Clin. exp. Immunol. (1991) 86, 124 133

New Zealand white rabbits immunized with RNA-complexed total histones develop an autoimmune-like response C. ATANASSOV, J.-P. BRIAND, D. BONNIER, M. H. V. VAN REGENMORTEL & S. MULLER Laboratoire dImmunochimie, Institut de Biologie Mokculaire et Cellulaire, CNRS, Strasbourg, France

(Acceptedc fr publication 8 MaY 1991)

SUMMARY The antibody response of rabbits immunized with a total histone mixture containing randomly coiled H1 /H5, H2A, H2B, H3 and H4 devoid of DNA was investigated in direct and competitive ELISA. The antisera were tested with isolated histones and chromatin and with a series of overlapping synthetic peptides covering the entire sequences of the four core histones and two peptides of H 1. It was found that the New Zealand (NZ) white rabbits immunized with the total histone (TH) mixture complexed with RNA produced IgG antibodies reacting with histones and with a number of histone peptides but not with chromatin. The antisera also contained IgG antibodies which bound components that correspond to common target antigens in autoimmune diseases such as native dsDNA, peptides of Sm-D antigen, ubiquitin, branched peptides of ubiquitinated H2A and poly(ADP-ribose). By competition experiments, it was shown that these antibodies corresponded to non-crossreacting antibody populations. New Zealand rabbits immunized with TH in the absence of RNA or random outbred rabbits immunized with the RNA-complexed histone fraction produced antibodies reacting with histone, chromatin and very few histone peptides, while no activity with nonrelated antigens was observed. The pattern of reactivity of antisera raised in NZ rabbits with RNAcomplexed TH was found to be very similar to that observed in sera of patients with systemic lupus erythematosus while, in contrast, the antibody response was very different in NZ or outbred rabbits immunized with various native nuclear particles and with individual histones. Altered nucleosome particles rather than native nucleosomes may represent the antigenic stimulus giving rise to autoantibodies.

Keywords anti-histone antibodies histone synthetic peptides animal model

INTRODUCTION In recent years, substantial experimental data have been accumulated concerning the possible involvement of chromatin structures as immunogens in systemic lupus erythematosus (SLE) and lupus-like disorders as well as in the murine grafttersus-host disease model (Hardin & Thomas, 1983; Gohill et al., 1985; Portanova, Claman & Kotzin, 1985; Portanova et al., 1987; Craft et al., 1987). Antibodies able to bind nucleosomes but not individual histones have been described in the serum of SLE patients (Rekvig & Hannestad, 1981) and antichromatin antibodies have been found in SLE mice (Fisher et al., 1989). It has also been suggested that extracellular DNA circulating in the form of DNA bound to nuclear proteins could play a role in the pathogenesis of SLE nephritis (Fourni&, 1988; Brinkman et a!., 1990; Rumore & Steinman, 1990).

In view of the possible role played by chromatin components in the appearance of autoantibodies, it would clearly be of interest to study carefully the immunogenic properties of nucleosomal particles. In a recent study of the fine specificity of

antibodies induced in rabbits following immunization with chicken erythrocyte nucleosomes, it was found that the antibodies reacted with nucleosomes and condensed chromatin but not with individual histones nor with native DNA (Muller et al., 1989). It was also found that these antibodies recognized the same histone peptides as the autoimmune histone antibodies found in SLE sera. In contrast, histone antibodies raised by immunizing rabbits with free histones recognized different regions of the histone molecules (Muller et al., 1989, 1991). To further explore the immunogenicity of nuclear particles, we decided to examine the antibody response in rabbits immunized with a total histone mixture containing HI1H5, H2A, H2B, H3 and H4 and devoid of DNA. Since it has been shown previously that individual histones are immunogenic in the absence of carrier RNA (Muller et al., 1991), the mixture was injected either in the presence or in the absence of complexing

Correspondence: Sylviane Muller, Laboratoire d'Immunochimie, Biologie Mol&culaire et Cellulaire du CNRS, 15 rue Ren& Descartes, 67084 Strasbourg cedex, France. Institut de

