Journal of Autoimmunity (1992) 5, 1-14
D o u b l e R e a c t i v i t y o f M o n o c l o n a l and P o l y c l o n a l R h e u m a t o i d Factors for IgG and Histones: M a p p i n g o f B i n d i n g Sites by m e a n s o f H i s t o n e S y n t h e t i c Peptides and Anti-Id Antibodies
Nadine Tuaillon, Thierry Martin,* Anne-Marie Knapp,* Jean-Louis Pasquali* and Sylviane Muller Laboratoire d' Immunochimie, Institut de Biologie Mol~culaire et Cellulaire, Strasbourg and *Laboratoire du Service de M~decine lnterne A, Hopital Central, Strasbourg, France (Received 11 June 1991 and accepted 13 September 1991)
Polyreactive antibodies able to bind various apparently unrelated structures represent a frequent antibody population in autoimmune diseases. In this work, the structural basis of the double reactivity of such autoantibodies was investigated using as models polyclonal and monoclonal human rheumatoid factors (RF) reacting with histones. Both direct ELISA binding and competitive inhibition experiments were performed. A more precise delineation of the histone regions r e c o g n i z e d b y t h e R F s w a s m a d e b y m e a n s o f 27 s y n t h e t i c p e p t i d e s o f these proteins. Anti-idiotope (Id) murine antibodies were used to map the binding sites involved on RF in the interaction with IgG and hist o n e s . A m o n g t h e 13 p o l y c l o n a l a n d s i x m o n o c l o n a l R F s t e s t e d , f o u r a n d t w o r e s p e c t i v e l y w e r e f o u n d to c r o s s - r e a c t w i t h I g G a n d h i s t o n e s . T h e fragments shown to be the most frequently recognized by RFs were l o c a t e d i n r e s i d u e s 1-16 a n d 204-218 o f i l l , 1-20 a n d 65-85 o f H 2 A , a n d 1 21 o f H3. T h e r e s u l t s o b t a i n e d b y c o m p e t i t i v e E L I S A a s s a y s u s i n g I g G , histone peptides and anti-Id monoclonal antibodies led us to confirm and characterize more precisely our previous finding suggesting the existence of topographically distinct binding sites for the different targets recognized by RFs.
Correspondence to: Sylviane Muller, Institut de Biologie Mol6culaire et Cellulaire, CNRS, 15 rue Descartes, 67084 Strasbourg cedex, France. Fax: (33) 88 61 06 80. 1
0896-8411/92/010001 + 14 $03.00/0
9 1992 Academic Press Limited
2
Nadine Tuaillon e t al. Introduction
A number of investigators have demonstrated the existence of an intriguingly high frequency of autoantibodies characterized by extensive 'multispecific' properties. In particular, this kind of cross-reactivity has been described in the case of rheumatoid factors (RFs) [1-3], anti-DNA antibodies which cross-react with phospholipids and cytoskeletal proteins [4-6] and for an anti-poly (ADPribose) monoclonal autoantibody [7]. The first question raised by the crossreactivity phenomenon concerns the definition of this apparent double or 'multi' specificity: do the 'multireactive' autoantibodies react at the level of a unique antibody site with related structures present at the surface of two distinct antigenic complexes or do they interact with two different antigenic structures using two distinct binding sites? The first possibility would be involved in the case of anti-DNA antibody cross-reacting with phospholipids, for example, and is a consequence of molecular mimicry. The second mechanism has been recently demonstrated to occur in the case of a particular RF, lef, reacting with IgG and histone [3]. The second question raised by the cross-reactivity phenomenon is related to its physiological significance. Monoclonal RF, in the same way as numerous multireactive autoantibodies, are frequently directly germ line encoded [8, 9], which could signify that multireactivity has a physiological function. Is this question related to ancestral properties of low affinity immunoglobulinbased immune responses, or is this function associated with the B cell ontogeny by forming a network of self reacting antibodies? In order to answer these questions, it is first necessary to define the structures of the recognized autoepitopes at a molecular level. RFs are particularly suitable for studying these questions. They allow the fine specificity of Ig to be investigated at the molecular level because (1) RFs exist in humans in both monoclonal (benign and malignant lymphoproliferations) and polyclonal (autoimmune diseases) forms; (2) monoclonal RFs and polyclonal RFs are mainly idiotypically and structurally different [10, 11] allowing the study of the structural basis of multireactivity. In the present study both polyclonal and monoclonal RFs reacting with IgG and histones were studied. The data obtained in inhibition assays using IgG, short histone peptides and anti-idiotope monoclonal antibodies led to the localization of particular RF binding sites. Various situations were found depending on the RF studied and the antigen used for testing. These involve distinct, partially overlapping or similar binding sites on the RF variable regions.
