Download pdf - Partial deficiency of the fourth component of human complement (C4) and autoantibody directed against C4 in a patient with SLE

Ann. Immunol. (Inst. Pasteur) 1983, 134 D, 233-245

P A R T I A L D E F I C I E N C Y OF T H E F O U R T H C O M P O N E N T

OF H U M A N C O M P L E M E N T (C4)

AND A U T O A N T I B O D Y D I R E C T E D

A G A I N S T C4 IN A P A T I E N T W I T H SLE

by J. Ripoche (*), M. Fontaine (*), M. Godin (**), G. Hauptmann (***) and J. Goetz (***)

(*) INSERM U-78, 543, Chemin de la Breldque, 76230 Bois-Guillaume, (**) Centre Hospilalier Universilaire de Rouen, Service de Ndphrologie (Pr Fillaslre), Rouen,

and (***) Inslitut d'Hdmalologie and Centre de Trans/usion Sanguine, 67085 Slrasbourg (France)

SUMMARY

The finding of dramatically depressed levels of C4 in a 17-year old patient with severe systemic lupus erythematosus (SLE) prompted a genetic s tudy of her family. This s tudy revealed the existence of a partial C4 deficiency; we found the presence of a C4A3,C4BQo haplotype which was transmit ted by the mother to each of her children. This pat ient pos- sessed, in her serum, an autoantibody with anti-C4 specificity. The immunochemical characterization of this autoant ibody revealed tha t it was IgM and belonged to the immunoconglutinin family. We have studied the effects of this autoantibody on the formation and dissociation kinetics of classical C3-convertase.

KEY-WORDS: Complement, SLE; C4, Genetic deficiency, IgM, Immuno- conglutinin.

INTRODUCTION

The synthesis of the fourth component of human complement (C4) is controlled by two closely linked loci, C4A and C4B [3, 21, 22]. These loci are located on the short arm of the sixth chromosome, t ight ly associated with the genes of the major histocompatibili ty complex [23]. At

Manuscrit regu le 23 mars 1983, accept6 le 13 septembre 1983.

Correspondence to Dr M. Fontaine.

234 J. RIPOCHE AND COLL.

least seven alleles at locus A and at least eight at locus B have thus far been recognized [10, 23]. These alleles include null alleles designated as C4AQo and C4BQo.

C4AQo and C4BQo alleles are common, with a frequency of 0.140 and 0.185, respectively [11]. In such a system with two loci, there may be carriers for one, two, three or four ~c silent ~ genes [4]. Complete C4 deficiency related to homozygous state C4AQo,C4BQo/C4AQo,C4BQo is very rare. Twelve cases have been described, eight of which presented systemic lupus erythematosus (SLE).

The C4A and C4B loci are closely associated with HLA A, B, and DR genes, as well as genes which code for the second component of complement (C2) and for factor B (Bf); this accounts for the transmission ~ en bloc ~, of all these genes. No particular association of the C4AQo,C4BQo haplotype has been found with HLA A, B, DR, C2 and Bf genes. In contrast the C2-defieient gene (C2Qo) has been found to be remarkably associated with the haplotype HLA A10 (A25) B18 BfS C4A4 C4B2 HLA DR2 Dw2 [12].

Two types of autoantibodies directed against proteins of the complement system have been described in SLE. Immunoconglutinins (IK) are autoantibodies directed against bound C3 or C4. These autoantibodies are synthesized in all situations where complement activation is believed to occur, especially in acute and chronic infections and autoimmune diseases such as SLE [16, 17]. Most of them are IgM immunoglobulins but IgG and IgA have been demonstrated [13]. Their exact role in vivo is unknown.

