution of NEF antibody sera was also investigated by means of protein A-Sepharose and using monoclonal antibodies against human IgG subclasses. NEF activity was mediated by IgG3 alone in 3 patients and by a combination of IgG1 and IgG3 in 2 patients.

Quantitation of C3 nephritic factor of alternative complement pathway by an enzyme-linked immunosorbent assay

We have developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of C3 nephritic factor of the alternative pathway of complement (NeFA). Incubation of the NeFA-positive serum (patient KS serum) with normal human serum (NHS) in Mg-EGTA resulted in the formation of C3-B-IgG complex. No complex was formed in EDTA. At first this was detected as three types of complexes: C3-IgG, B-IgG and B-C3, by the combination of antibodies. The reaction mixture in Mg-EGTA was filtered through an ACA 22 column, from which the complexes were eluted in the same part as the first protein peak. When IgG purified from KS serum was incubated with NHS in Mg-EGTA, B-C3 complex increased in proportion to the dose of IgG. These results indicated that only one kind of complex consisting of IgG, C3 and B (IgG-C3-B) was generated by the addition of NeFA to NHS. Serum NeFA could be quantified as the titer of B-C3 complex formed after its incubation with NHS in Mg-EGTA. Using the ELISA method, NeFA was positive in five out of six patients with membranoprolifirative glomerulonephritis (MPGN) type II and in only one of 17 with MPGN type I. Titers obtained by the new method were in good accordance with those by C3 conversion and C3bBb stabilization assays for NeFA, and the new method was more exact and simple than the conventional methods.

Novel C3 Nephritic Factor Activity in the Glomerulonephritis of Staphylococcal Endocarditis

A case of glomerulonephritis complicating staphylococcal endocarditis is presented. Hypocomplementaemia and a C3-activating factor in the serum suggested that the patient might have mesangiocapillary glomerulonephritis in association with C3-nephritic factor. Renal biopsy showed that this was not so and further examination of the serum factor showed that it differed from classical C3-nephritic factor because it was not an immunoglobulin. It is postulated that complement activation and glomerulonephritis in staphylococcal endocarditis may be the direct result of a bacterial product. A substance in the serum which activates C3 should be confirmed to be an immunoglobulin before the presence of classical C3-nephritic factor is assumed.

C3 nephritic factor determination: A comparison between two methods

C3 nephritic factor (NEF), an IgG autoantibody to the alternative pathway C3 convertase, is usually measured by crossed immunoelectrophoresis (CI) but recently a reliable haemolytic assay (HA) was described by Rother (1982). This method is more specific than CI because it is negative in sera with immune complexes, SLE and sera incubated with IgG aggregates. The haemolytic assay is sensitive enough to detect NEF antibody in serum from patients with only slightly low C3 levels and NEF negatives by CI. The haemolytic assay is easy to perform and reproducible, the interassay coefficient of variation being 10.7% compared to 64% in the CI. The intra-assay coefficient of variation in CI was 28% compared to 5.5% in the haemolytic assay. The haemolytic method enabled us to study the kinetic effects of NEF on C3b.Bb bound to sheep erythrocytes, and the lysis mediated by ShE.C3b.Bb.NEF complex. Also the C and NEF binding to sheep erythrocytes was studied.

Clinical evaluation and characterization of a unique C3 breakdown factor detected in a patient with acute glomerulonephritis.

A unique factor causing breakdown of the third component of complement (C3) was detected in a patient with acute glomerulonephritis (AGN). The activity was detected by conversion of C3 in a mixture with normal human serum using crossed immunoelectrophoresis. This factor capable of C3 breakdown was not blocked in the presence of ethylenediaminetetraacetic acid (EDTA), heat-pretreatment (56 degrees C, 30 min) und dialysis against 0.1 M phenylmethylsulfonyl fluoride (PMSF). The factor was isolated and characterized by immunochemical and physicochemical techniques. It had beta-mobility on electrophoresis and was separated at a lighter sedimentation coefficient than 7S by sucrose density gradient ultracentrifugation. The activity was absorbed on a DEAE-cellulose column and was eluted under definite conditions of relative salt concentration. These data appear to suggest that this factor is distinct from C3 nephritic factor (C3NeF), immunoglobulins, immune complexes, and other C3 convertases previously described. Moreover, it is clear that the factor was mainly responsible for the C3 activation observed in the patient with acute glomerulonephritis.

