- Email: [email protected]
Histones interact with immunoglobulins and give them polyspecificity
Immunology
Letters 53 (1996) 95-99
Histones interact with immunoglobulins Dmitri
N. Abakushin”,
Alexander
and give them polyspecificity M. Poverenny
Department of Radiation Biochernistr)t, Medical Radiological Research Centre, Russian Academy qf‘ Medical Sciences. Korolec :s str.. 4. Obninsk 249020. Kaluga region. Russia
Received 4 June 1996; accepted 31 July 1996
Abstract The presence of antihistone antibodies was determined in different commercial immunoglobulin preparations. The ability of histones to perform the function of cofactor in interaction of immunoglobulin preparations and sera of healthy people with charged molecules such as DNA, cardiolipin and phosphatidylcholine, was determined. The addition of histones to immunoglobulin preparations or serum samples is accompanied by forming the stable complex, components of which interact with the charged molecules. The capacity of histones to play the role of cofactor is less evident in the case when they are preincubated with DNA or cardiolipin. The interaction of natural antibodies with histones can explain their polyspecificity in some cases. Keywords:
Anti-DNA
antibodies:
Anticardiolipin
antibodies:
Antihistone
antibodies
: Antiphospholipid
antibodies:
Cofactor;
Histone
1. Introduction The data
on possible
role of cofactors
in immunolog-
in 1990. It was shown that /I,-glycoprotein I (/?,GPI) performs the function of cofactor in interaction of antibodies (Abs) with cardiolipin [l-3]. This phenomenon was subjected to careful analysis in numerous publications [4-61. Assumptions were made that mAb derived from patients with the antiphospholipid syndrome have specificity for positive charged epitopes on the fifth domain of /&GPI that acts as a primary antigen for these Abs [7]. It is questionable whether the participation of cofactors in interactions with other charged antigens is possible. In previous studies we have shown that the sera of healthy people and preparations of Ig, contain Abs to cardiolipin and DNA in a hidden state and their activity is displayed after chromatography at acidic pH on Sephadex G-200 or after ion-exchange chromatography [8,9]. Studying this phenomenon we have found that
ical reactions
were obtained
* Corresponding [email protected]
author.
for the first time
Fax:
+ 7
095
2302326;
e-mail:
preparations of Ig isolated from a pool of healthy donors sera by alcohol fractionation contain a non-immunoglobulin component that can play the role of cofactor in interactions of Ab with native DNA [lo]. We have suggested that histones can carry out this function. In the present paper, we validate the data obtained with indicated hypotheses.
2. Materials
2.1. Commerciul
prepurations
and reugents
Two different commercial preparations of normal human immunoglobulins were used as initial material. The first one represented 10% normal human gammaglobulin solution packed into the ampoules and intended for intramuscular infusion (Moscow Research Institute of Epidemiology and Microbiology, Russia). Technological procedures of manufacturing involved the alcohol fractionation of serum proteins according to Cohn. Sandoglobulin - normal human immunoglobulin for intravenous infusion (Sandoz, Switzerland) was
0165-2478/961$12.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved PII SO165-2478(96)02626-O
and methods
96
D.N. Abakushin. A.M. Pocerenny
I Inmunoltigy Lertrrs 53 (1996) 95-99
used. Total calf thymus histones (Calbiochem Behring, USA); native calf thymus DNA (Calbiochem Behring, USA); cardiolipin from bovine heart (Sigma); L-crphosphatidylcholine type III-B from bovine brain (Sigma); peroxidase-coupled antibodies to human IgG (Sevac, Czhecho-Slovakia) were used. 3H-DNA was isolated from Escherichia coli W3110 thv- grown in “H-thymidine supplemented media as described previously [ 111.
ence/absence of histones. SDS-PAGE was performed under non-reducing conditions. The gel was stained with Coomassie Blue to visualize the proteins.
2.2. Serum samples
2.8. Anti-phospholipid ELBA
Blood was obtained from three healthy individuals. It was then allowed to clot. Serum was then recovered and decomplemented.
Anti-phospholipid ELISA was carried out using phospholipids adsorbed to polystyrene as described to detect anticardiolipin Abs [4].
