Immunoglobulin G Autoantibody Directed Against the Classical Pathway C3 Convertase Enzyme

Nephritic Factor of the Classical Pathway of Complement: IMMUNOGLOBULIN G AUTOANTIBODY DIRECTED AGAINST THE CLASSICAL PATHWAY C3 CONVERTASE ENZYME L. Halbwachs, … , S. Wattel, J. Leibowitch J Clin Invest. 1980;65(6):1249-1256. https://doi.org/10.1172/JCI109787. A factor, functionally characterized by its capacity to stabilize the normally labile classical pathway C3-converting complex of the classical pathway of complement, has been isolated from the serum of one patient with a case of acute glomerulonephritis, subsequent to a cutaneous infection. The factor confers long-lived stabilization of classical pathway C3 convertase complexes formed both in the solid (sensitized sheep erythrocytes bearing activated C1̄ ̄ and the classical pathway C3 convertase) and fluid phase. The half-life of such stabilized C3-cleaving enzymes extended beyond several hours at 37°C. The stabilizing activity was associated with a protein fraction immunochemically identified as immunoglobulin (Ig)G, a sizeable population of which exhibited a gamma chain of 60,000 daltons. The IgG-associated stabilizing activity was found to bind to the classical pathway C3 convertase enzyme via a fragment bearing the antigen- binding site of the molecule [F(ab)2 and F(ab)]. Such binding was demonstrable for classical pathway and not for alternative pathway C3 convertase. Thus, the stabilizing factor behaves like an autoantibody to the C3-converting complex of the classical pathway of complement. The binding of the antibody to the enzyme affords protection of the latter against decay-degradation. By analogy with the nephritic factor of the alternative pathway situation where IgG autoantibodies specifically bind to alternative pathway C3 convertase enzymes and protect them from degradation, the functionally unusual IgG in our […] Find the latest version: https://jci.me/109787/pdf Nephritic Factor of the Classical Pathway of Complement IMMUNOGLOBULIN G AUTOANTIBODY DIRECTED AGAINST THE CLASSICAL PATHWAY C3 CONVERTASE ENZYME L. HALBWACHS, M. LEVEILLA, Ph. LESAVRE, S. WATTEL, and J. LEIBOWITCH, Departement de Nephrologie et Institut National de la Sante et de la Recherche Medicale U 25, Groupe de Recherche sur le Complement en Pathologie Humnaine, Hopital Necker, 75015 Paris, France A B S T R A C T A factor, functionally characterized by enzymes and protect them from degradation, the its capacity to stabilize the normally labile classical functionally unusual IgG in our patient was desig- pathway C3-converting complex of the classical path- nated as the nephritic factor of the classical pathway of complement, has been isolated from the serum way. Indirect evidence suggests that nephritic factor of one patient with a case of acute glomerulonephritis, of the classical pathway-IgG mnight be of the IgG3 subsequent to a cutaneous infection. The factor confers subclass. long-lived stabilization of classical pathway C3 convertase complexes formed both in the solid (sensitized INTRODUCTION sheep erythrocytes bearing activated Cl and the classi- The complement system schematically comprises two cal pathway C3 convertase) and fluid phase. The half- major enzymatic complexes, both endowed with simi- life of such stabilized C3-cleaving enzymes extended lar cleaving functions on the third (C3) and the fifth beyond several hours at 370C. The stabilizing activity (C5) native complement proteins. was associated with a protein fraction immunochemi- These C3/C5 convertases differ in their molecular cally identified as immunoglobulin (Ig)G, a sizeable pop- composition according to the events that have initiated ulation of which exhibited a gamma chain of 60,000 dal- their formation: (a) Immune complexes, which activate tons. The IgG-associated stabilizing activity was found the first component, Cl, will lead to the formation of to bind to the classical pathway C3 convertase enzyme a classical pathway C3/C5 convertase, consisting in the via a fragment bearing the antigen-binding site of the active forms of the fourth and the second components molecule [F(ab)2 and F(ab)]. Such binding was demon- bound together as classical pathway C3 convertase strable for classical pathway and not for alternative path- (C4b2a)l c(mplexes (1). (b) "Activators" of the alterna- way C3 convertase. Thus, the stabilizing factor be- tive pathway will allow factor B and C3 to form al- haves like an autoantibody to the C3-converting ternative pathway C3 convertase (C3bBb) complexes complex of the classical pathway of complement. The endowed with C3/C5 convertase activity (2). binding of the antibody to the enzyme affords pro- Both classical and tection of the latter against decay-degradation. By alternative pathway bimolecular an-alogy with the nephritic factor of the alternative convertases are subjected in serum to potent homeo- pathway situation where autoantibodies IgG specifi- 'Abbreviations used in this paper: 83-1H, /8-1H globulin; cally bind to alternative