An IgG autoantibody which inactivates C1-inhibitor.

Antibodies are considered to play a specific pathogenic role in certain disease states such as myasthenia gravis, Graves' disease and autoimmune haemolytic anaemia. Autoantibodies which interfere with the function of enzyme cascade systems have also been described in diseases such as acquired haemophilia (anti-factor VIII antibodies) and glomerulonephritis (C3 nephritic factor). The identification of these autoantibodies is crucial to an understanding of the aetiology of such diseases and is also of importance in revealing the inter-relationships of the immune system with other biological pathways. This is the first report of an immunoglobulin G (IgG) autoantibody reactive with C1-inhibitor (C1-Inh), a pivotal inhibitor of the inflammatory response which is known to inactivate proteins of the complement, kinin, fibrinolytic and 'contact phase' systems. This autoantibody was isolated from a patient with a novel variant of acquired angioedema and C1-Inh dysfunction. This finding highlights the involvement of the immune system in the pathogenesis of disorders characterized by the presence of dysfunctional inflammatory response proteins.

Two cases of Mesangiocapillary Glomerulonephritis that show defective C3b receptor on erythrocyte

We examined CR 1 on the red blood cell in the patients with primary glomerulonephritis using immune adherence haemagglutination (IAHA) method, and we found 2 patients of Mesangiocapillary Glomerulonephritis (MCGN) with CR 1 deficiency on the red blood cell. The two patients were Nephrotic syndrome. One patient's serum was found to contain anti Nuclear antibody (ANA), and C3 Nephritic factor (C3NeF) causing hypocomplementaemia was detected in the other patient. The deficiency of CR 1 was found in one of the parents of both these MCGN patients. It is thought that the cause of CR 1 deficiency on the red blood cell is hereditary and that it might be influencing the pathogenesis of these MCGN patients.

MOLECULAR PROPERTIES OF C3 CONVERTING FACTOR (C3 COF)

We have recently identified a yet undescribed factor in serum of an 8 year old girl with meningococcal septicemia. This factor caused hypercatabolism of the third component of complement, C3, in vivo and in vitro even in the absence of divalent cations and was termed C3 converting factor C3 CoF. Studies were undertaken to characterize this factor. On gel filtration using HPLC equipment and a TSK SW 4000 blue column two protein-containing fraction of serum were found to be able to induce C3 conversion in vitro: In the IgG-region significant activation of normal C3 occured in the presence of Mg-EGTA but not in EDTA. C3 CoF activity, in contrast, was cofiltrated with IgM. The IgG fraction was isolated from the patient's serum by ammonium sulphate precipitation, anion exchange chromatography and affinity chromatogra phy to apparent homogeneity on immunoelectrophoresis. It had all the properties required to define patient's IgG as C3 nephritic factor C3 NeF and, thus, no longer raised our interest. For the approximately 900 kD protein further biochemical data could be obtained: It is a β2-globulin, precipitable by low ionic strength buffer (=euglobulin), partly heat-resistant at 56°C, requires neither Mg nor Ca and cannot be inhibited by soy bean trypsin inhibitor at 5 mg/ml. This is, to our knowledge, the first case where two different activators of C3 simultaneously occur, one with known properties (C3 NeF) and one yet undescribed (C3 CoF).

A circulating inhibitor of fluid-phase amplification. C3 convertase formation in systemic lupus erythematosus.

C3 nephritic factor (C3NeF) was used to assess the formation of the fluid-phase amplification convertase, C3b,Bb, in 37 serum specimens from 24 patients with systemic lupus erythematosus (SLE). C3b,Bb formation was measured by the concentration of Ba, released when C3b,B is activated. Incubation of normal human serum (NHS) with C3NeF accelerates C3b amplification loop turnover with the formation of large quantities of C3b,Bb. In contrast, sera from 22 of 24 patients with SLE formed little or no convertase when incubated with C3NeF. C3 conversion to C3b was commensurately reduced. The inhibition could not be attributed to depressed serum concentrations of C3, factor B, or classical pathway components. Inhibitor present in excess could be demonstrated in 23 of 34 specimens of SLE serum by mixing experiments. The spontaneous convertase formation that occurs when a portion of the serum H is inactivated with F(ab')2 anti-H was also shown to be inhibited in SLE serum. The inhibition was found, however, to be H dependent in that convertase formation was normal in SLE serum depleted of H. It is concluded that the C3b in most SLE sera is unusually susceptible to inactivation by H, but a functional abnormality was not demonstrable in either C3 or H isolated from SLE serum. The inhibition could be simulated in NHS by addition of heparin, 100 micrograms/ml. In vivo, inhibition of convertase formation could interfere with the solubilization and disposal of immune complexes by reducing the deposition of C3b on the immune complex lattice.