2.3. Antihistone ELISA
2.9. Farr assa_vprocedure
The levels of antihistone antibodies were measured by ELISA as previously described [12] with some modifications. Total calf thymus histones were diluted to 2.5 mg/ml in 0.02 M HCl and subsequently to 10 pg/ml in carbonate buffer pH 9.5. The diluted histones (100 ~1) were added to the wells of polystyrene 96-wells ELISA plates (96F. Nunc, Denmark).
The levels of DNA-binding proteins were determined by Farr assay as previously described [14]. Briefly, about 80 ng ( - 25 000 DPM) of 3H-DNA was added to each sample.
2.7. Anti-DNA
ELISA
The levels of anti-native DNA antibodies were determined by ELISA as described previously [13].
3. Results 2.4. Preparation of histone-immunoglobulin
complexes
Normal human Ig preparations (gammaglobulin or ‘Sandoglobulin’) without anti-DNA and anticardiolipin activity diluted to 40 /dg/ml in 0.5% gelatin/PBS, containing 0.1% tween 20, were mixed (v/v) with different amounts of histones in the same medium for ELISA. Serum samples without anti-DNA and anticardiolipin activity diluted (1:50) in PBS were mixed (v/v) with different amounts of histones in the same medium for Farr assay. The mixtures were incubated for 2 h at 37°C and then tested for anti-DNA or anticardiolipin activity and were compared with control samples (without added histones). 2.5. Fractionation of histone-immunoglobulin by geljiltration on Sephacryl S-200
The presence of antihistone Ab in different preparations of Ig was determined by ELISA (Fig. 1). We found that the preparations do not interact with native DNA, cardiolipin and phosphatidylcholine. Using
complexes
0.1 ml of normal human Ig (10 mg/ml) preincubated with 1.25 ml of histones (2 mg/ml) in PBS pH 7.2 were chromatographed on Sephacryl S-200 (Pharmacia) in PBS pH 7.2 at 4°C on a 1.5 cm x 70 cm column. Eluted fractions (4 ml) were collected by LKB 7000 Ultrorac collector. Protein concentration was determined in eluted fractions by BCA Protein Assay Kit (Pierce, USA) according to the manufacturer’s protocol.
000 01
Concentrafion
2.6. SDS-PAGE Some eluates from the Sephacryl S-200 column were analyzed by SDS-PAGE (7-22%) to determine pres-
elf Yg, pglsample
100
Fig. 1. Binding of human Ig preparations to the immobilized histones (I pgg/sample) determined by ELISA. Anti-human IgG conjugate used to visualize the Abs to histones in gammaglobulin for intramuscular infusion, (0): and Ig for intravenous infusion, (W).
D.N. Abakushin, A.M. Poverentg
hmunology
Letters 53 (1996) 95-99
19
20
21
97
22
24
25 Histone
Ig
Histone
k 00
,,(
I
I
I11111,
0 01
I
II,
0 10
Concentration
of histones, pglsample
Fig. 2. Binding of human Ig, for intramuscular infusion, preincubated with histones. to immobilized native DNA determined by ELISA. Anti-human IgG conjugate is used to visualize the Abs bound with DNA. Gammaglobulin (2 pglsample) preincubated with different amounts of histones, (0); gammaglobulin (2 pg/sample) was added after different amounts of histones were preincubated with immobilized DNA, (U); different amounts of histones without added gammaglobulin. (A ): background of gammaglobulin (2 pgg/sample). (-).
ELISA technique, we demonstrated that only normal Ig preincubated with histones show anti-DNA activity (Fig. 2). The same results were obtained for determination of anticardiolipin Abs by ELISA (Fig. 3). Subsequent addition of Ig (after washing) to histones
‘Oi 08
Fig. 4. SDS-PAGE of six fractions eluted from the Sephacryl S-200 column (Fig. 5) and initial preparation of histone and lg. Position of IgM. IgG and histone are shown. The presence of histones in all fractions eluted from the Sephacryl S-200 column is shown.