Nephritic factor of the classical pathway (C4 Nef) has recently been described in two patients with SLE [5] and in a patient with acute post- infectious glomerulonephritis [9]. C4 Nef is an autoantibody of the IgG class which is directed against the cell-bound classical C3-convertase E A C h . It has the property of binding to classical convertase and stabilizing it. This stabilization can be achieved by two means: prevention of intrinsic decay, or resistance to the regulatory action of C4bp [8, 9]. This protein has been termed C4 Nef because of similarities with the struc- ture and functions of the C3 Nef of the alternative complement pathway. The determinant which is recognized by C4 Nef has not yet been determined. C4 Nef binds to the EACI~I~2a complex, and no detectable binding was found on the EACI~5 complex.

In this communication, we report the observation of a young patient (D. S.) with severe SLE. This patient has a partial genetic C4 deficiency, as revealed by the family study. She possesses, in her serum, an IK of

DGVBS = VBS containing 2.5% dextrose and 0.1% gelatin.

SLE = systemic lupus erythematosus . IK = immunoconglut inin. C4 Nef = nephri t ic factor of the classical

pa thway .

C3 Nef = nephri t ic factor of the alter- nat ive pathway.

Es = ery throcyte (sheep). OD = optical density. SDS-PAGE = sodium dodecyl sulphate poly-

acrylamide gel electrophoresis. VBS = veronal buffer solution.

C4 DEFICIENCY AND AUTOANTIBODY IN SLE 235

the IgM class at a very high titre, which recognizes cell-bound C4. We have purified this IK and tested its effects on the classical convertase of complement.

MATERIALS AND METHODS

Blood samples.

Blood samples from the patient were collected three times during the course of the disease. One sample was available for every member of the family. Each sample was allowed to clot at room temperature for 2 h and sera were stored at --80 ~ C until used.

Proteins determinations.

Protein determination of C3, C4 and B were measured by radial immuno- diffusion with Partigen plates (Behring Institute). The results were expressed as rag/100 ml. C1 inhibitor (C1 INH) was immunochemically measured with an anti-C1 INH (Behring Institute). Concentration of H was determined by rocket electrophoresis [18] using an anti-H rabbit immune serum which was prepared m our laboratory.

Cellular intermediates.

Sheep erythrocytes (Es) were sensitized (EsA) with rabbit anti-sheep erythro- cyte antibodies (Institut Pasteur). EsACI~ were made with an R3 reagent [6]. E~ACT4 were obtained by allowing EsACI~-2 to decay 30 rain at 37 ~ C. EsAC] were made with an R4 reagent [1].

H~emolytic assays for C2, C3 and C4 were performed by one-step methods using R2 [15], R3 and R4 reagents.

Antisera.

Rabbit anti-C4 was prepared in our laboratory and adsorbed against sheep red blood cells. Anti-whole human serum was obtained f rom, Organon Teknika 7). Precipitating anti-IgM, anti-IgG, anti-K and anti-8 chains were obtained from the (( Centre R6gional de Transfusion Sanguine 77 (Bois-Guillaume, France).

C4 typing.

For C4 typing, serum samples with the addition of EDTA (final concentration, 0.002M) were incubated with neuraminidase from Closlridium per]ringens (type VIII, Sigma Chemical Co., St Louis, MO., USA) at a concentration of 10 milli- units/~l of plasma for 18 h at room temperature. The desialated material was then subjected to electrophoresis in 0.9% (w/v) agarose (Serva, Heidelberg, W. Germany for 1/3 and ICN Pharmaceuticals for 2/3) using a Tris/glycine/ barbital discontinuous buffer system [3, 22] with addition of Na~EDTA (0.0005 M) followed by immunofixation with anti-human C4 antiserum.

Anti-human C4 antisera were purchased from ICL Scientific, Fountain Valley, CA., USA.

Anti-Chido and anti-Rodgers antibody.

The serum of the patient was tested for the presence of anti-Chido and anti- Rodgers antibodies by the indirect antiglobulin test against C4-coated red cells


prepared according to the method of Jenkins el al. [14]. Red cells were coated with C4 from Ch(a-~-)Rg(a-f-), Ch(a+)Rg(a--) and Ch(a--)Rg(a-}-) individuals selected by C4 typing.

