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Detection of C3bBb-stabilizing activity (C3 nephritic factor) in the serum from patients with membranoproliferative glomerulonephritis
Journal of Immunological Methods, 131 (1990) 71-76
71
Elsevier JIM 05620
Detection of C3bBb-stabilizing activity (C3 nephritic factor) in the serum from patients with membranoproliferative glomerulonephritis H i r o y u k i Ohi, S h i z u h i k o W a t a n a b e , T a k a y u k i Fujita, M a s a t o Seki a n d M i c h i n o b u H a t a n o Department of Internal Medicine II, Nihon University School of Medicine, 30-10yaguchi Kamimachi, Itabashi-ku, Tokyo, Japan
(Received 1 November 1989, revised received22 November 1989, accepted 27 March 1990)
It is known that membranoproliferative glomerulonephritis (MPGN), hypocomplementaemia and C3 nephritic factor (C3NeF) are closely related to each other, and the presence or absence of C3NeF in the serum is important for evaluating the nature of MPGN. However, some difficulties have been encountered in detecting this factor and therefore a new assay permitting the direct detection of C3NeF without purifying IgG from the patient's serum has been devised. Using this assay method, C3bBb-stabilizing activity was observed even in sera from M P G N patients who were non-hypocomplementaemic. Furthermore, among 98 cases with hypocomplementaemia, C3NeF was found to be absent in 66 cases. Key words: C3 nephritic factor; C3bBb-stabilizingactivity; Membranoproliferativeglomerulonephritis
Introduction
C3 nephritic factor (C3NeF), an auto-antibody of the IgG class against the C3 convertase C3bBb of the alternative complement pathway, has been shown to stabilize C3bBb and to induce complement activation by inhibiting the activity of control protein (Daha et al., 1976, 1977; Weiler et al., 1976; Davis et al., 1977; Scott et al., 1978). However, some difficulties have been encountered in detecting this factor due to the need to isolate IgG from the serum. For this purpose, a sufficient amount of serum is required and it takes a long time for the measurement of C3NeF to be performed. Consequently, it is usually more convenient to measure C3 splitting factor in hypocom-
Correspondence to: H. Ohi, Department of Internal Medicine II, Nihon University School of Medicine, 30-10yaguchi Kamimachi, Itabashi-ku, Tokyo, Japan.
plementaemic serum, instead of C3NeF (Vallota et al., 1972). We describe here a simple procedure which permits the assay of C3NeF in small amounts of serum.
Materials and methods Buffer solution
Gelatin veronal buffer containing Ca 2÷ and Mg 2÷ (GVB2+), D-glucose veronal buffer containing Ca 2÷ and Mg 2+ (D-GVBZ+), 0.01 M E D T A GVB, 0.04 M E D T A GVB, and phosphatebuffered saline (PBS) as described by Lachmann (1978). Serum samples
Control serum (NHS) was collected from 21 normal apparently healthy subjects and pooled before use. Serum was sampled from 11 patients with membranoproliferative glomerulonephritis (MP-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
72
GN), including three C3NeF-positive patients and eight C3NeF-negative patients. IgG was isolated from these patients' sera and the C3bBb-stabilizing activity was determined in the IgG fraction. Assays for C3NeF were also performed on serum collected from 202 M P G N patients in many hospitals and stored at - 70 o C.
Purification of IgG IgG was isolated from the serum of an M P G N patient and a control serum by ion-exchange chromatography using D E A E cellulose (DE52, Whatman). The break-through peak volume was concentrated to the starting volume and then dialysed with PBS. The IgG so obtained was employed as the test IgG.
Complement components Factors B and D were purified by the method reported by Lachmann et al. (1978) and Martin et al. (1976). Factors C2 and C3 were purchased from the Chemo, Sero, Therapeutic Research Institute. C-EDTA (C3-C9 reagent) was prepared by diluting guinea-pig serum 25-fold with 0.04 M EDTA GVB.