Materials and methods
Patients' RFs
Serum polyclonal RFs were purified from seropositive rheumatoid arthritis (RA) patients as previously described [12]. The RFs were eluted from a human IgGsepharose column in acid (A: 0.1 M glycine HCI, pH 2.8) or basic (B: 0.02 M diethylamine, pH 11.2) conditions [ 12]. In both cases, the eluates were rapidly neutralized with 1 M Tris-HCl, pH 7.0, then dialysed against PBS and concentrated in a Micro-Pro Di Con (Bio Molecular Dynamics, Beaverton, OR, USA).
D o u b l e reactivity o f r h e u m a t o i d factors
3
Serum monoclonal RFs were purified from patients suffering from mixed cryoglobulinemia as previously described [3].
Anti-idiotope monoclonal antibodies A 75 and BII-2. I A75 is a monoclonal murine antibody (IgG1) which was raised against a human monoclonal RF (Alt). It was shown to recognize an idiotope (Id) mapped on the Alt K light chain [13]. BII-2.1 is a murine monoclonal antibody (IgG1) which was raised against human polyclonal bireactive RFs and recognizes an idiotope apparently linked to the H3 binding site of the immunizing RFs [ 12].
Synthetic peptides of histones Twenty-seven synthetic peptides covering the entire sequences of calf histones H2A, H2B and H3 as well as two peptides corresponding to the N- and C-terminus domains 1-16 and 204-218 of human spleen H l b variant were used in this study. Their preparation and" purification were previously described [14]. Their final purity, was measured by high-pressure liquid chromatography, as > 85%. Amino acid compositions of peptides were verified using a Pico-Tag analyser (Millipore, USA) and were found to correspond to the expected composition.
E L I S A for rheumatoid factor ( R F ) measurement RF activity was detected by E L I S A using 25 pg/ml heat aggregated human IgG (Sigma, 1 4506) in 0.1 M sodium carbonate buffer (pH 9.5) for coating microtiter plates (Falcon, USA; Ref. 3912). Blocking of remaining sites on the plastic was achieved by incubation for 30 min at 37~ with 10 mg/ml bovine serum albumin in phosphate-buffered saline (PBS) pH 7.4 containing 0.05% Tween 20 (PBS-TB SA). After repeated washings with PBS-T, monoclonal (1 ~tg/ml equivalent RF) or polyclonal (1.5 ~tg/ml equivalent RF) RFs diluted in PBS-T were added for I h at 37~ RF binding was visualized by addition of goat anti-human IgM conjugated to horseradish peroxidase (Jackson, Bar Harbor, ME, USA, Ref. 109.035.043) for 30 min at 37~ The final reaction was detected by incubation with 3,3',5,5'tetramethylbenzidine (TMB, Ref. 22.92836, Janssen Chimica, Beerse, Belgium) in the presence of H202. The reaction was stopped by addition of HCI (final dilution 0.25 M) and OD values were measured at 450 nm.