124

Induction of autoreactive antibodies in New Zealand white rabbits RNA. The reactivity of the total histone (TH) antisera was analysed with isolated histones in direct and competitive ELISA. The antisera were also tested with chromatin and with a series of overlapping synthetic peptides covering the entire sequences of the four core histones and two peptides of H 1. The specificity of the reaction with peptides was checked by inhibition experiments with homologous and heterologous peptides. Furthermore, the antisera were tested in ELISA with various antigens known to be recognized by autoantibodies present in human or murine lupus sera, namely native dsDNA, peptides of Sm-D polypeptide, ubiquitin, peptides of ubiquitinated H2A and poly(ADP-ribose). It was found that the New Zealand (NZ) white rabbits immunized with the TH mixture in the presence of complexing RNA produced antibodies reacting with histones and a number of histone peptides but not with chromatin. In addition, the antisera also reacted strongly with the other components that are common target antigens in autoimmune diseases. In contrast to these latter results, NZ rabbits immunized with TH without RNA or random outbred rabbits immunized with RNA-complexed TH fraction produced antibodies reacting with histones and chromatin as expected but with only very few histone fragments. Furthermore, these animals did not develop an immune response to unrelated target nuclear antigens. MATERIALS AND METHODS Preparation of chromatin, histones and histone peptides Polynucleosome chains (20-40 nucleosomes) were prepared from purified calf thymus nuclei as previously described (Huletsky et al., 1989). The preparations were routinely characterized by agarose gel electrophoresis. The histone content was checked by electrophoresis on SDS-18% polyacrylamide slab gel. Total histone mixture, HI, H2B, H3 and H4 were prepared from chicken erythrocytes. Histone H2A was isolated from calf thymus. They were purified by the acid method of Johns slightly modified by Michalski-Scrive et al. (1982). Chicken erythrocyte histone H5 was a gift from Dr A. Mazen (Strasbourg). Thirtyfour synthetic peptides (6-29 residues long) covering the entire sequences of H2A, H2B, H3 and H4 molecules (calf thymus sequences) and two peptides corresponding to the N- and Cterminus domains of human spleen H 1 b variant (Ohe, Hayashi & Iwai, 1986) were tested. The peptides used are shown in Table 2. Between calf and chicken the amino acid exchanges are one in H3 (135 residues), four in H2A (129 residues), six in H2B (125 residues) and none in H4 (102 residues) (Von Holt et al., 1989). Between chicken and human HI the sequence of fragment 1- 16 differs by six amino acid residues while only one exchange is found in the sequences of fragment 16 from calf thymus and human H 1. Fragment 204-218 shows two amino acid exchanges between human H I b and chicken H 1. The C-terminal sequence of calf HI is not yet available (Von Holt et al., 1989). The reactivity of synthetic peptides with rabbit antisera and with human autoimmune sera has been described previously (Muller et al., 1986, 1987, 1991; Tuaillon et al., 1990). They were synthesized using a modified stepwise solid phase method of Merrifield as previously described (Briand et al., 1989) on a NPS 4000 multichannel peptide synthesizer (Neosystem Laboratories, Strasbourg, France). Amino acid composition and net peptide content were determined with a Waters Pico Tag system (Waters Corp., Milford, MA).

125

Ubiquitin and ubiquitin peptides

Ubiquitin was a commercial preparation from Sigma Chemical Co. (St Louis, MO; ref. U 6253). Synthetic peptide CYS 22-45 of ubiquitin was described previously (Muller, Briand & Van Regenmortel, 1988). Two synthetic peptides, Tl and T2, corresponding to the branched region of ubiquitinated H2A (UH2A) were described by Plau&, Muller & Van Regenmortel (1989). A peptide T3 was prepared using the same procedure and corresponds to T2 with an additional cysteine residue at the NH2-terminal leucine 73 (in replacement of a tyrosine residue in the T2 peptide). No additional residue was added at the NH2terminal lysine. This latter residue was acetylated in the T3 peptide. Peptides of Sm-D polypeptide Two peptides corresponding to residues 1-20 and 44-67 of the polypeptide D of Sm antigen were used. These two peptides have been shown previously to contain dominant antigenic regions of Sm-D polypeptide (Barakat et al., 1990; Muller et al., 1990). Antisera Antisera induced with individual histones complexed to RNA were those previously described (Muller et al., 1991). Two outbred rabbits (P, C; Group 1, Table 1) were injected with 150 yg chicken erythrocyte TH mixture in the presence of RNA (histone: RNA 3: 1 w/w; Stollar & Ward, 1970) and complete Freund's adjuvant for the first injection, or with incomplete Freund's adjuvant for the subsquent injections. A series of biweekly injections was administered over 6 months. After three injections, the animals were bled 1 week after each injection and the antibody level was measured in ELISA. After the fifth bleeding rabbit C died accidentally. In parallel, two NZ white rabbits (M, V) received an identical immunization schedule (Group 2); two NZ rabbits (L, R) received TH fraction complexed with RNA in the absence of adjuvant (Group 3) whereas two other NZ rabbits (A, H) received TH fraction in the absence of RNA and with adjuvant (Group 4); NZ rabbits S and N received TH alone (Group 5). As control, two NZ rabbits (J, G) received saline solution emulsified with Freund's adjuvant (Group 6). All rabbits immunized with TH in the absence of adjuvant (Groups 3 and 5) received i.v. injections; the other rabbits received TH mixture by i.m. injections. New Zealand rabbits were from Charles River France (Cleon). Table 1. Composition of the immunogen preparations used to immunize the outbred and New Zealand (NZ) rabbits