Determination of complement components C3 was measured by the single radial immunodiffusion method using anti-C3c serum (Behringwerke) (Mancini et al., 1964). C3d was measured by the so-called double-dec k e r rocket method employing anti-C3c and antiC3d sera (Bio-Rad) (Brandslund et al., 1981). C3 conversion was evaluated by antigen antibody-crossed immunoelectrophoresis in the sera of M P G N patients with added 0.01 M EDTA added (Laurell, 1965). Ao Patient's IgG+PBS I diluted in 0.01M EDTA-GVB
Intermediate cells (EAC4b3bBb cells) EAC14 cells were prepared by the method of Nagaki et al. (1974). C2 and C3 were added to the EAC14 cells in DGVB 2÷, and the mixture incubated at 20 ° C for 20 min. The resultant cells were washed twice with 0.01 M EDTA-GVB and incubated at 37 ° C for 90 min in 0.01 M EDTAGVB. The EAC4b3b cells so obtained were washed with DGVB 2+ and then adequate amounts of B and D were added. The mixture was incubated at 3 0 ° C for 5 min to give EAC4b3bBb cells. The minimum time required to form the EAC4b3bBb cells was established by kinetic analysis of lytic C5b-9 formation on the cells. An incubation period of 5 min at 3 0 ° C was chosen for C3bBb formation on the EAC4b3b cells because 100% haemolysis was attained after 5 min.
Assay of C3bBb-stabilizing activity The C3bBb-stabilizing activity was assayed in
Bo
C°
I Patient's serum 2 C3NeFIgG+NHS
I Patient's serum 2 C3NeFIgG+NHS V diluted in 0.O1M EDTA-GVB
--
I
diluted in 0.01M EDTA-GVB
1 heat treated (56 C,30min)
1 add EAC4b3bBb 30 C,15min
add EAC4b3bBb
in ice,15min
add EAC4b3bBb in ice, ]5rnin
add 0.04M EDTA-GVB 30 C,30min
add C-EDTA 37 C,60min
1 0D 414nm
Fig. 1. The determination of C3bBb-stabilizing activity in five different samples. A: patient's IgG; B-I: patient's serum; B-2: C 3 N e F I g G + NHS; C-1: patient's serum after heat treatment; C-2: C 3 N e F I g G + N H S after heat treatment.
73
serum from patients and in NHS, using purified IgG (from the serum samples) in PBS solution, 0.01 M EDTA-added serum (0.01 M E D T A serum), and heated 0.01 M E D T A serum (56 ° C, 30 min) (Figs. 1A, B-l, and C-1, respectively). A 10 /tl aliquot of patient serum which had been previously treated using the conditions outlined above and 50/~1 of EAC4b3bBb cells (2.5 × 108 cells/ml) were mixed and incubated at 0 4 ° C for 15 rain. 50/~1 of 0.04 M EDTA-GVB was then added. The mixture was incubated at 30 ° C for 30 min, and 50 /tl of C-EDTA added. After reaction at 37 ° C for 60 rain, 600 ~tl of ice-cooled DGVB 2÷ were added to stop the reaction and the mixture centrifuged at 2000 rpm for 5 min. The absorbance of the supernatant fluid so obtained was measured (OD414). The percentage haemolysis was calculated on the basis of counting complete lysis as 100%. When treated patient's serum and EAC4b3bBb cells were incubated at 3 0 ° C for 5 min, decay of the EAC4b3bBb cells and nonspecific haemolysis of cells were observed, and we could not detect C3bBb-stabflizing activity. However, when the mixture was incubated at 0 - 4 ° C for 15 rain, this phenomenon was not encountered, and we were able to detect C3bBb-stabilizing activity.
Detection of the C3bBb-stabilizing activity of NHSmixed with purified C3NeF lgG IgG isolated from C3NeF-positive serum was mixed with NHS in a ratio of 1 : 1. C3NeF IgG was mixed with PBS (Fig. 1A mixture) and with N H S (Figs. 1B-2, and 1C-2). The C3bBb-stabilizing activity of the purified C3NeF IgG was measured in these two mixtures and in N H S alone both without heating and after heating at 56 ° C for 30 min.