EL I S A for histones and histone peptides The binding of antibodies to individual histones was measured by E L I S A using 100 ng/ml histone or 1 laM histone peptide in 0.05 M carbonate buffer (pH 9.6) for coating plates. After saturation of plates with 1% BSA as described above and repeated washings with PBS-T, monoclonal and polyclonal RFs (respectively 1 and 1.5 pg/ml) were added. The reaction was visualized as described above for RF measurement. The hexapeptide 130-135 was used after conjugation to ovalbumin using 1% glutaraldehyde.
4
Nadine
Table
et al.
Tuaillon
1. Reactivity with histones of R F IgM elutedfrom the IgG-sepharose column with
acid ( A ) and basic ( B) buffer Reactivity (OD 450 rim) R F (A) Polyclonal human R F
H1
Hag Leh Ste Kre Kra Nor Buh
0.44 -. . . . . . 0.60 -. . . . . . . . .
J
a
c
Ack Bou Dal Swi Sch
.
. . .
H2A
.
.
. . .
. . .
R F (B)
H2B
H3
-.
.
H2A
H2B
H3
0.74
--
0.61
0.62 .
0.73
H4 B
m
--
--
-0.71
-0.82
B
m
. . .
. . .
H1
0.58 -1.02 . . . . . . 0.62 . . . "'----
. -. .
H4
.
. . . ---
D
B
D
Polyclonal RF (1.5 ~tg Ig/ml) were allowed to react with histones (100 ng/ml). OD values ~ 2.00 . . . .
. . . . . . .
H2B 1-25 21-38 36-50 48-63 60-77 75-92 92-110 110-125
m
m
B
m
m
m
H3 1-21 18-32 30-45 40-55 53-70 68-85 83-100 98-112 111-130 130-135
0.40 ----__ __ -__ ~
0.38 -0.40 --w __ -__ --
0.56 ----__ m
-m
--
Polyclonal R F s (1.5 ~tg/ml) were allowed to react w i t h 1 ~M hi s t one peptides. F o r E L I S A , the h e x a p e p tide 130-135 of H 3 was conjugated to B S A u s i n g 1% g l u t a r a l d e h y d e (c oupl i ng mol a r ratio of p e p t i d e / carrier calculated from amino acid analysis =9:1). O D values ~
D o u b l e R e a c t i v i t y o f M o n o c l o n a l and P o l y c l o n a l R h e u m a t o i d Factors for IgG and Histones: M a p p i n g o f B i n d i n g Sites by m e a n s o f H i s t o n e S y n t h e t i c Peptides and Anti-Id Antibodies
Nadine Tuaillon, Thierry Martin,* Anne-Marie Knapp,* Jean-Louis Pasquali* and Sylviane Muller Laboratoire d' Immunochimie, Institut de Biologie Mol~culaire et Cellulaire, Strasbourg and *Laboratoire du Service de M~decine lnterne A, Hopital Central, Strasbourg, France (Received 11 June 1991 and accepted 13 September 1991)
Polyreactive antibodies able to bind various apparently unrelated structures represent a frequent antibody population in autoimmune diseases. In this work, the structural basis of the double reactivity of such autoantibodies was investigated using as models polyclonal and monoclonal human rheumatoid factors (RF) reacting with histones. Both direct ELISA binding and competitive inhibition experiments were performed. A more precise delineation of the histone regions r e c o g n i z e d b y t h e R F s w a s m a d e b y m e a n s o f 27 s y n t h e t i c p e p t i d e s o f these proteins. Anti-idiotope (Id) murine antibodies were used to map the binding sites involved on RF in the interaction with IgG and hist o n e s . A m o n g t h e 13 p o l y c l o n a l a n d s i x m o n o c l o n a l R F s t e s t e d , f o u r a n d t w o r e s p e c t i v e l y w e r e f o u n d to c r o s s - r e a c t w i t h I g G a n d h i s t o n e s . T h e fragments shown to be the most frequently recognized by RFs were l o c a t e d i n r e s i d u e s 1-16 a n d 204-218 o f i l l , 1-20 a n d 65-85 o f H 2 A , a n d 1 21 o f H3. T h e r e s u l t s o b t a i n e d b y c o m p e t i t i v e E L I S A a s s a y s u s i n g I g G , histone peptides and anti-Id monoclonal antibodies led us to confirm and characterize more precisely our previous finding suggesting the existence of topographically distinct binding sites for the different targets recognized by RFs.