preincubated with Ag did not lead to a positive result. However, the subsequent addition of substantially higher amounts of histones to Ig can lead to positive results in anticdrdiolipin ELISA but not in anti-DNA ELISA. After gel filtration of Ig preparation or histones on Sephacryl S-200, it was shown that Ig was determined in fractions 18-26 and histones was determined in fractions 34436 (data not shown). However, preincubation of Ig with histones was accompanied by the formation of an Ig-histone complex that was testified by electrophoresis of the histones in all fractions which react with DNA and phospholipids (Fig. 4). It was shown that histones formed a complex with the Ig which was obtained by fractionation on Sephacryl S200. Anti-DNA, anticardiolipin and antiphosphatidylcholine activities were determined in all follow-through (18-26) fractions (Fig. 5). Initial preparation of normal
06
04
02
00 0.1
Concentration
10
of histones, Kg/sample
Fig. 3. Binding of human Ig, for intravenous infusion, preincubated with histones. to immobilized cardiohpin. determined by ELISA. Anti-human IgG conjugate is used to visualize the Abs bound with cardiolipin. Ig (I pg/sample) preincubated with different amounts of histones. (0): Ig (I pg/sample) was added after different amounts of histones were preincubated with immobilized cardiolipin, (N); different amounts of histones without added lg. (A): background of Ig (I /(g/sample). ( - 1.
Fig. 5. Elution profile of gel filtration on Sephacryl S-200 (absorbance at 280 nm) and anti-DNA. (0): anticardiolipin. (B); and antiphosphatidylcholine reactivities (lsl). of follow-through fractions of histone-Ig complexes (4 Icgjsample) as detected by ELISA (absorbance at 492 nm). The backgrounds of human tg (4 Aigjsample) were 0.025 for anti-DNA; 0.046 for anticardiolipin; and 0.054 for antiphosphatidylcholine ELISAs. Histones were determined in fractions 34~ 36.
98
D.N. Abakushin, A.M. Poverenny / Immunology Letlers 53 (1996) 95-99
1000
Concktration of histone~~ng/sample Fig. 6. 3H-DNA-binding activity of the serum samples (1:lOO) of three healthy individuals preincubated with different amounts of histones as detected by Farr assay.
human Ig and the preparation after gel filtration on Sephacryl S-200 were used as the negative control. Gel filtration on Sephacryl S-200 has no effect on the properties of Ig. It was shown that the histone-Ig complex is able to interact with charged Ag. We found that normal Ig preincubated with histones acquires the ability to interact not only with anionic phospholipids (cardiolipin), but also some of these fractions (18-22) can interact with zwitterionic phospholipids (phosphatidylcholine) (Fig. 5). In the case of sera of healthy people we found that the addition of histones increases the serum ability to bind DNA as detected by the Farr assay (Fig. 6). The same results were obtained for determination of DNA-binding proteins in the case of normal human Ig (data not shown).
4. Discussion While researching the phenomenon of hidden Ab reacting with DNA, we have shown by the Farr assay and ELISA that the presence of non-immunoglobulin cofactor was necessary in some cases for interaction of Abs with native DNA. It was shown that preincubation of Ig or sera of healthy people with histones, gives them the ability to interact with native DNA. It was determined that the cause of this is the interaction of histones with Ig and the stability of the resulting complex formed. These complexes can be isolated by gel filtration on Sephacryl S-200. SDS-PAGE revealed the presence of histones in Ig fractions which were able to react with DNA and phospholipids. The derived preparations of Ig are able in this case to interact not only with DNA but also with cardiolipin. In previous work it was