Group I 2 3 4

5 6

Rabbits Outbred NZ NZ NZ NZ NZ

P M L A S J

C V R H N G

TH

RNA

Adjuvant*

+ + + + + -

+ + + -

+ + + +

* Complete Freund's adjuvant for the first injection and incomplete Freund's adjuvant for the subsequent immunizations. TH, Total histone.

C. Atanassov et al.

126

Antiserum to poly(ADP-ribose) was a kind gift of Dr Y. Kanai (Tokyo). Anti-ubiquitin antiserum was previously described (Muller et al., 1988). ELISA The ELISA procedure used to measure the binding of rabbit antibodies was as follows. Microtitre plates (Falcon Co., Oxnard, CA; ref. 3912) were coated overnight at 37°C with the various antigens, namely TH mixture or individual histone (100 ng/ml), chromatin (50 ng/ml), ubiquitin (500 ng/ml), and peptides corresponding to various sequences of histones, ubiquitin and Sm-D polypeptide (0-1-4 gM). For ELISA, the hexapeptide 130-135 of H3 was conjugated to bovine serum albumin (BSA) using 1% glutaraldehyde (coupling molar ratio of peptide/carrier calculated from amino acid analysis= 9: 1). The antigens were diluted in 0 05 M carbonate-bicarbonate buffer, pH 9-6, except for chromatin which was suspended in phosphate-buffered saline, pH 7 4, containing 0-05% Tween-20 (PBS-T). After three washings with PBS-T, antisera diluted in PBS-T containing 1% BSA were added for 1 h at 37°C. The plates were washed three times and then incubated for 30 min at 37°C with AffiniPure goat anti-rabbit IgG (H + L) conjugated to horseradish peroxidase (Jackson Laboratories, Bar Harbor, ME; ref. 111-035-003). The final reaction was visualized by the addition of 3,3',5,5' tetramethylbenzidine (Janssen Chimica, Beerse, Belgium; ref. 22 928 36) and H202. After 15 min at 37°C, the reaction was stopped by the addition of HCl (final concentration 0-25 M) and optical density values were measured at 450 nm. Antibodies to native DNA were detected as previously described (Muller et al., 1989). Measurement of antibodies reacting with poly(ADP-ribose) was performed with polymer kindly provided by G. Poirier (Quebec). Poly(ADP-ribose) (20 ng/ml, i.e. 40 pmol/ml) was coated overnight at 37°C on polyvinyl plates (Falcon 3912) that had been precoated for 30 min at 37°C with 200 ng/ml poly-L-lysine in PBS-T. After washing the plates, antisera diluted 1:1000 were added. The subsequent steps of the test were as described above. For inhibition experiments, inhibitor antigens were incubated with diluted antisera in PBS-T-BSA for 1 h at 37°C and then overnight at 4°C. The mixture was added to wells precoated with the appropriate antigens and incubated for 1 h at 37°C. Further steps of the assay were as described above.

Dot-immunobinding assay For dot-immunoassay, aliquots of I jpl of purified histone or peptide in PBS-T were applied to nitrocellulose (NC) sheets (Serva, Heidelberg, Germany; ref. 71243, pore size 0-2 ym). After blocking remaining binding sites of the membrane with PBS-T containing 2% BSA, individual strips were incubated overnight at room temperature with antisera diluted 1:1000 in PBS-T- 1% BSA. Following extensive rinsing with PBS-T, the reaction was revealed by addition of '251-protein A (30-50 mCi/ mg; Amersham International, Amersham, UK; Ref. IM 144) diluted in PBS-T to a final concentration of 350-500 nCi/ml. For poly(ADP-ribose) Immunodyne immunoaffinity membranes (Pall, Saint-Germain-en-Laye, France; pore size 0-2 pm) were used instead of NC membrane. Dotted membrane strips were dried and saturated with PBS-T containing 3% BSA for 3 h at room temperature. The subsequent steps were as described above except that protein A was diluted in PBS-T containing 2% BSA

as indicated by

the supplier.