Correspondence to: Sylviane Muller, Institut de Biologie Mol6culaire et Cellulaire, CNRS, 15 rue Descartes, 67084 Strasbourg cedex, France. Fax: (33) 88 61 06 80. 1
0896-8411/92/010001 + 14 $03.00/0
9 1992 Academic Press Limited
2
Nadine Tuaillon e t al. Introduction
A number of investigators have demonstrated the existence of an intriguingly high frequency of autoantibodies characterized by extensive 'multispecific' properties. In particular, this kind of cross-reactivity has been described in the case of rheumatoid factors (RFs) [1-3], anti-DNA antibodies which cross-react with phospholipids and cytoskeletal proteins [4-6] and for an anti-poly (ADPribose) monoclonal autoantibody [7]. The first question raised by the crossreactivity phenomenon concerns the definition of this apparent double or 'multi' specificity: do the 'multireactive' autoantibodies react at the level of a unique antibody site with related structures present at the surface of two distinct antigenic complexes or do they interact with two different antigenic structures using two distinct binding sites? The first possibility would be involved in the case of anti-DNA antibody cross-reacting with phospholipids, for example, and is a consequence of molecular mimicry. The second mechanism has been recently demonstrated to occur in the case of a particular RF, lef, reacting with IgG and histone [3]. The second question raised by the cross-reactivity phenomenon is related to its physiological significance. Monoclonal RF, in the same way as numerous multireactive autoantibodies, are frequently directly germ line encoded [8, 9], which could signify that multireactivity has a physiological function. Is this question related to ancestral properties of low affinity immunoglobulinbased immune responses, or is this function associated with the B cell ontogeny by forming a network of self reacting antibodies? In order to answer these questions, it is first necessary to define the structures of the recognized autoepitopes at a molecular level. RFs are particularly suitable for studying these questions. They allow the fine specificity of Ig to be investigated at the molecular level because (1) RFs exist in humans in both monoclonal (benign and malignant lymphoproliferations) and polyclonal (autoimmune diseases) forms; (2) monoclonal RFs and polyclonal RFs are mainly idiotypically and structurally different [10, 11] allowing the study of the structural basis of multireactivity. In the present study both polyclonal and monoclonal RFs reacting with IgG and histones were studied. The data obtained in inhibition assays using IgG, short histone peptides and anti-idiotope monoclonal antibodies led to the localization of particular RF binding sites. Various situations were found depending on the RF studied and the antigen used for testing. These involve distinct, partially overlapping or similar binding sites on the RF variable regions.
Materials and methods
Patients' RFs
Serum polyclonal RFs were purified from seropositive rheumatoid arthritis (RA) patients as previously described [12]. The RFs were eluted from a human IgGsepharose column in acid (A: 0.1 M glycine HCI, pH 2.8) or basic (B: 0.02 M diethylamine, pH 11.2) conditions [ 12]. In both cases, the eluates were rapidly neutralized with 1 M Tris-HCl, pH 7.0, then dialysed against PBS and concentrated in a Micro-Pro Di Con (Bio Molecular Dynamics, Beaverton, OR, USA).
D o u b l e reactivity o f r h e u m a t o i d factors
3
Serum monoclonal RFs were purified from patients suffering from mixed cryoglobulinemia as previously described [3].
Anti-idiotope monoclonal antibodies A 75 and BII-2. I A75 is a monoclonal murine antibody (IgG1) which was raised against a human monoclonal RF (Alt). It was shown to recognize an idiotope (Id) mapped on the Alt K light chain [13]. BII-2.1 is a murine monoclonal antibody (IgG1) which was raised against human polyclonal bireactive RFs and recognizes an idiotope apparently linked to the H3 binding site of the immunizing RFs [ 12].