determined that histones can play the role of cofactor in interactions. of monoclonal antihistone Abs with cardiolipin [15]. According to Pereira et al., histones can bind to anionic phospholipids (cardiolipin and phosphatidylserine) with high avidity, but not to zwitterionic phospholipids (phosphatidylcholine) and upon forming a complex, interact with monoclonal antihistone Abs. The addition of histones to the sera of SLE patients containing antihistone Abs often enhances their anticardiolipin activity. We have found in the present study that the preparations of normal human Ig isolated by alcohol fractionation from sera of healthy donors and the preparation of Ig for intravenous infusion, contain Abs that interact with histones and, as a result of preincubation with histones, their ability to interact with DNA is revealed. We have found that interaction of histones with Ig permits the formation of an immune complex more active in its interaction with cardiolipin and DNA than such interaction upon pre-incubation of histones with those antigens. The Ig-histone complex keeps activity at ionic strength corresponding to 2.4 M NaCl. It has been previously reported, that the complex of cardiolipin and histones loses the ability to interact with antihistone Abs under these conditions [IS]. Thus, it was shown that histones can play the role of cofactor in the interaction of Abs with such antigens as DNA, anionic and zwitterionic phospholipids. The presence of Abs in serum or within a preparation of Ig that can interact with histones is the determining condition in this case. Though in previous studies, it was stated that their presence in serum is the hallmark of drug-induced lupus [16- 181, it is necessary to emphasize that the Abs reacting with histones are rather widely occurring and they can be determined in preparations of Ig. In fact, the addition of histones to different preparations of Ig and to serum samples of donors is accompanied by their ability to interact with DNA and phospholipids. The biological significance of this phenomenon demands further study. It is necessary to discuss the works devoted to the study of the structures of epitopes which monoclonal Abs to P,GPI interact with. It was shown that anticardiolipin Abs recognize /?,GPI in the absence of phospholipid and ,!3,GPI acts as a primary antigen for these Abs [19-211. The experiments with synthetic peptides representing the segments of the fifth C-terminal domain of pIGPI, that contains the major phospholipid binding site and a region critical for the binding of anticardiolipin Abs, confirm this. It was shown that one of the peptides (Cys-Lys-Asn-Lys-GluLys-Lys-Cys), with which Abs interact, has a high positive charge [7]. The ability of a considerable population of Ig to interact with epitopes through the Coulomb forces may provide the possibility of participation as cofactor of histones or other compounds bearing positive charge in interaction with various negatively
D.N. Abakushin,
A.M.
Poverenn?
charged biopolymers. The well-known fact of polyspecificity of some natural Ig populations is indeed explained by this hypothesis [22,23]. As is assumed, such immune complexes can have nephritogenic activity [2426].
I Immunology
[IO] Saenko,
[ll] [12]
[13]
Acknowledgements The authors thank Professor Yehuda Shoenfeld, TelHashomer, Israel, for his useful discussions.
References
[14] [15]
[16] [17] [18]
111McNeil, H.P., Simpson, R., Chesterman, C.N. and Krilis, S.A. (1990) Proc. Natl. Acad. Sci. USA 87, 4120.
[19]
121Galli, M., Comfurius, P., Maassen, C., Hemker, H.C., de-Baets, M.H., van Bredo-Vriesman, P.J.C., Barbui, T., Zwaal, R.F.A. and Bevers, E.M. (1990) Lancet 335, 1544. 131Matsuura, E., Igarashi, Y., Fujimoto, M., Ichikawa, K., Koike, T. (1990) Lancet 336, 177. 141Kra-Oz. Z., Lorber, M., Shoenfeld, Y., Scharff, Y. (1993) Clin. Exp. Immunol. 93, 265. 151Koike, T. (1994) Clin. Immunol. Immunopathol. 72, 187. WI Harris, E.N., Pierangeli, S.S. (1994) J. Clin. Immunoassay 17, 1. 171Wang, M.X., Kandiah, D.A., Ichikawa, K., Khamashta, M., Hughes, G., Koike, T., Roubey, R. and Krihs, S.A. (1995) J. Immunol. 155, 1629. PI Poverenny, A.M., Rott, G.M., Sulaeva, N.I., Lekakh, IV., Saenko, V.A., Podgorodnichenko, V.K., Nasonov, E.L., Cebecauer. L. and Nasonova, V.A. (1989) Mol. Biol.-Engl. Tr. 23. 153. 191Saenko, V.A., Rott, GM. and Poverenny, A.M. (1989) Bull. Exp. Biol. Med.-Engl. Tr. 2, 217.