RESULTS

Histone synthetic peptides Thirty-six peptides of histones H I, H2A, H2B, H3 and H4 were used in this study (Table 2). Their preparation and reactivity with rabbit antisera have been described elsewhere (Muller et al., 1991). The degree of purity of these peptides assessed by HPLC was at least 85%. The net peptide content was used as a basis for molarity calculation. Amino acid analysis of two samples of each peptide showed that the purified products had the expected composition. Binding of TH antisera with histones in ELISA Two outbred rabbits (labelled P, C) and eight NZ white rabbits (M, V, L, R, A, H, S and N, see Table 1) were immunized with a randomly coiled TH mixture, containing the four core histones H2A, H2B, H3 and H4 in equal proportion and H1/H5 (ratio 1:3). In the conditions used for immunization, the histone mixture is probably not octameric since the octamer is only stable in 2 M salt. The content of this mixture was checked by gel electrophoresis on 18% polyacrylamide SDS gel as well as by ELISA and dot-immunobinding assays using TH as antigen and various control antisera including anti-ubiquitin, anti-Sm D peptide and anti-poly(ADP-ribose) antisera. As expected, following the purification procedure, the TH mixture was found to be free of these compounds and of any contaminants within the sensitivity limit of the techniques used (Fig. 1). The reactivity of successive bleedings was first tested in ELISA in optimized conditions using sera diluted 1:1000 and histones (100 ng/ml) directly adsorbed to microtitre plates. The eight rabbits from Groups 1-4 developed IgG antibodies reacting with TH mixture. No reaction was shown when TH were injected without RNA and adjuvant (Group 5). When tested with isolated histones instead of TH mixture a very low reaction with each isolated histone was found in the case of rabbit C while, in contrast, a strong reaction was observed with H2A, H3, H5 and H I in the case of rabbit P (Fig. 2a). For NZ rabbits M and V, the extent of reactivity was in the order H2A > H3 > H4, H2B, H5, H I (Fig. 2b) and for rabbit L, it was in the order H2A>H1, H2B, H3 and H4 (Fig. 2c). Reaction was found to a lower extent in sera from rabbit R which only possessed antibodies binding H I and H2A, in rabbit A with H2A, HI and H2B and in rabbit H with H2A and H2B. No reactivity was found in sera from NZ rabbits S and N immunized with TH without RNA and adjuvant or in control rabbits J and G injected with adjuvant without histone. In homologous competition ELISA using the same histone molecule both as coated antigen and as inhibitor free in solution, 47-740 inhibition was found for a 100 molar excess inhibitor. No significant inhibition (i.e. < 25%) was found when heterologous inhibition systems were tested using different histone molecules as antigen and inhibitor. Binding of TH antisera to histone peptides in ELISA In an attempt to identify which part of each histone was recognized by TH antisera, a series of 34 overlapping synthetic peptides covering the entire sequences of the four calf thymus core histones and two peptides of human H I sequence were tested in ELISA. In a preliminary study, each peptide was tested with 10 different control normal rabbit sera in order to define the optimal concentration of each peptide showing no false positive -iM of each reactivity. Depending on the peptide used, 14

127

Induction of autoreactive antibodies in New Zealand white rabbits Table 2. ELISA binding of histone peptides with total histone antisera raised in New Zealand white (M, V, L, R, A, H, S, N) and in outbred (P. C) rabbits Group 2

Peptides

PM

M

V

Group 3

Group 4

Group 5

L

A

S

R

H

N

Group 1 P

C

HI 1-16 204-218

4 2

_-

H2A 1-20 18-35 28-42 44-61 56-70 65-85 85-100 90-105 103-120 116-129

2 2 2 4 2 2 4 2 4 4

H2B 1-25 21-38 36-50 48-63 60-77 75-92 92-110 110-125

2 4 2 4 4 4 2 4

+

+

+

-

+

+

+

H3 1-21 18-32 30-45 40-55 53-70 68-85 83-100 98-112 111-130 130-135

1 1 1 1 1 1 2 2 2 2

+

+

_

+

+

_

_

-

-

+ +

+

+

+

-

+

-

-

-

+

+

-

_

_

_

H4 1-29 27-44 42-59 57-74 72-89 85-102

1 1 2 2 2 2

Total no. peptides recognized

++

17

11

19

1

4

2

2

2

3

4

The third bleeding from each rabbit was tested in ELISA with histone peptides used at a concentration of 1-4 ym. Antisera were diluted 1: 1000 and allowed to react with plates coated with peptides. Absorbence values were measured after a hydrolysis time of 15 min and an antiserum was arbitrarily considered positive (+) when optical density values at 450 nm were . 0 30 under these ELISA conditions. Values 2 1 5 were considered as strongly positive (+ +). For each peptide, binding of normal rabbit serum was insignificant. In order to be tested in ELISA, hexapeptide 130-135 of H3 was conjugated to bovine serum albumin (BSA) using 1% glutaraldehyde (BSA: peptide ratio was 1:9).