Synthetic peptides of histones Twenty-seven synthetic peptides covering the entire sequences of calf histones H2A, H2B and H3 as well as two peptides corresponding to the N- and C-terminus domains 1-16 and 204-218 of human spleen H l b variant were used in this study. Their preparation and" purification were previously described [14]. Their final purity, was measured by high-pressure liquid chromatography, as > 85%. Amino acid compositions of peptides were verified using a Pico-Tag analyser (Millipore, USA) and were found to correspond to the expected composition.
E L I S A for rheumatoid factor ( R F ) measurement RF activity was detected by E L I S A using 25 pg/ml heat aggregated human IgG (Sigma, 1 4506) in 0.1 M sodium carbonate buffer (pH 9.5) for coating microtiter plates (Falcon, USA; Ref. 3912). Blocking of remaining sites on the plastic was achieved by incubation for 30 min at 37~ with 10 mg/ml bovine serum albumin in phosphate-buffered saline (PBS) pH 7.4 containing 0.05% Tween 20 (PBS-TB SA). After repeated washings with PBS-T, monoclonal (1 ~tg/ml equivalent RF) or polyclonal (1.5 ~tg/ml equivalent RF) RFs diluted in PBS-T were added for I h at 37~ RF binding was visualized by addition of goat anti-human IgM conjugated to horseradish peroxidase (Jackson, Bar Harbor, ME, USA, Ref. 109.035.043) for 30 min at 37~ The final reaction was detected by incubation with 3,3',5,5'tetramethylbenzidine (TMB, Ref. 22.92836, Janssen Chimica, Beerse, Belgium) in the presence of H202. The reaction was stopped by addition of HCI (final dilution 0.25 M) and OD values were measured at 450 nm.
EL I S A for histones and histone peptides The binding of antibodies to individual histones was measured by E L I S A using 100 ng/ml histone or 1 laM histone peptide in 0.05 M carbonate buffer (pH 9.6) for coating plates. After saturation of plates with 1% BSA as described above and repeated washings with PBS-T, monoclonal and polyclonal RFs (respectively 1 and 1.5 pg/ml) were added. The reaction was visualized as described above for RF measurement. The hexapeptide 130-135 was used after conjugation to ovalbumin using 1% glutaraldehyde.
4
Nadine
Table
et al.
Tuaillon
1. Reactivity with histones of R F IgM elutedfrom the IgG-sepharose column with
acid ( A ) and basic ( B) buffer Reactivity (OD 450 rim) R F (A) Polyclonal human R F
H1
Hag Leh Ste Kre Kra Nor Buh
0.44 -. . . . . . 0.60 -. . . . . . . . .
J
a
c
Ack Bou Dal Swi Sch
.
. . .
H2A
.
.
. . .
. . .
R F (B)
H2B
H3
-.
.
H2A
H2B
H3
0.74
--
0.61
0.62 .
0.73
H4 B
m
--
--
-0.71
-0.82
B
m
. . .
. . .
H1
0.58 -1.02 . . . . . . 0.62 . . . "'----
. -. .
H4
.
. . . ---
D
B
D
Polyclonal RF (1.5 ~tg Ig/ml) were allowed to react with histones (100 ng/ml). OD values ~ 2.00 . . . .
. . . . . . .
H2B 1-25 21-38 36-50 48-63 60-77 75-92 92-110 110-125
m
m
B
m
m
m
H3 1-21 18-32 30-45 40-55 53-70 68-85 83-100 98-112 111-130 130-135
0.40 ----__ __ -__ ~
0.38 -0.40 --w __ -__ --
0.56 ----__ m
-m
--
Polyclonal R F s (1.5 ~tg/ml) were allowed to react w i t h 1 ~M hi s t one peptides. F o r E L I S A , the h e x a p e p tide 130-135 of H 3 was conjugated to B S A u s i n g 1% g l u t a r a l d e h y d e (c oupl i ng mol a r ratio of p e p t i d e / carrier calculated from amino acid analysis =9:1). O D values ~