Letters
[20] [21] [22]
[23]
[24] [25] [26]
53 (1996) 95-99
99
V.A. and Poverenny, A.M. (1993) Bull. Exp. Biol. Med.-Engl. Tr. 6, 624. Saenko, V.A., Kabakov, A.E. and Poverenny, A.M. (1992) Immunol. Lett. 34, 1. Subiza, J.L.. Caturla, A., Pascual-Salcedo, D., Chamorro, M.J., Gazapo, E., Figueredo, M.A., and de la Concha, E.G. (1989) Arthritis Rheum. 32, 406. Lekakh, I.V., Rott, G.M. and Poverenny, A.M. (1991) Mol. Biol.-Engl. Tr. 25, 1391. Word, R.T., Young, F.E., Tan, E.M. and Farr. R.S. (1968) Science 161, 806. Pereira, L.F., Marco, F.M., Boimorto, R., Caturla, A., Bustos, A., de la Concha. E.G. and Subiza, J.L. (1994) Clin. Exp. Immunol. 97. 175. Fritzler, M.J. and Tan, E.M. (1978) J. Chn. Invest. 62, 560. Fishbein, E.. Alarcon-Segovia, D. and Vega, J.M. (1979) Clin. Exp. Immunol. 36, 145. Rubin, R.L., Burlingame, R.W., Arnott, J.E.. Totoritis, M.C., Mcnally, E.M. and Johnson, A.D. (1995) J. Immunol. 154, 2483. Arvieux, J., Roussel, B., Jacob, M.C. and Colomb, M.G. (1991) J. Immunol. Methods 143, 223. Kouts, S., Bunn, C.L.. Steinkasserer, A. and Krilis, S. (1993) FEBS Lett. 326, 105. Roubey, R.A.S., Eisenberg, R.A., Harper, M.F. and Winfield, J.B. (1995) J. Immunol. 154, 954. Avrameas, S. (1993) in: Natural Autoantibodies: Their Physiological Role and Regulatory Significance (Shoenfeld Y. and Isenberg D.A., Ed.) pp. l-14, CRC Press, Boca Raton. Abu-Shakra, M. and Shoenfeld, Y. (1993) in: Natural Autoantibodies: Their Physiological Role and Regulatory Significance (Shoenfeld Y. and Isenberg D.A., Ed.) pp. 15-33, CRC Press, Boca Raton. Brinkman, K., Termaat, R., Berden. J.H.M. and Smeenk, R.J.T. (1990) Immonol. Today 11, 232. Bernstein, K.A., Divalerio, R. and Lefkowith. J.B. (1995) J. Immunol. 154. 2424. Divalerio, R., Bernstein, K.A., Varghese, E. and Lefkowith, J.B. (1995) J. Immunol. 155, 2258.
Letters 53 (1996) 95-99
Histones interact with immunoglobulins Dmitri
N. Abakushin”,
Alexander
and give them polyspecificity M. Poverenny
Department of Radiation Biochernistr)t, Medical Radiological Research Centre, Russian Academy qf‘ Medical Sciences. Korolec :s str.. 4. Obninsk 249020. Kaluga region. Russia
Received 4 June 1996; accepted 31 July 1996
Abstract The presence of antihistone antibodies was determined in different commercial immunoglobulin preparations. The ability of histones to perform the function of cofactor in interaction of immunoglobulin preparations and sera of healthy people with charged molecules such as DNA, cardiolipin and phosphatidylcholine, was determined. The addition of histones to immunoglobulin preparations or serum samples is accompanied by forming the stable complex, components of which interact with the charged molecules. The capacity of histones to play the role of cofactor is less evident in the case when they are preincubated with DNA or cardiolipin. The interaction of natural antibodies with histones can explain their polyspecificity in some cases. Keywords:
Anti-DNA
antibodies:
Anticardiolipin
antibodies:
Antihistone
antibodies
: Antiphospholipid
antibodies:
Cofactor;
Histone
1. Introduction The data
on possible
role of cofactors
in immunolog-
in 1990. It was shown that /I,-glycoprotein I (/?,GPI) performs the function of cofactor in interaction of antibodies (Abs) with cardiolipin [l-3]. This phenomenon was subjected to careful analysis in numerous publications [4-61. Assumptions were made that mAb derived from patients with the antiphospholipid syndrome have specificity for positive charged epitopes on the fifth domain of /&GPI that acts as a primary antigen for these Abs [7]. It is questionable whether the participation of cofactors in interactions with other charged antigens is possible. In previous studies we have shown that the sera of healthy people and preparations of Ig, contain Abs to cardiolipin and DNA in a hidden state and their activity is displayed after chromatography at acidic pH on Sephadex G-200 or after ion-exchange chromatography [8,9]. Studying this phenomenon we have found that
ical reactions
were obtained
* Corresponding [email protected]
author.
for the first time
Fax:
+ 7
095
2302326;
e-mail:
preparations of Ig isolated from a pool of healthy donors sera by alcohol fractionation contain a non-immunoglobulin component that can play the role of cofactor in interactions of Ab with native DNA [lo]. We have suggested that histones can carry out this function. In the present paper, we validate the data obtained with indicated hypotheses.