Agglutination assays.

Assays were performed in microtitration plates. Intermediates at 108 cells/ml were mixed with an equal volume of different dilutions of the sample to be tested and distributed on the plate. A reading was made after 2 h of storage at 4 ~ C.

Electrophoresis.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Weber and Osborn's method [26]. Phosphorylase, albumin, ovalbumin, carbonic anhydrate, trypsin inhibitor and alpha-lactal- bumin (Pharmacia, Uppsala) were used as internal markers. Immunoelectropho- reses were carried out on 1.3% agar noble (Difco) in 0.05 M barbital buffer, pH 8.6.

Chromatography experiments.

Ion exchange chromatography was performed on DEAE-Sephaeel (Pharmacia) equilibrated with a 5 mM Tris, 5 mM EDTA, 10 mM r acid, 1 mM benzamidine buffer pH 7.3. Gel filtrations were performed on a Sephacryl S-300 column (1.5• cm) equilibrated with 0.15 M NaC1, 0.05 M r acid and 0.02 M EDTA pH 7.2.

EA C i l b ~ stabilization assay.

EACi~-2 were used immediately for the stabilization assay according to Gigli et al. [8], slightly modified. Thus, samples (5 ml) of EACI-~2 (108 cells/ml) were incubated alone or with 100 or 200 ~l of samples to be tested and incubated at 37 ~ C. At time zero and intervals thereafter, aliquots of 0.5 ml were removed and 0.5 ml guinea-pig complement (diluted 1/40 in veronal buffer solution (VBS), pH 7.2, 10 mM EDTA) was added to each of them. After further incubation at 37 ~ C for 1 h, the reaction was stopped with ice-cold DGVBS (VBS containing 2.5% dextrose 0.1% gelatin). After centrifugation, lysis was measured by OD at 415 nm.

Formation o/ classical pathway conoertase E A C ~

To determine the effect of anti-C4 on formation of classical pathway convertase, three samples of 5 ml of EA (10 s cells/ml) and R3 (1/40) were mixed in DGVBS alone or in DGVBS containing 100 ~1 or 200 ~1 of the sample to be tested, and were incubated at 37 ~ C. Aliquots of 0.5 ml were removed at time zero and at intervals thereafter; lysis was developed by addition of 0.5 ml guinea-pig complement (diluted 1/40 in VBS, pH 7.2, 10 mM EDTA) and subsequent incubation for 60 rain at 37 ~ C.

HLA typing.

HLA A and B typing was performed by a two-step lymphocytotoxicity micro- technique on plates using the FT 25 battery provided by (c France Transplant ,). HLA DR typing was performed as described [24].

Anti-DNA antibody research.

Anti-DNA antibody was titrated by the Farr technique, as described [19].


R E S U L T S

Complement studies.

Complement component t i trat ions are shown in table I. Three samples were available for D. S. and the third was collected after she bad unAergone cortieosteroid t rea tment . D. S. had neither functional C4 nor functional C2. Her antigenic C4 was very low but not zero; her C3 was also very reduced. H and B were within the normal range. C1 inhibitor was found to be normal. Although the SLE of D. S. was very active, the dramatic fall in antigenic C4 led us to search for a genetic C4 deficiency.

TABLE I. - - Serum complement component titrations.

C4h C4p C2h C3h C3p Bp Hp

D. S. (Patient) 0 3.5 0 12 22 12 59.2 (3 samples) 0 3.0 0 15 26 13 ND

9 ND 10 ND ND N D ND R. S. (Father) 100 35.0 56 126 83 16 45 M. S. (Mother) 32.5 23.0 54 89 83 16 56 F. S. (Brother) 58 33.0 74 100 ND 18 41 I. S. (Sister) 41 25.0 65 71 ND 18 33 M. S. (Sister) 45 31.5 61 95 ND 17 37

Normal range 60-140 20-50 60-140 60-140 50-120 10-40 20-60

h = haemolytic t i t rat ion; results are expressed as percentage versus a normal h u m a n serum used as standard.

p = immunochemical t i t ra t ion (mg/100 ml). ND = not done.