ment-activating factor but no C3bBb-stabilizing activity (C3NeF-negative patients), and normal subjects (see Fig. 2). Treatment of the serum only by dilution with E D T A resulted in such a large reduction in the percentage haemolysis in the C3NeF-positive sesrum that distinction between C3NeF-positive serum and normal or C3NeFnegative serum became impossible. However, heating the EDTA-diluted serum at 56 ° C for 30 min permitted a clear differentiation to be made. When the present C3NeF assay was tested, the following average titers were obtained: C3NeFpositive cases (n = 7), 79.6 _ 35% (mean _ SD); C3NeF-negative cases (n = 43), 12.9 + 9.7%; and normal human serum (n = 9), 17.5 + 5.1%. In sera from two C3NeF-positive patients (cases 1 and 2), which were diluted 2 0 - 160-fold with EDTA-GVB and then heated at 56 ° C for 30 rain, C3bBb-stabilizing activity was detected in a dosedependent manner (Fig. 3), as determined by the conventional technique using IgG from the serum. However, in normal or C3NeF-negative serum, no such activity was found. This clearly demonstrated (n=11) 100
°-]C3NeF-positive cases l~ n=3 o C3NeF-negative cases n=8 * Normal human serum u~
E .C
so-
+
Results
O
Detection of C3NeF in serum Comparisons between the conventional technique for assaying C3NeF using IgG from serum and the present method were made in three patients confirmed to have C3bBb-stabilizing activity in the IgG fraction of their serum (C3NeFpositive patients), eight patients having comple-
IgG
Serum Heated in O.01M serum EDTA in O.01M EDTA
Fig. 2. A clear discrimination was observed for both IgG and heated serum in E D T A when EAC3bBb-stabilizing activity was determined using three C3NeF-positive cases, N H S and eight C3NeF-negative cases.
74
that the new assay method was able to distinguish C3NeF-positive serum from C 3 N e F negative serum. After a mixture of C3NeF I g G plus N H S (1 : 1) had been heated at 5 6 ° C for 30 rain, C3bBbstabilizing activity was also detected dose-dependently. However, when normal human I g G or normal human serum was heated at 56 ° C for 30 rain together with EDTA-GVB, the C3bBb was no longer functionally active and no haemolysis was noted.
Serum from MPGN patients Among the 202 M P G N patients, 98 patients had C3 levels of less than 40% of normal and 104 had levels which were more than 40%. The new method was applied to assay C 3 N e F in these 202 patients, and, as a result, C 3 N e F was detected in 21 patients (see Fig. 4). The detection of C 3 N e F was not related to the C3 values: it was detected
/
2.0
Case I (IgG)
~
Z
w
Case I (~.~,t~) Case 2 (~,:t~)
• Heated ~Heated -- NHS
xl~ x~O x~
--Hypocomplementaemia-- i (C3~40%) | 98
L-C3NeF(--)77
MPGN (n=202) --C3NeF(+)
I --Normocomplementaemia-~ (C3>40%) 104
6
| L__ C3NeF ( - ) 98
Fig. 4. C3bBb-stabilizing activity (C3NeF)-positive cases among 202 hypo- and normocomplementaemic MPGN patients. even in serum samples with a low normal C3 level (58 - 92%). These serum samples still had C3NeF activity even after isolation of IgG. In E D T A plasma from four patients with normal C3 levels, no conversion of C3 was noted and C3d was not detected. Furthermore, the results were unchanged when determinations were made using I g G isolated from 21 cases of C 3 N e F activity-positive sera and 10 cases of C3NeF-negative hypocomplementaemic sera (C3 < 10%).
Case 2(IgG)
Discussion
1.0
-"
~- C 3 N e F ( + ) 2 1
NHS C3NeF(--)
serum
IgG
x26
Fig. 3. In sera obtained from two C3NeF-positive patients (cases 1 and 2), which were diluted 20 -160-fold with EDTAGVB and then heated at 56 ° C for 30 min, C3bBb-stabilizing activity was detected in a dose-dependent manner. In contrast C3NeF-negative serum and N H S revealed no such activity.