128

C. Atanassov et al. Lanes l-7

Lanes

ng/dot

ng/dot

8-10

100

1160

50

580

25

290

12'5

145

6.2

72

Fig. 1. Analysis of the reaction between total histone (TH) mixture and homologous and heterologous antisera in dot immunoassay. Antisera diluted 1:1000 were allowed to react with serial dilutions of TH mixture (lanes 1-6), peptide 1-20 of Sm-D (lane 7) and poly(ADP-ribose) (lanes 8- 10). Antibody binding was revealed after incubation with 1251-protein A. Exposure for autoradiography at -70OC was for 1 h. Antisera were: anti-TH antisera from rabbits P (lanes 1 and 9) and V (lanes 2 and 10), control serum from rabbit G (lane 3), antiserum to peptide 22-45 of ubiquitin (lane 4), antiserum to peptide 1-20 of Sm-D (lanes 6 and 7) and antiserum to poly(ADP-ribose) (lanes 5 and 8).

peptide were found suitable for coating ELISA microtitre plates. The overall reactivity pattern of sera from rabbits in Groups 1-5 is shown in Table 2. No reactivity was found in the sera of NZ rabbits J and G in Group 6. The appearance of antibodies reacting with some peptides following immunization with TH mixture is illustrated in Fig. 3. Although the absorbence values obtained in such ELISA conditions are not strictly comparable on a quantitative basis, it can be seen that certain peptides were strongly recognized by antibodies developed by rabbits M, V and L whereas, in contrast, very few histone peptides were bound by antibodies from the seven other rabbits R, A, H, S, N, P and C. Antibodies from the three NZ rabbits M, V and L recognized the following peptides: 204-218 of H 1, 1 -20 of H2A, 1 -25 of H2B, 1-21, 40-55, 53-70 and 130-135 of H3 and 1-29 and 27-44 of H4. Ten peptides, namely 65-85, 103-120 and 116-129 of H2A, 21-38, 60-77 and 110-125 of H2B,83-100,98-112 and 111- 130 of H3 and 42-59 of H4 were recognized by antibodies from two of the three antisera. In contrast to these results, the serum from rabbit R only reacted with peptide 1-25 of H2B. The sera from rabbits A, H (Group 4) and S, N (Group 5) also reacted with very few peptides, namely peptides 65-85 of H2A, 1-25 and 60-77 of H2B, 111-130 and 130-135 of H3 and 27-44 of H4. Only four peptides were recognized by antibodies from outbred rabbits P and C, namely peptides 65-85 of H2A and 83100, 111-130 and 130-135 of H3. The four peptides recognized by antibodies from outbred rabbits were also recognized by antibodies from NZ rabbits. The specificity of the reaction obtained in the direct ELISA format with peptide coated on plastic microtitre plates was checked by inhibition assays using peptides as antigen and homologous or heterologous peptides as competitors free in solution. Depending on the peptide tested and using a 200 molar excess of homologous inhibitor, 27-73% of inhibition was

found. In parallel experiments, no or very low (< 100,4) inhibition was found with heterologous peptides. Reactivity of TH antisera with chromatin in ELISA Serum from rabbits M, V, J and G did not react in ELISA with chromatin purified from calf thymus nuclei. In contrast, a strong reaction was shown with the antisera from rabbit P from the third bleeding onward (Fig. 4). A much lower reaction was observed in the case of rabbit C.

Reactivity of TH antisera with non-histone-related antigens in ELISA The antisera of the 10 rabbits immunized with TH as well as those of control rabbits J and G were tested with several antigens known to be recognized by autoantibodies from lupus mice and lupus patients. Native double-stranded (ds)DNA and Sm antigen are markers for SLE whereas ubiquitin and poly(ADP-ribose) represent probes frequently recognized by SLE sera but seldom bound by antibodies present in other rheumatic diseases (Morrow et al., 1982; Tauchi et al., 1986; Plaue et al., 1989) As shown in Figs 5a and Sb, antiserum from rabbits V and L contained antibodies reacting with dsDNA in ELISA. A low level of reactivity with dsDNA was found in rabbit M while no reactivity was found in the other rabbits. Rabbits V, L and R also possessed a high level of antibodies reacting with regions 1-20 and 44-67 of polypeptide D of Sm antigen (Figs 5c and 5d). A lower but significant reactivity with Sm peptides was found in the serum of rabbit M while no reaction was found in the other rabbits. The sera of rabbits M, V, L and R also reacted with ubiquitin and peptide 22-45 of ubiquitin (Figs 5e and 5f) as well as with the branched peptides T1, T2 and T3 of ubiquitinated H2A. All bleedings of NZ rabbits M and V reacted strongly with