2. Materials
2.1. Commerciul
prepurations
and reugents
Two different commercial preparations of normal human immunoglobulins were used as initial material. The first one represented 10% normal human gammaglobulin solution packed into the ampoules and intended for intramuscular infusion (Moscow Research Institute of Epidemiology and Microbiology, Russia). Technological procedures of manufacturing involved the alcohol fractionation of serum proteins according to Cohn. Sandoglobulin - normal human immunoglobulin for intravenous infusion (Sandoz, Switzerland) was
0165-2478/961$12.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved PII SO165-2478(96)02626-O
and methods
96
D.N. Abakushin. A.M. Pocerenny
I Inmunoltigy Lertrrs 53 (1996) 95-99
used. Total calf thymus histones (Calbiochem Behring, USA); native calf thymus DNA (Calbiochem Behring, USA); cardiolipin from bovine heart (Sigma); L-crphosphatidylcholine type III-B from bovine brain (Sigma); peroxidase-coupled antibodies to human IgG (Sevac, Czhecho-Slovakia) were used. 3H-DNA was isolated from Escherichia coli W3110 thv- grown in “H-thymidine supplemented media as described previously [ 111.
ence/absence of histones. SDS-PAGE was performed under non-reducing conditions. The gel was stained with Coomassie Blue to visualize the proteins.
2.2. Serum samples
2.8. Anti-phospholipid ELBA
Blood was obtained from three healthy individuals. It was then allowed to clot. Serum was then recovered and decomplemented.
Anti-phospholipid ELISA was carried out using phospholipids adsorbed to polystyrene as described to detect anticardiolipin Abs [4].
2.3. Antihistone ELISA
2.9. Farr assa_vprocedure
The levels of antihistone antibodies were measured by ELISA as previously described [12] with some modifications. Total calf thymus histones were diluted to 2.5 mg/ml in 0.02 M HCl and subsequently to 10 pg/ml in carbonate buffer pH 9.5. The diluted histones (100 ~1) were added to the wells of polystyrene 96-wells ELISA plates (96F. Nunc, Denmark).
The levels of DNA-binding proteins were determined by Farr assay as previously described [14]. Briefly, about 80 ng ( - 25 000 DPM) of 3H-DNA was added to each sample.
2.7. Anti-DNA
ELISA
The levels of anti-native DNA antibodies were determined by ELISA as described previously [13].
3. Results 2.4. Preparation of histone-immunoglobulin
complexes
Normal human Ig preparations (gammaglobulin or ‘Sandoglobulin’) without anti-DNA and anticardiolipin activity diluted to 40 /dg/ml in 0.5% gelatin/PBS, containing 0.1% tween 20, were mixed (v/v) with different amounts of histones in the same medium for ELISA. Serum samples without anti-DNA and anticardiolipin activity diluted (1:50) in PBS were mixed (v/v) with different amounts of histones in the same medium for Farr assay. The mixtures were incubated for 2 h at 37°C and then tested for anti-DNA or anticardiolipin activity and were compared with control samples (without added histones). 2.5. Fractionation of histone-immunoglobulin by geljiltration on Sephacryl S-200
The presence of antihistone Ab in different preparations of Ig was determined by ELISA (Fig. 1). We found that the preparations do not interact with native DNA, cardiolipin and phosphatidylcholine. Using
complexes
0.1 ml of normal human Ig (10 mg/ml) preincubated with 1.25 ml of histones (2 mg/ml) in PBS pH 7.2 were chromatographed on Sephacryl S-200 (Pharmacia) in PBS pH 7.2 at 4°C on a 1.5 cm x 70 cm column. Eluted fractions (4 ml) were collected by LKB 7000 Ultrorac collector. Protein concentration was determined in eluted fractions by BCA Protein Assay Kit (Pierce, USA) according to the manufacturer’s protocol.