It is known that M P G N , hypocomplementaemia and C 3 N e F are closely related to each other, and the presence or absence of C 3 N e F in the serum is important for evaluating the nature of the disease (West et al., 1965; Peters et al., 1972; Vallota et al., 1974; Williams et al., 1974). C3NeF (C3bBb-stabilizing factor) has been reported in M P G N , but its frequency varies among different studies. This appears to be related to the different methods of detection used. Many reports have been based on measurement of the C3 splitting activity instead of evidence concerning whether or not C3bBb is stabilized on F_A, or confirmation of C3bBb-stabilizing activity. The presence of a C3 splitting factor which differs from C3NeF has been reported in the serum of M P G N patients (Ohi et al., 1982; W a t a n a b e et al., 1987). It is clearly of importance to confirm C3NeF, as well
75
as other C3 splitting factors, in MPGN when evaluating the nature of this disease, since it appears that C3NeF participates in the onset o f hypocomplementaemia in MPGN, because C3NeF itself is an auto-antibody, and also because C3NeF induces complement activation. The present new assay procedure permitted the direct detection of C3bBb-stabilizing activity without purifying IgG from serum, and also allowed comparisons of the C3bBb-stabilizing activities of many sera. Heating serum diluted 20-fold in EDTA at 56 ° C for 30 min made it possible to detect C3bBb-stabilizing activity, in addition to performing determinations by the conventional technique using purified IgG. Furthermore, based on the fact that this activity could be detected even after mixing C3NeF IgG with NHS, the new method can be effectively employed as a C3NeF screening assay. Heating at 56 ° C for 30 min resulted in a clear differentiation between the serum of an M P G N patient and normal serum. This appeared to result from the inactivation, by such heat treatment, of a factor which influences the complement pathway. Employing the new assay method, C3bBbstabilizing activity was even observed in sera from MPGN patients who were non-hypocomplementaemic. This is because using previous assay methods only hypocomplementaemic serum could be assayed. However, there is a need to determine whether any particular observed activity differs from C3NeF. It was noteworthy that out of 98 cases of hypocomplementaemia, C3NeF was absent in 66 cases. Among these 66 cases, C3 splitting factor was present in 51 cases (data not shown). Studies should therefore be undertaken into the mechanisms underlying the hypocomplementaemia of such sera. In addition there is a need to investigate the presence or absence of activated P, the physiological C3bBb stabilizing factor.
Acknowledgements The authors are indebted to the many hospitals which kindly provided patients' sera for evaluation.
References Brandslund, I., Siersted, H.C., Svebag, S.E. and Teisner, B. (1981) Double-decker roket immunoelectrophoresis for direct quantitation of complement C3 split products with C3d specificities in plasma. J. Immunol. Methods 44, 63. Daha, M.R., Fearon, D.T. and Austen, K.F. (1976) C3 nephritic factor (C3 NeF): Stabilization of fluid phase and cell-bound alternative pathway convertase. J. Immunol. 116, 1. Daha, M.R., Austen, K.F. and Fearon, D.T. (1977) The incorporation of C3 nephritic factor (C3NeF) into a stabilized C3 convertase, C3bBb (C3NeF), and its release after decay of convertase function. J. Immunol. 119, 812. Davis III, A.E., Ziegler, J.B., Gelfand, E.W. Rosen, F.S. and Alper, C.A. (1977) Heterogeneity of nephritic factor and its identification as an immunoglobulin. Proc. Natl. Acad. Sci. U.S.A. 74, 3980. Harrison, R.A. and Lachmann, P.J. (1978) Complement Technology In: D.M. Weir (Ed.), Handbook of Experimental Immunology, 3rd edn., 23. Blackwell Scientific Publications, Oxford, 5A, 1-5A, 23. Laurell, C.B. (1965) Antigen-antibody crossed electrophoresis. Analyt. Biochem. 10, 358. Mancini, G., Vaerman, J.P., Carbonara, A.O. and Heremans, J.F. (1964) A single-radial diffusion method for the immunological quantitation of proteins. In: H. Peeters (Ed.), Prot. Biol. Fluids l l t h Coll. Bruges. Pergamon Press, Oxford, pp. 370-373. Martin, A., Lachmann, P.J., Halwachs, L. and Hobart, M.J. (1976) Haemolytic diffusion plate assays for factors B and D of the alternative pathway of complement activation. Immunochemistry 13; 317. Nagaki, K., Iida, K. and Inai, S. (1974) A new method for the preparation of EAC14 cell with human or guinea-pig serum. J. Immunol. Methods 5, 307. Ohi, H., Amos, N., Schifferli, J.A. and Peters, D.K. (1982) Characterization of an alternative pathway activating factor in serum of a patient with MPGN. Mol. Immunol. 