Induction of autoreactive antibodies in New Zealand white rabbits

129

DISCUSSION

E 0

I') 0 0

2

3

4

5

6

7

8

9

Bleeding

Fig. 2. Test in ELISA of the antibody response to isolated histones in (a) outbred rabbit and NZ rabbits (b) V and (c) L immunized with total histone mixture. Antisera were diluted 1: 1000 and allowed to react with 100 ng/ml of HI (A); H2A (O); H2B (A); H3 (0) and H4 (-).

poly(ADP-ribose) (Fig. 5g). Activity to polymer was also observed in bleedings from rabbit L and, surprisingly, in rabbits P and C. No or very low reactivity was shown in rabbits R, A, H, S, N, J and G. The specificity of the observed reactions was tested by ELISA inhibition experiments. As shown in Table 3, preincubation of antiserum from rabbits M and V with heterologous antigens had no effect on antibody binding to the five different histones. Likewise, dsDNA was unable to inhibit the binding of antibodies to ubiquitin or peptide 1-20 of Sm-D while ubiquitin and poly(ADP-ribose) did not inhibit the reaction of antibodies with dsDNA or peptide 1-20 of Sm-D antigen.

Systemic lupus erythematosus is a complex systemic disease in which sera from patients contain antibodies reacting with a variety of proteins and nucleic acids in the cell nucleus (Tan, 1989). Although many of the target antigens have been identified, sometimes at the level of a short sequence of amino acid residues, the aetiology of autoimmunity remains unknown. Several possibilities have been proposed, involving, for example, the presentation of an altered or delocalized self-antigen or the capacity of a foreign antigen to induce the production of antibodies crossreacting with a self constituent. Other mechanisms which are not mutually exclusive could be involved in the activation of resting autoimmune B cells. Examples are the action of endogenous or exogenous non-specific polyclonal cell activators in bringing about a generalized stimulation and the appearance of natural autoantibodies, or the effect of T cellmediated regulation or idiotype network regulation (Dziarski, 1988; Klinman, Ishigatsubo & Steinberg, 1988; Schwartz & Datta, 1989). In the case of anti-histone autoantibodies, several investigators have shown that IgG autoantibodies are unlikely to arise only by polyclonal B cell activation and that they are probably selectively induced by self-antigens such as nucleosomal structures (Hardin & Thomas, 1983; Gohill et al., 1985; Portanova et al., 1985). This conclusion is supported by the observation that the epitopes recognized by autoantibodies are mostly located in accessible regions of the nucleosome. This has led to the suggestion that the immune system is probably stimulated by the outside surface of the nucleoprotein particles rather than by the individual histones (Hardin, 1986). Recently, we have tested sera from patients with SLE for their ability to react with synthetic peptides of core histones and compared them with the reactivity of sera raised in rabbits against nucleosomes and histones (Muller et al., 1989). It was found that autoimmune sera and antisera to mononucleosomes reacted with the same peptides in contrast to the antibodies induced with histones either complexed to RNA or free of carrier RNA, which recognized epitopes located in different histone domains (Muller et al., 1991). For instance, fragment I21 of H3 was strongly recognized by autoantibodies in SLE as well as by anti-nucleosome antibodies but not by anti-H3 antisera, whereas peptides 110-125 of H2B and 85-102 of H4 were recognized by anti-histone antibodies (induced with complexed or uncomplexed histones) but not by anti-nucleosome and autoimmune sera. The aim of the present study was to examine the immunogenic properties of a random histone mixture either free or complexed to RNA. The choice of this type of immunogen was initially motivated by two sets of observations. Firstly, although the mechanism of in vivo nucleosome assembly is still controversial (Wu et al., 1986; Park, Kim & Paik, 1987; Sugasawa et al., 1989; Jackson, 1990), there is evidence for a small pool of histones in the cytoplasm that undergoes exchange with histones in the nuclear compartments. Several conservative, semi-conservative or heterogeneous models of chromatin assembly have been proposed in which various histone complexes initially devoid of DNA are later complexed with DNA during replication in nuclei. A basal level of histone exchange could also exist in the absence of replication and transcription. The fact that complexed histones exist in nuclei and cytoplasm in the absence

130

C. Atanassov et al.

E 0

In d

0

I old,

0

2

1

3

4

5

67

8

9

0

3

2

1

45

6

8

7

I

9

Bleeding

Fig. 3. Test in ELISA of the antibody response to histone peptides in (a) outbred rabbit P and in NZ rabbits (b) V; (c) L and (d) A immunized with total histone mixture. Antisera were diluted 1: 1000 and allowed to react with 2 yM 204-218 H 1 (0), 1 gM 1-21 H3 (*), 2 yM 1-25 H2B (A), 2 ,M bovine serum albumin-conjugated peptide 130-135 H3 (0) and 1 gM 1-29 H4 (-).