C3 nephritic factor protects bound C3bBb from cleavage by factor I and human erythrocytes

C3 nephritic factor is an autoantibody to the alternative-pathway C3 convertase (C3bBb) which increases the half-life of the convertase both in the presence and absence of serum regulatory proteins. Human erythrocytes contain membrane proteins which also can regulate C3bBb. One of these proteins, the C3b/C4b receptor (CR1), plays an important role in the processing of soluble immune complexes. C3b which is fixed to immune complexes binds to CR1 and is cleaved by factor 1 to C3c and C3dg. We have tested the effectiveness of the nephritic factor in protecting bound C3b from cleavage by factor I and human erythrocytes. Sheep erythrocyte intermediates EAC1423b were prepared using 125I-labeled C3 and incubated with factors B and D in the presence and absence of nephritic factor. Breakdown of C3b was measured by release of 125I-C3c following incubation with human erythrocytes and factor I. Purified IgG from two patients with nephritic factor prevented C3c release in a dose-dependent manner. Normal human IgG was ineffective as was nephritic factor in the absence of factor B. Factor P also inhibited the release of C3c in the presence of factor B with equivalent activity at approx. 20-fold higher concns than nephritic factor. These results indicate that nephritic factor can impair human erythrocyte dependent degradation of C3b in alternative-pathway-activating immune complexes.

Complement activation by heat-killed human kidney cells: formation, activity, and stabilization of cell-bound C3 convertases.

Heat-killed kidney cells (HKKC) incubated with serum or purified C3 and factors B and D stain intensely for C3 by immunofluorescence. Binding of complement (C) did not occur if ethylenediaminetetraacetic acid (EDTA) was added to the C source or if factor B or D was omitted from the mixture of alternative pathway components. When the C-treated cells were washed, then incubated in purified C3 containing 10 mM EDTA, C3 conversion ensued, confirming the presence of a cell-bound C3 convertase. C3-reactive cells were not generated if EDTA was present during formation of the convertase. The addition of C3 nephritic factor (C3NeF) during formation of the C3 convertase from alternative pathway components greatly enhanced subsequent C3 cleavage, presumably by stabilizing labile C3bBb sites; however, binding of C3NeF did not appear to extend the half-life of the convertase, which was 35 min at 37 degrees C in each case. In contrast, little or no effect on C3 conversion could be detected after the addition of equivalent C3NeF to serum and HKKC. Thus, C activation by HKKC involves formation and activity of a cell-bound C3 convertase, which under certain circumstances can be stabilized by C3NeF.

C3 metabolism in acute glomerulonephritis: Implications for sites of complement activation

Immunochemical and metabolic studies of complement were performed in 11 patients with acute poststreptococcal glomerulonephritis (AGN) to determine the mechanism(s) of hypocomplementemia. Four patients with profound reduction in serum C3 showed metabolic changes comparable to those seen in hypocomplementemic mesangiocapillary GN (MCGN), that is, nonlinear plasma disappearance of 125I.C3 and a gross (that is, 30-fold) reduction in C3 synthesis (0.01 to 0.02 mg/kg/hr); fractional catabolic rate (FCR) and extravascular/intravascular distribution (EV/IV) ratio were also increased (3.02 to 6.99%/hr; 1.14 to 2.96, respectively). The remaining seven patients had less (or no) reduction in C3 and showed normal or elevated values for all three metabolic parameters; six had linear plasma disappearance curves. Metabolic data in five simultaneously studied control subjects were FCR: 1.56 to 2.12%/hr; EV/IV ratio: 0.12 to 0.41 and synthesis rate: 0.30 to 0.52 mg/kg/hr. C3 nephritic factor (NeF) could not be detected in any sera and a significant reduction in serum C5 accompanied the changes in C3 (r = 0.89; P less than 0.001). Previous studies of C3 NeF-associated MCGN show that the fluid phase alternative pathway convertase seen in this condition has little or no C5 cleaving ability. We propose, therefore, that complement activation in AGN occurs via a surface-bound convertase which is capable of cleaving both C3 and C5. The glomerular capillary could provide such a site for activation.