pathway C3 convertase C3bBb, alternative pathway C3 convertase; C4b2a, classical pathway C3 convertase; DGVB, dextrose gelatin veronal buffer; DGVB++, DGVB containing calciumil and mnag- This work was presented in part at the Soci6t6 Frangaise nesium; EAC14b2a sensitized sheep erythrocytes bearing de N6phrologie (May 1979), and at the Eighth International activated Cl and the complex C4b2a; EAC3bB, sensitized Complement Workshop (October 1979). sheep erythrocytes bearing the complex C3bBb; NFA, Ne- Dr. Leveille was the recipient of a grant from the phritic factor of the alternative pathway; NFc, NF of the College de M6dicne des H6pitaux de Paris. classical pathway; NHS, normal human serum; PBS, phos- Received for publication 14 September 1979 and in revised phate-buffered saline; SDS-PAGE, sodium dodecyl sulfate form 27 November 1979. polyacrylamide gel electrophoresis. J. Clin. Inivest. (D The American Society for Clinical Investigation, Inc. 0021-9738/80/06/1249/08 $1.00 1249 Volume 65 June 1980 1249-1256 static control, and will only acquire an efficient the main serological abnormalities together with the finding C5 cleaving activity provided: (a) the respective of a circulating C3 cleaving complex. All the blood samples obtained from the patient contained similar levels of stabiliz- C4b2a and C3bBb complexes are deposited and/or ing factor. formed on a suitable solid surface, and not if they are Normal human serum (NHS). This was a pool obtained present in a "fluid-phase" form (3, 4). (b) the com- from normal healthy blood donors. Normal and patient's sera plexes are not degraded by intrinsic decay dissociation were separated after clotting 2 h at room temperature, and and/or by the action of control proteins (5). These are kept frozen at -80°C. C2-deficient serum. These sera were obtained from patients the C4 binding protein (6), the f3IH globulin (,f1H) with demonstrated homozygous C2 deficiency. (7, 8), and the C3b inactivator, which inactivates both Purified components. Purified Cls, the enzymatically ac- C3b (9, 10) and C4b (11). tive subcomponent of C1, was obtained by the method of The nephritic factor of the alternative pathway of Takahashi et al. (23). Purified C4 was kindly donated by complement (NFA) was first reported in 1969 (12) Dr. M. G. Colomb, Laboratoire de Biochimie, Centre d'Etudes Nucl6aires, Grenoble, France. C2 was purified by the method in a patient with chronic glomerulonephritis, and of Kerr and Porter (24) in which the CNBr-Sepharose step described as a factor that would lead to the formation was replaced by a chromatography on CM-cellulose in of a fluid-phase, magnesium-dependent C3-cleaving 0.01 M P04 0.005 M EDTA, pH 5.9. C2 hemolytic activity enzyme when added to normal human serum. Over the was titrated according to Lachmann et al. (25). In our condi- tions, a normal human serum pool contains 2.5 x 104 C2 next decade the occurrence of this factor was un- hemolytic U/ml. Factor B and factor D were purified according equivocally established in dense deposit disease to Lachmann et al. (25) and Lesavre et al. (26), respectively. membranoproliferative glomerulonephritis (type II) NFA was purified according to Daha et al. (21) from the (13) and partial lipodistrophy (14, 15). serum of one patient with membranoproliferative glomerulo- NFA has been identified as an autoantibody specifi- nephritis (type II). Preparation of EAC14T2a. EAC14b, sensitized sheep cally directed against the C3bBb enzyme, (16-19) erythrocytes bearing activated Cl and C4 were prepared as which confers a prolonged half-life upon this labile described by Lachmann et al. (25); 25 x 108 EA in DGVB++ complex. The enhanced longevity of NFA stabilized were incubated with 400 gl of C2-deficient serum for 30 min C3bBb complex is caused by retardation of Bb f:actor B at 37°C, then washed several times in DGVB++. EAC14b2a were prepared by incubating 109 EAC14b with 2,000 hemo- release from the complex and protection against lytic U of C2 for 5 min at 30°C in 2 ml DGVB++. They were ,B1H-mediated accelerated decay dissociation (20, 21). immediately used for the stabilization assay. The existence of an analogue in the classical pathway EAC14b2a stabilization assay. 200 ,tl of EAC14b2a cells for the NFA has not yet been reported. (5 x 108/ml) in DGVB++ were mixed with 50 ,ul of twofold In this paper, we describe a serum factor functionally dilutions of the patient's serum, starting at 1/10, or dilutions of the purified factor in DGVB-EDTA 0.02 M, and incubated characterized by its unique capacity to stabilize for 30 min at 37°C. 10 ul of normal guinea pig serum were C4b2a complexes in both solid and fluid phase. This then added as a source of C3-C9 and, after further factor is indistinguishable from immunoglobulin (Ig)G, incubation at 37°C for 1 h, the reaction was stopped by the and it is shown to act as an antibody. It was isolated addition of 2 ml cold DGVB++. After centrifugation, lysis was measured by OD at 415 nm.

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