19, 1392 (abstract). Peters, D.K., Martin, A., Weinstein, A., Barratt, T.M., Cameron, J.S., Ogg, C.S. and Lachmann, P.J. (1972) Complement studies in membranoproliferative glomerulonephritis. Clin. Exp. Immunol. 11,311. Scott, D.M., Amos, N., Sissons, J.G.P., Lachmann, P.J. and Peters, D.K. (1978) The immunoglobulin nature of nephritic factor (NeF). Clin. Exp. Immunol. 32, 12. Vallota, E.H., Forristal, J., Davis, N.C., Davis, N.C. and West, C.D. (1972) The C3 nephritic factor and membranoproliferative nephritis: Correlation of serum levels of the nephritic factor with C3 levels, with therapy, and with progression of the disease. J. Pediatr. 80, 947. Vallota, E.H., Gotze, O., Spiegelberg, H.L., Forristal, J., West, C.D. and Muller-Eberhard (1974) A serum factor in chronic hypocomplementemic nephritis distinct from immunoglobulins and activating the alternate pathway of complement. J. Exp. Med. 139, 1249. Watanabe, S., Ohi, H., Seki, M., Fujita, T. and Hatano, M. (1987) Long-term observation of persistent hypocom-
76 plementaemia in three patients with membrano proliferative glomerulo nephritis. J. Jap. Sec. Intern. Med. 76, 106. Weiler, J.M., Daha, M.R., Austen, K.F. and Fearon, D.T. (1976) Control of the amplification convertase of complement by the plasma protein 1H. Prec. Natl. Acad. Sci. U.S.A. 73, 3268. West, C.D., McAdams, A.J., McConville, J.M., Davis, J.M., Davis, N.C. a n d Holland, N.H. (1965) Hypocomplemen-
temic and normocomplementemic persistent (chronic) glomerulonephritis: Clinical and pathologic characteristics. J. Pediatr. 67, 1089. Williams, D.G., Peters, D.K., Fallows, J., Petrie, A., Kourilsky, O., Morlmaroger, L. and Cameron, J.S. (1974) Studies of serum complement in the hypocomplementemic nephritides. Clin. Exp. Immunol. 18, 391.
71
Elsevier JIM 05620
Detection of C3bBb-stabilizing activity (C3 nephritic factor) in the serum from patients with membranoproliferative glomerulonephritis H i r o y u k i Ohi, S h i z u h i k o W a t a n a b e , T a k a y u k i Fujita, M a s a t o Seki a n d M i c h i n o b u H a t a n o Department of Internal Medicine II, Nihon University School of Medicine, 30-10yaguchi Kamimachi, Itabashi-ku, Tokyo, Japan
(Received 1 November 1989, revised received22 November 1989, accepted 27 March 1990)
It is known that membranoproliferative glomerulonephritis (MPGN), hypocomplementaemia and C3 nephritic factor (C3NeF) are closely related to each other, and the presence or absence of C3NeF in the serum is important for evaluating the nature of MPGN. However, some difficulties have been encountered in detecting this factor and therefore a new assay permitting the direct detection of C3NeF without purifying IgG from the patient's serum has been devised. Using this assay method, C3bBb-stabilizing activity was observed even in sera from M P G N patients who were non-hypocomplementaemic. Furthermore, among 98 cases with hypocomplementaemia, C3NeF was found to be absent in 66 cases. Key words: C3 nephritic factor; C3bBb-stabilizingactivity; Membranoproliferativeglomerulonephritis
Introduction
C3 nephritic factor (C3NeF), an auto-antibody of the IgG class against the C3 convertase C3bBb of the alternative complement pathway, has been shown to stabilize C3bBb and to induce complement activation by inhibiting the activity of control protein (Daha et al., 1976, 1977; Weiler et al., 1976; Davis et al., 1977; Scott et al., 1978). However, some difficulties have been encountered in detecting this factor due to the need to isolate IgG from the serum. For this purpose, a sufficient amount of serum is required and it takes a long time for the measurement of C3NeF to be performed. Consequently, it is usually more convenient to measure C3 splitting factor in hypocom-
Correspondence to: H. Ohi, Department of Internal Medicine II, Nihon University School of Medicine, 30-10yaguchi Kamimachi, Itabashi-ku, Tokyo, Japan.
plementaemic serum, instead of C3NeF (Vallota et al., 1972). We describe here a simple procedure which permits the assay of C3NeF in small amounts of serum.