All members of the family were tes ted for antigenic C3, C4, R and H, and for functional C2, C3 and C4. Every haemolyt ic t i t rat ion was performed three times; results given are average values.

Functional C4 was 32.5% in the mother, M. S., and about 50% in the other children. Antigenic C4 was within the normal range, as were C3, B and H concentrations in every member of the family. Surprisingly, C2 was found to be diminished in all family members.

DNA antibodies.

Levels of DNA binding were > 9 0 % in D. S., no ant i -DNA antibodies were found in other family members.

C4 typing.

Results are given in table II. C4 was not detectable by immunoelec- trophoresis in the pat ient ' s serum; therefore, her C4 type could not be

Ann. Immunol. (Inst. Pasteur), 134 D, n ~ 2, 1983. 16


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established. Every child in the family was found to be heterozygous for the haplotype C4A3, C4BQo; this haplotype was transmitted by the mother, M. S., who was found to be homozygous C4A3,C4BQo/C4A3,C4BQo. D. S.'s father (R. S.) was homozygous for the haplotype C4A3,C4B1. D. S. had to be heterozygous and C4A3,C4B1/C4A3,C4BQo.

H L A tgping.

The results of HLA typing are given in table II. D. S. was HLA A2 A3, B7 B12, DR W2W4. Her mother was homozygous for the haplotype A2 B12 DR W4. Children F. S. and M. S. had the same phenotype as D. S.

Absence of anti-Chido and anti-Rodgers in D. S. serum.

A research for anti-Chido and anti-Rodgers antibodies in D. S.'s serum was performed as described in ~c Materials and Methods ~. No antibody of this type was found.

Presence of anti-C4 autoantibodg in D. S.'s serum.

After adsorption on sheep erythrocytes, D. S.'s serum agglutinated E~ACi~-at a dilution of 1/8. No agglutination was found with E~AC1, nor with EsA.

Isolation of the autoanlibodg and its immunochemical characterization.

D. S.'s serum (14 ml) was subjected to ammonium sulphate precipitation (40~o saturation). The precipitate was dissolved and dialysed against 5 mM Tris, 5 mM EDTA, 10 mM ~-amino-caproic acid, 1 mM benzamidine buffer pH 7.3. The supernatant was applied to a DEAE-Sephacel column. The anti-C4 activity was found in the fraction eluted with 0.2 M NaC1 in the buffer solution. This fraction was subsequently subjected to gel- filtration on a Sephacryl S-300 column calibrated with IgM, IgG and albumin. Three fractions were pooled as indicated in figure 1 by solid bars, concentrated and tested for anti-C4 activity. Anti-C4 activity was found in the void volume of the column. Further purification to homogeneity of anti-C4 was obtained by a second gel filtration on the Sephacryl S-300 column. The fraction with anti-C4 activity was finally concentrated to 3 ml (final concentration 2 mg/ml). It agglutinated EsAC-14 at a 1/64 dilution. SDS-PAGE analysis under reducing conditions (fig. 2) revealed the presence of two bands with molecular weights of 75,000 and 25,000 daltons. Upon Ouchterlony analysis, anti-C4 exhibited a precipitation line with anti-~ only. Precipitating arcs were observed with anti-K and anti-z chains, indicating oligoclonality for the anti-C4 autoantibody.

Action of anti-C4 autoantibodg on formation and decag of EsA-CI~b-~.

EsACT4b--~ formation and decay assays were performed as described in (c Materials and Methods )~. In each experiment, E s A ~ intermediates

240 J. B I P O C H E AND COLL.

_e ~ 2

0

20 30 40 V ml FIa. 1. - - Sephacryl S-300 elution profile of the D E A E eluale

F r a c t i o n s w e r e p o o l e d as i n d i c a t e d b y sol id ba r s . , - - - ,

containing anti-C4 activity.