K

b

E r_

0

to Cu

I

I

0

0

I

2

3

4

-__=Eu 5

6

7

B

Bn-

9 Blood ing

1

fsir'. 2

3

i i

U

4

5

6

7

8

9

Fig. 4. Test in ELISA of the antibody response to calf thymus chromatin in (a) New Zealand and (b) outbred rabbits (bleedings 1-9). Antisera from rabbits P (0), C (U), M (U) and V (0) were diluted 1:1000 and allowed to react with 50 ng/ml calf thymus chromatin (expressed in terms of DNA).

of DNA could provide an explanation for their involvement in the anti-histone autoimmune response. By contrast nucleosomes per se do not exist as a free physiologically stable entity separate from chromatin fibre in either nuclei or cytoplasm. Secondly, it is possible that in pathological situations and after cell death, abnormal nucleosome particles are released into the circulation thence stimulating the immune system. The histone mixture used in the present study was tested as a model antigen for altered nucleosomes and for heterogeneous histone complexes. All rabbits from Groups 1-4 developed IgG antibodies that reacted in ELISA with TH mixture and individual histones. However, the same histone regions were not equally recognized

by all animals (Table 1). Antisera from outbred rabbits of Group 1 recognized a very few fragments that were mainly located in the C-terminal end of H3. Likewise, very few peptides were recognized by antibodies from rabbits immunized by TH in the absence of RNA (Groups 4, 5) and one rabbit (R) of Group 3. In contrast, a large number of peptides were recognized by antibodies from NZ rabbits injected with TH in the presence of RNA (two rabbits of Group 2, M, V, and one rabbit, L, of Group 3). It is interesting to observe that while some antisera reacted rather weakly with particular individual histones, they showed a stronger reaction with related histone peptides. The fact that different antibody populations are revealed when

Induction of autoreactive antibodies in New Zealand white rabbits

131

b

d

c

2

E 0

f 0

II

I 0

I

I

1

2

3

4

5

6

_- + -+ I 7 8 9

I 0

1

2

3

4

rIos 5 6 7 8 9

Bleeding

Fig. 5. Test in ELISA of the antibody response to (a, b) double-stranded (ds)DNA; (c, d) peptide 44-67 of Sm-D antigen; (e, f) ubiquitin and (g, h) poly(ADP-ribose) in outbred and New Zealand rabbits. Antisera from rabbits M (0), V (-), L (*), R (0), P (-) and C (+) were diluted 1: 1000 and allowed to react with the various antigens, namely 150 ng/ml dsDNA, 0 25 pM Sm-D peptide, 500 ng/ml ubiquitin, and 20 ng/ml poly(ADP-ribose). Binding of antibodies from rabbits P and C with dsDNA, Sm-D peptides and ubiquitin was insignificant (not shown).

histone peptides instead of complete molecules are tested has been observed and discussed previously in the case of autoimmune human sera (Tuaillon et al., 1990). When antisera were tested with unrelated antigens, unexpected reactions shown to correspond to non-crossreacting populations of antibodies were observed in the case of NZ rabbits M, V and L but not in other rabbits. The three NZ rabbits immunized with the TH mixture in the presence of RNA produced IgG antibodies that reacted with native DNA, peptides of Sm-D antigen, ubiquitin, branched peptides of ubiquitinated H2A and poly(ADP-ribose). In general, the highest titres were obtained in the early bleedings. The occurrence of artefactual reactions in the ELISA tests is unlikely since several controls were performed to ensure that

there were no contaminating nuclear components in the TH fraction used as antigen and for immunization. Natural crossreactivity in the early immune response might occur when antibodies of low avidity are produced, but the same phenomenon should have been observed with sera from other rabbits. As an additional control, NZ rabbits were injected with adjuvant in the absence of TH mixture and sera collected from these animals showed no reactivity with any of the test antigens. When the patterns of reactivity of the various antisera with histones, histone peptides, chromatin and non-related nuclear antigens are compared, the similarity in antibody response in terms of antigen recognition in SLE sera and in antisera raised in NZ rabbits with RNA-complexed TH is striking (Table 4). In particular, the inability of the rabbits to produce antibodies

C. Atanassov et al.

132

Table 3. Inability of heterologous antigens to inhibit antibody reactivity of total histone antisera

Components used as inhibitor with serum from Rabbit M (bleeding 8)

Rabbit V (bleeding 7) Test antigens

Hi H2A H2B H3 H4 dsDNA

Ubiquitin Peptide 1-20 Sm-D

dsDNA (%)

Ubiquitin

P(ADP-ribose)

dsDNA (O%,)

Ubiquitin (0/)

No.