Materials and methods Buffer solution
Gelatin veronal buffer containing Ca 2÷ and Mg 2÷ (GVB2+), D-glucose veronal buffer containing Ca 2÷ and Mg 2+ (D-GVBZ+), 0.01 M E D T A GVB, 0.04 M E D T A GVB, and phosphatebuffered saline (PBS) as described by Lachmann (1978). Serum samples
Control serum (NHS) was collected from 21 normal apparently healthy subjects and pooled before use. Serum was sampled from 11 patients with membranoproliferative glomerulonephritis (MP-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
72
GN), including three C3NeF-positive patients and eight C3NeF-negative patients. IgG was isolated from these patients' sera and the C3bBb-stabilizing activity was determined in the IgG fraction. Assays for C3NeF were also performed on serum collected from 202 M P G N patients in many hospitals and stored at - 70 o C.
Purification of IgG IgG was isolated from the serum of an M P G N patient and a control serum by ion-exchange chromatography using D E A E cellulose (DE52, Whatman). The break-through peak volume was concentrated to the starting volume and then dialysed with PBS. The IgG so obtained was employed as the test IgG.
Complement components Factors B and D were purified by the method reported by Lachmann et al. (1978) and Martin et al. (1976). Factors C2 and C3 were purchased from the Chemo, Sero, Therapeutic Research Institute. C-EDTA (C3-C9 reagent) was prepared by diluting guinea-pig serum 25-fold with 0.04 M EDTA GVB.
Determination of complement components C3 was measured by the single radial immunodiffusion method using anti-C3c serum (Behringwerke) (Mancini et al., 1964). C3d was measured by the so-called double-dec k e r rocket method employing anti-C3c and antiC3d sera (Bio-Rad) (Brandslund et al., 1981). C3 conversion was evaluated by antigen antibody-crossed immunoelectrophoresis in the sera of M P G N patients with added 0.01 M EDTA added (Laurell, 1965). Ao Patient's IgG+PBS I diluted in 0.01M EDTA-GVB
Intermediate cells (EAC4b3bBb cells) EAC14 cells were prepared by the method of Nagaki et al. (1974). C2 and C3 were added to the EAC14 cells in DGVB 2÷, and the mixture incubated at 20 ° C for 20 min. The resultant cells were washed twice with 0.01 M EDTA-GVB and incubated at 37 ° C for 90 min in 0.01 M EDTAGVB. The EAC4b3b cells so obtained were washed with DGVB 2+ and then adequate amounts of B and D were added. The mixture was incubated at 3 0 ° C for 5 min to give EAC4b3bBb cells. The minimum time required to form the EAC4b3bBb cells was established by kinetic analysis of lytic C5b-9 formation on the cells. An incubation period of 5 min at 3 0 ° C was chosen for C3bBb formation on the EAC4b3b cells because 100% haemolysis was attained after 5 min.
Assay of C3bBb-stabilizing activity The C3bBb-stabilizing activity was assayed in
Bo
C°
I Patient's serum 2 C3NeFIgG+NHS
I Patient's serum 2 C3NeFIgG+NHS V diluted in 0.O1M EDTA-GVB
--
I
diluted in 0.01M EDTA-GVB
1 heat treated (56 C,30min)
1 add EAC4b3bBb 30 C,15min
add EAC4b3bBb
in ice,15min
add EAC4b3bBb in ice, ]5rnin
add 0.04M EDTA-GVB 30 C,30min
add C-EDTA 37 C,60min
1 0D 414nm
Fig. 1. The determination of C3bBb-stabilizing activity in five different samples. A: patient's IgG; B-I: patient's serum; B-2: C 3 N e F I g G + NHS; C-1: patient's serum after heat treatment; C-2: C 3 N e F I g G + N H S after heat treatment.