= an t i -C4 a c t i v i t y .

A B

FIG. 2. - - S D S - P A G E in 7.5% polyacrulamide gel. A = m o l e c u l a r w e i g h t m a r k e r s . B ~ p u r i f i e d an t i -C4 a n t i b o d y . S a m p l e s w e r e r e d u c e d be fo re e l e c t r o p h o r e s i s .

N

I I I i i I i ] ~ / /

5 1 5 3 0 6 0

time min.

N

i

5 1 5 3 0 6 0

time min. FIG. 3. - - D i s soc ia l ion k ine t i cs of the c lass ical C3 convertase.

(a) Kine t ics of decay a t 37 o C in DGVBS ++ of ce l l -bound C4b2a alone (o), wi th 100 lzl (o), and wi th 200 ~I (m) of purified anti-C4.

(b) Kine t ics of fo rma t ion a t 37 ~ C of cel l-bound C4-5~ in DGVBS ++ alone (o) or in t h e presence of purified anti-C4, 100 ~1 (o ) and 200 ~1 (m).


were mixed with 100 and 200 ~l of purified anti-C4 as described. Aliquots were removed at times 0, 1, 2, 5, 10, 15, 30 and 60 min and lysis was developed with guinea-pig complement as described in (( Materials and Methods )). The number of haemolytic sites, Z------Ln ( l - -Y) , was plotted versus the incubation t ime (where y is the percentage of lysis).

Figure 3b shows results obtained for the convertase formation experiment; we observed neither enhancement of the Tmax (which represents the point of max imum haemolysis) nor stabilization of the const i tuted convertase. At a high dose of autoant ibody (200 ~l), a delaying phenomenon of the Tmax was observed. Figure 3a shows results obtained for the C3 convertase dissooiation experiment. A slightly accentuated initial decay was observed with a high dose of autoant ibody, but the phenomenon did not seem to be specific. In any case, the overall dissociation of the convertase at t ime 30 min was equivalent to tha t obtained with buffer alone.

DISCUSSION

Decreased levels of C4, C2 and C3 are often found in patients with active SLE, and there is some correlation between reduced levels and the severi ty of the disease. Levels of functional and immunochemical C4 in D. S.'s serum were dramatical ly low. This suggested an underlying genetic deficiency. Partial C4 deficiency was affirmed by C4 typing, which revealed the presence of one cc silent gene ,), CgBQo, at the C4A locus. However, interpretat ion of C4 t i trat ions in the family must take into account three facts:

- - considerable variat ion is found in C4 plasma concentration, and there is no direct relationship between the level and the C4 phenotype [2, 20];

- - products of the C4A and C4B loci do not have the same haemolyt ic efficiency; those of the C4B locus are more efficient than those of the C4A locus [2];

- - t h e r e exists a variable consumption of complement components in SLE. It is believed tha t immune complexes in the circulation may init iate act ivat ion of classical pa thway convertase by deposition of Clq on immune complexes.

Indeed, in the S. family, the mother, M. S., who is homozygous for the double silent gene C4BQo, and the children F. S., Ma. S. and I. S., who are heterozygous, have antigenic C4 within the normal range. If 35 mg/dl is t aken as the 100% theoretical value for antigenic C4 (medium value, Behring), then M. S. is 76~o, F. S. 93~/o, Ma. S. 89% and I. S. 70~/o. These values are quite different from the theoretical values of 75~/o and 50~o which are expected for carriers of one and two silent genes, respectively. Differences in the haemolyt ic efficiency of the C4A and C4B loci explain the functional C4 results: 25~/o in the mother, and about 50~o in F. S., Ma. S. and I. S. Individuals who a re homozygous for the C4AQo locus do not have significantly reduced functional C4 levels [11]. However,


partial C4 deficiency does not completely explain D. S.'s level of C4. In this patient, there must be major consumption via the classical pathway, which explains the very low levels of C4, C2 and C3. Low levels of functional C2 in the other members of the family are not clearly explained. These results were confirmed by haemolytic titration using a C2-deficient serum. Low levels of haemolytic C2 are often observed in patients with C4 deficiency (G. Hauptmann, unpublished results), and this has thus far not been clearly understood. However, this fact does not explain the low C2 level in the father, R. S. One possibility is that there exi~S an underlying partial C2 deft- ciency in this family. Unfortunately, i twas not possible to perform C2typing.