(%NI)

No. (%)

P(ADP-ribose)

(%t)

(O%,)

(O%,)

100 94 97 100 96 28* 100 100

97 97 95 100 94 92 32* 98

97 100 98 97 100 100 100 100

100 (0-95) 100 (0 56) 100 (1 50) 100 (1-65) 100 (0 78) 100 (0 56) 100 (0 82) 100 (0-69)

100 100 100 100 NC 37* 100 93

91 97 96 100 NC 100 26* 100

96 100 100 100 NC

100 (0 89) 100 (0-91) 100 (0-71) 100 (0 97) 100 (0 25) 100 (0 42) 100 (0-48) 100 (063)

90 89 97

Microtitre plates were coated with 100 ng/ml histone, 150 ng/ml double-stranded (ds)DNA, 500 ng/ml ubiquitin or 250 nm peptide 1-20 of Sm-D antigen. According to the antigen tested, antisera were diluted 1: 500 to 1:8000. The antisera were preincubated with the various inhibitors at a molar excess of250 over the listed antigens. Anti-rabbit conjugate was diluted 1: 40 000. Results are expressed as the % of antibody binding obtained without inhibitor. Optical density values corresponding to 100% binding are indicated in brackets. Homologous competition values are indicated by an asterisk. NC, Not calculable. Table 4. Similarity in antibody response in systemic lupus erythematosus (SLE) sera and in antisera raised in New Zealand (NZ) and outbred rabbits with total histone mixture, nucleosome particles and chromatin

Reactivity with Source of sera

Immunogen

Not defined Histone mixture+ RNA Histone mixture Nucleosomet Outbred rabbit Histone mixture+ RNA Core particles

SLE, human NZ rabbit

Non-related Individual histones Histone peptides Chromatin nuclear antigens* + + + +

++ ++ + ++ +

-

+ +

+ +

Chromatin * DNA, peptides of Sm-D antigen, ubiquitin, peptides of ubiquitinated H2A. t From Muller et al. (1989).

reacting with chromatin is mirrored by the low frequency of autoantibodies reacting with chromatin in SLE sera (Bonnier & Muller, unpublished data). In contrast, for NZ rabbits immunized with mononucleosomes and at least for one outbred rabbit immunized with TH (Fig. 4), a strong reaction with chromatin was seen. In conclusion, although the number of rabbits described in this study is rather small, our results show that SLE sera and antisera from rabbits immunized with a TH mixture in the presence of RNA contain antibodies that react in ELISA with similar nuclear targets. The phenomenon was seen in the absence of adjuvant but not in the absence of RNA. Therefore, it seems that a structure equivalent to altered nucleosome particles (containing histones and nucleic acid) but not histone mixture alone nor native nucleosome per se may play a role as antigenic stimulus giving rise to autoantibodies. Several questions remain to be answered. Firstly, it should be of interest to

understand why this autoimmune response was observed only with NZ white rabbits (Table 4). It is worth noting that NZ rabbits have been shown to develop antigen arthritis under certain conditions (Burch et al., 1991). Further studies are needed to determine the extent to which genetic factors are responsible for the observed phenomenon. Secondly, in our hands, both core particles and chromatin chains were found to be non-immunogenic when injected in outbred rabbits (Table 4); the experiment should be repeated in NZ rabbits. Finally, we have observed severe weight loss and signs of sickness in NZ rabbits, particularly for rabbit M. Additional studies concerning the major pathological features of these and other immunized NZ rabbits will be performed, in particular at the level of the kidneys to ascertain possible glomerulonephritis. New Zealand rabbits which develop autoreactive antibodies following immunization with histone-RNA complexes may be a useful experimental model for studying syndromes resembling SLE.

Induction of autoreactive antibodies in New Zealand white rabbits ACKNOWLEDGMENTS We are most grateful to Dr G. Poirier (Quebec) for a gift of poly(ADPribose), to Dr Y. Kanai (Tokyo) for antiserum to poly(ADP-ribose), to Dr A. Mazen (Strasbourg) for a gift of histone H5 and to Dr S. Plau6 (Neosystem Laboratories, Strasbourg) for the synthesis of peptide T3 of U-H2A. We also thank L. Thibeault for help with chromatin preparation. This study was supported in part by grants to M.H.V.V.R. from Le Fond Regional de Recherche-Developpement d'Alsace and to S.M. from the Association pour la Recherche sur le Cancer (ARC). C.A. was supported by fellowships from the Federation of European Biochemical Societies (FEBS) and ARC.

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