73
serum from patients and in NHS, using purified IgG (from the serum samples) in PBS solution, 0.01 M EDTA-added serum (0.01 M E D T A serum), and heated 0.01 M E D T A serum (56 ° C, 30 min) (Figs. 1A, B-l, and C-1, respectively). A 10 /tl aliquot of patient serum which had been previously treated using the conditions outlined above and 50/~1 of EAC4b3bBb cells (2.5 × 108 cells/ml) were mixed and incubated at 0 4 ° C for 15 rain. 50/~1 of 0.04 M EDTA-GVB was then added. The mixture was incubated at 30 ° C for 30 min, and 50 /tl of C-EDTA added. After reaction at 37 ° C for 60 rain, 600 ~tl of ice-cooled DGVB 2÷ were added to stop the reaction and the mixture centrifuged at 2000 rpm for 5 min. The absorbance of the supernatant fluid so obtained was measured (OD414). The percentage haemolysis was calculated on the basis of counting complete lysis as 100%. When treated patient's serum and EAC4b3bBb cells were incubated at 3 0 ° C for 5 min, decay of the EAC4b3bBb cells and nonspecific haemolysis of cells were observed, and we could not detect C3bBb-stabflizing activity. However, when the mixture was incubated at 0 - 4 ° C for 15 rain, this phenomenon was not encountered, and we were able to detect C3bBb-stabilizing activity.
Detection of the C3bBb-stabilizing activity of NHSmixed with purified C3NeF lgG IgG isolated from C3NeF-positive serum was mixed with NHS in a ratio of 1 : 1. C3NeF IgG was mixed with PBS (Fig. 1A mixture) and with N H S (Figs. 1B-2, and 1C-2). The C3bBb-stabilizing activity of the purified C3NeF IgG was measured in these two mixtures and in N H S alone both without heating and after heating at 56 ° C for 30 min.
ment-activating factor but no C3bBb-stabilizing activity (C3NeF-negative patients), and normal subjects (see Fig. 2). Treatment of the serum only by dilution with E D T A resulted in such a large reduction in the percentage haemolysis in the C3NeF-positive sesrum that distinction between C3NeF-positive serum and normal or C3NeFnegative serum became impossible. However, heating the EDTA-diluted serum at 56 ° C for 30 min permitted a clear differentiation to be made. When the present C3NeF assay was tested, the following average titers were obtained: C3NeFpositive cases (n = 7), 79.6 _ 35% (mean _ SD); C3NeF-negative cases (n = 43), 12.9 + 9.7%; and normal human serum (n = 9), 17.5 + 5.1%. In sera from two C3NeF-positive patients (cases 1 and 2), which were diluted 2 0 - 160-fold with EDTA-GVB and then heated at 56 ° C for 30 rain, C3bBb-stabilizing activity was detected in a dosedependent manner (Fig. 3), as determined by the conventional technique using IgG from the serum. However, in normal or C3NeF-negative serum, no such activity was found. This clearly demonstrated (n=11) 100
°-]C3NeF-positive cases l~ n=3 o C3NeF-negative cases n=8 * Normal human serum u~
E .C
so-
+
Results
O
Detection of C3NeF in serum Comparisons between the conventional technique for assaying C3NeF using IgG from serum and the present method were made in three patients confirmed to have C3bBb-stabilizing activity in the IgG fraction of their serum (C3NeFpositive patients), eight patients having comple-
IgG
Serum Heated in O.01M serum EDTA in O.01M EDTA
Fig. 2. A clear discrimination was observed for both IgG and heated serum in E D T A when EAC3bBb-stabilizing activity was determined using three C3NeF-positive cases, N H S and eight C3NeF-negative cases.
74
that the new assay method was able to distinguish C3NeF-positive serum from C 3 N e F negative serum. After a mixture of C3NeF I g G plus N H S (1 : 1) had been heated at 5 6 ° C for 30 rain, C3bBbstabilizing activity was also detected dose-dependently. However, when normal human I g G or normal human serum was heated at 56 ° C for 30 rain together with EDTA-GVB, the C3bBb was no longer functionally active and no haemolysis was noted.