Important variations in HLA A, B and DR frequency have been found in patients with SLE. Results of HLA typing for D. S. show the presence of the haplotype A2 B12, which was inherited from her mother. The haplotype A2 B12 was found in 11 of 106 patients with SLE in a recent study [25]. This same study also found an increased frequency of DR2 antigen. However, all the children in family S. bear the antigens HLA A2 B12 DR2, together with the C4 phenotype C4A3,C4BQo/C4A3, C4B1, and only one of them has developed SLE. Furthemore, anti-DNA antibodies were not found in other family members.

IK are autoantibodies directed against cell-bound components C3 and/or C4 of the complement system. Agglutination experiments clearly demonstrated the presence of such an autoantibody directed against C4 in the serum of the propositus. Purification of this antibody has permitted its identifica- tion. SDS-PAGE and Ouchterlony analysis demonstrated that it was IgM.

In an effort to compare this autoanti-C4 with the nephritic factor of the classical pathway, we studied its effects on the kinetics of formation and dissociation of classical C3 convertase. As shown in the results, no stabilizing effect of autoanti-C4 on classical convertase was found. We found no destabilizing effect on classical convertase; we observed only a delaying effect on the Tmax; however, this phenomenon was observed at very high quantities of IK (80 ~g/108 cells) and was certainly related to a steric effect rather than to a specific effect.

The function of IK in complement-dependent reactions is poorly understood. We have demonstrated that IK of the propositus had no ~, nephritic ~ activity. The very low levels of immunochemical C4 suggest tha t IK could interfere with the physiological catabolism of C4. We are currently examining the influence of IK on the inactivating system of C4b (C4bp, I) [7].

RI~SUMI~

DEFICIT PARTIEL EN QUATRIEME COMPOSANT DU COMPLI~MENT (C4)

ET PRt~SENCE D'UN AUTOANTICORPS DIRIGI~ CONTRE LE C4

CHEZ UNE PATIENTE ATTEINTE D'UN LUPUS I~RYTHI~MATEUX DISSI~MINI~

La mise en 6vidence d'un effondrement de la teneur en C4 chez une patiente de 17 ans, atteinte d'un lupus 6ryth~mateux diss~min~ s~v~re,


nous a conduit ~ effectuer une 6tude familiale. Cette ~tude a r~v616 l'existence d 'un d6ficit partiel en C4; presence d 'un haplotype C4A3,C4BQo transmis par la m~re h chacun de ses enfants. Cette pat iente poss6dait un autoant icorps anti-C4 dans son s6rum. Sa caract6risation immunochi- mique a montr6 qu'il s 'agit d 'une IgM appar tenant ~ la famille des immuno- conglutinines. Nous avons ~tudi6 les effets de cet autoanticorps sur les cin~tique de formation et de dissociation de la convertase classique.

MOTS-CL~S : Compl6ment, LED ; C4, D6ficit g6n6tique, IgM, Immuno- conglutinine.

ACKNOWLEDGEMENTS

We wish to thank Mrs M. Daveau for anti-DNA research, Mrs B. Cavelier for HLA typing, Mrs M. Dutheil for technical assistance and Mrs M. Picard for preparing the manuscript.

This work was supported by CRL INSERM n ~ 82.10.40 (to M. F.) and n ~ 82.10.15 (to G. H.) and by the University of Rouen.

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