000 01
Concentrafion
2.6. SDS-PAGE Some eluates from the Sephacryl S-200 column were analyzed by SDS-PAGE (7-22%) to determine pres-
elf Yg, pglsample
100
Fig. 1. Binding of human Ig preparations to the immobilized histones (I pgg/sample) determined by ELISA. Anti-human IgG conjugate used to visualize the Abs to histones in gammaglobulin for intramuscular infusion, (0): and Ig for intravenous infusion, (W).
D.N. Abakushin, A.M. Poverentg
hmunology
Letters 53 (1996) 95-99
19
20
21
97
22
24
25 Histone
Ig
Histone
k 00
,,(
I
I
I11111,
0 01
I
II,
0 10
Concentration
of histones, pglsample
Fig. 2. Binding of human Ig, for intramuscular infusion, preincubated with histones. to immobilized native DNA determined by ELISA. Anti-human IgG conjugate is used to visualize the Abs bound with DNA. Gammaglobulin (2 pglsample) preincubated with different amounts of histones, (0); gammaglobulin (2 pg/sample) was added after different amounts of histones were preincubated with immobilized DNA, (U); different amounts of histones without added gammaglobulin. (A ): background of gammaglobulin (2 pgg/sample). (-).
ELISA technique, we demonstrated that only normal Ig preincubated with histones show anti-DNA activity (Fig. 2). The same results were obtained for determination of anticardiolipin Abs by ELISA (Fig. 3). Subsequent addition of Ig (after washing) to histones
‘Oi 08
Fig. 4. SDS-PAGE of six fractions eluted from the Sephacryl S-200 column (Fig. 5) and initial preparation of histone and lg. Position of IgM. IgG and histone are shown. The presence of histones in all fractions eluted from the Sephacryl S-200 column is shown.
preincubated with Ag did not lead to a positive result. However, the subsequent addition of substantially higher amounts of histones to Ig can lead to positive results in anticdrdiolipin ELISA but not in anti-DNA ELISA. After gel filtration of Ig preparation or histones on Sephacryl S-200, it was shown that Ig was determined in fractions 18-26 and histones was determined in fractions 34436 (data not shown). However, preincubation of Ig with histones was accompanied by the formation of an Ig-histone complex that was testified by electrophoresis of the histones in all fractions which react with DNA and phospholipids (Fig. 4). It was shown that histones formed a complex with the Ig which was obtained by fractionation on Sephacryl S200. Anti-DNA, anticardiolipin and antiphosphatidylcholine activities were determined in all follow-through (18-26) fractions (Fig. 5). Initial preparation of normal
06
04
02
00 0.1
Concentration
10
of histones, Kg/sample
Fig. 3. Binding of human Ig, for intravenous infusion, preincubated with histones. to immobilized cardiohpin. determined by ELISA. Anti-human IgG conjugate is used to visualize the Abs bound with cardiolipin. Ig (I pg/sample) preincubated with different amounts of histones. (0): Ig (I pg/sample) was added after different amounts of histones were preincubated with immobilized cardiolipin, (N); different amounts of histones without added lg. (A): background of Ig (I /(g/sample). ( - 1.
Fig. 5. Elution profile of gel filtration on Sephacryl S-200 (absorbance at 280 nm) and anti-DNA. (0): anticardiolipin. (B); and antiphosphatidylcholine reactivities (lsl). of follow-through fractions of histone-Ig complexes (4 Icgjsample) as detected by ELISA (absorbance at 492 nm). The backgrounds of human tg (4 Aigjsample) were 0.025 for anti-DNA; 0.046 for anticardiolipin; and 0.054 for antiphosphatidylcholine ELISAs. Histones were determined in fractions 34~ 36.