Serum from MPGN patients Among the 202 M P G N patients, 98 patients had C3 levels of less than 40% of normal and 104 had levels which were more than 40%. The new method was applied to assay C 3 N e F in these 202 patients, and, as a result, C 3 N e F was detected in 21 patients (see Fig. 4). The detection of C 3 N e F was not related to the C3 values: it was detected
/
2.0
Case I (IgG)
~
Z
w
Case I (~.~,t~) Case 2 (~,:t~)
• Heated ~Heated -- NHS
xl~ x~O x~
--Hypocomplementaemia-- i (C3~40%) | 98
L-C3NeF(--)77
MPGN (n=202) --C3NeF(+)
I --Normocomplementaemia-~ (C3>40%) 104
6
| L__ C3NeF ( - ) 98
Fig. 4. C3bBb-stabilizing activity (C3NeF)-positive cases among 202 hypo- and normocomplementaemic MPGN patients. even in serum samples with a low normal C3 level (58 - 92%). These serum samples still had C3NeF activity even after isolation of IgG. In E D T A plasma from four patients with normal C3 levels, no conversion of C3 was noted and C3d was not detected. Furthermore, the results were unchanged when determinations were made using I g G isolated from 21 cases of C 3 N e F activity-positive sera and 10 cases of C3NeF-negative hypocomplementaemic sera (C3 < 10%).
Case 2(IgG)
Discussion
1.0
-"
~- C 3 N e F ( + ) 2 1
NHS C3NeF(--)
serum
IgG
x26
Fig. 3. In sera obtained from two C3NeF-positive patients (cases 1 and 2), which were diluted 20 -160-fold with EDTAGVB and then heated at 56 ° C for 30 min, C3bBb-stabilizing activity was detected in a dose-dependent manner. In contrast C3NeF-negative serum and N H S revealed no such activity.
It is known that M P G N , hypocomplementaemia and C 3 N e F are closely related to each other, and the presence or absence of C 3 N e F in the serum is important for evaluating the nature of the disease (West et al., 1965; Peters et al., 1972; Vallota et al., 1974; Williams et al., 1974). C3NeF (C3bBb-stabilizing factor) has been reported in M P G N , but its frequency varies among different studies. This appears to be related to the different methods of detection used. Many reports have been based on measurement of the C3 splitting activity instead of evidence concerning whether or not C3bBb is stabilized on F_A, or confirmation of C3bBb-stabilizing activity. The presence of a C3 splitting factor which differs from C3NeF has been reported in the serum of M P G N patients (Ohi et al., 1982; W a t a n a b e et al., 1987). It is clearly of importance to confirm C3NeF, as well
75
as other C3 splitting factors, in MPGN when evaluating the nature of this disease, since it appears that C3NeF participates in the onset o f hypocomplementaemia in MPGN, because C3NeF itself is an auto-antibody, and also because C3NeF induces complement activation. The present new assay procedure permitted the direct detection of C3bBb-stabilizing activity without purifying IgG from serum, and also allowed comparisons of the C3bBb-stabilizing activities of many sera. Heating serum diluted 20-fold in EDTA at 56 ° C for 30 min made it possible to detect C3bBb-stabilizing activity, in addition to performing determinations by the conventional technique using purified IgG. Furthermore, based on the fact that this activity could be detected even after mixing C3NeF IgG with NHS, the new method can be effectively employed as a C3NeF screening assay. Heating at 56 ° C for 30 min resulted in a clear differentiation between the serum of an M P G N patient and normal serum. This appeared to result from the inactivation, by such heat treatment, of a factor which influences the complement pathway. Employing the new assay method, C3bBbstabilizing activity was even observed in sera from MPGN patients who were non-hypocomplementaemic. This is because using previous assay methods only hypocomplementaemic serum could be assayed. However, there is a need to determine whether any particular observed activity differs from C3NeF. It was noteworthy that out of 98 cases of hypocomplementaemia, C3NeF was absent in 66 cases. Among these 66 cases, C3 splitting factor was present in 51 cases (data not shown). Studies should therefore be undertaken into the mechanisms underlying the hypocomplementaemia of such sera. In addition there is a need to investigate the presence or absence of activated P, the physiological C3bBb stabilizing factor.
Acknowledgements The authors are indebted to the many hospitals which kindly provided patients' sera for evaluation.
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