98
D.N. Abakushin, A.M. Poverenny / Immunology Letlers 53 (1996) 95-99
1000
Concktration of histone~~ng/sample Fig. 6. 3H-DNA-binding activity of the serum samples (1:lOO) of three healthy individuals preincubated with different amounts of histones as detected by Farr assay.
human Ig and the preparation after gel filtration on Sephacryl S-200 were used as the negative control. Gel filtration on Sephacryl S-200 has no effect on the properties of Ig. It was shown that the histone-Ig complex is able to interact with charged Ag. We found that normal Ig preincubated with histones acquires the ability to interact not only with anionic phospholipids (cardiolipin), but also some of these fractions (18-22) can interact with zwitterionic phospholipids (phosphatidylcholine) (Fig. 5). In the case of sera of healthy people we found that the addition of histones increases the serum ability to bind DNA as detected by the Farr assay (Fig. 6). The same results were obtained for determination of DNA-binding proteins in the case of normal human Ig (data not shown).
4. Discussion While researching the phenomenon of hidden Ab reacting with DNA, we have shown by the Farr assay and ELISA that the presence of non-immunoglobulin cofactor was necessary in some cases for interaction of Abs with native DNA. It was shown that preincubation of Ig or sera of healthy people with histones, gives them the ability to interact with native DNA. It was determined that the cause of this is the interaction of histones with Ig and the stability of the resulting complex formed. These complexes can be isolated by gel filtration on Sephacryl S-200. SDS-PAGE revealed the presence of histones in Ig fractions which were able to react with DNA and phospholipids. The derived preparations of Ig are able in this case to interact not only with DNA but also with cardiolipin. In previous work it was
determined that histones can play the role of cofactor in interactions. of monoclonal antihistone Abs with cardiolipin [15]. According to Pereira et al., histones can bind to anionic phospholipids (cardiolipin and phosphatidylserine) with high avidity, but not to zwitterionic phospholipids (phosphatidylcholine) and upon forming a complex, interact with monoclonal antihistone Abs. The addition of histones to the sera of SLE patients containing antihistone Abs often enhances their anticardiolipin activity. We have found in the present study that the preparations of normal human Ig isolated by alcohol fractionation from sera of healthy donors and the preparation of Ig for intravenous infusion, contain Abs that interact with histones and, as a result of preincubation with histones, their ability to interact with DNA is revealed. We have found that interaction of histones with Ig permits the formation of an immune complex more active in its interaction with cardiolipin and DNA than such interaction upon pre-incubation of histones with those antigens. The Ig-histone complex keeps activity at ionic strength corresponding to 2.4 M NaCl. It has been previously reported, that the complex of cardiolipin and histones loses the ability to interact with antihistone Abs under these conditions [IS]. Thus, it was shown that histones can play the role of cofactor in the interaction of Abs with such antigens as DNA, anionic and zwitterionic phospholipids. The presence of Abs in serum or within a preparation of Ig that can interact with histones is the determining condition in this case. Though in previous studies, it was stated that their presence in serum is the hallmark of drug-induced lupus [16- 181, it is necessary to emphasize that the Abs reacting with histones are rather widely occurring and they can be determined in preparations of Ig. In fact, the addition of histones to different preparations of Ig and to serum samples of donors is accompanied by their ability to interact with DNA and phospholipids. The biological significance of this phenomenon demands further study. It is necessary to discuss the works devoted to the study of the structures of epitopes which monoclonal Abs to P,GPI interact with. It was shown that anticardiolipin Abs recognize /?,GPI in the absence of phospholipid and ,!3,GPI acts as a primary antigen for these Abs [19-211. The experiments with synthetic peptides representing the segments of the fifth C-terminal domain of pIGPI, that contains the major phospholipid binding site and a region critical for the binding of anticardiolipin Abs, confirm this. It was shown that one of the peptides (Cys-Lys-Asn-Lys-GluLys-Lys-Cys), with which Abs interact, has a high positive charge [7]. The ability of a considerable population of Ig to interact with epitopes through the Coulomb forces may provide the possibility of participation as cofactor of histones or other compounds bearing positive charge in interaction with various negatively
D.N. Abakushin,
A.M.
Poverenn?
charged biopolymers. The well-known fact of polyspecificity of some natural Ig populations is indeed explained by this hypothesis [22,23]. As is assumed, such immune complexes can have nephritogenic activity [2426].
I Immunology
[IO] Saenko,
[ll] [12]
[13]
Acknowledgements The authors thank Professor Yehuda Shoenfeld, TelHashomer, Israel, for his useful discussions.
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