Autoantibodies, lupus and the science of sabotage
A. Rahman
Centre for Rheumatology, Department of Medicine, University College London, London, UK
Correspondence to: Centre for Rheumatology, Arthur Stanley House, 4050 Tottenham Street, London W1T 4NJ, UK. E-mail: anisur.rahman{at}ucl.ac.uk
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Abstract
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Anti-double-stranded DNA antibodies (anti-dsDNA) and antiphospholipid antibodies (APL) are important in the pathogenesis of systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS) respectively. Not all anti-dsDNA or APL antibodies can cause clinical effects. Those that are particularly likely to cause tissue damage tend to be of IgG isotype and to possess particular binding properties. Rigorous statistical analysis of published sequences of human monoclonal anti-DNA and APL antibodies showed that IgG antibodies with binding properties characteristic of pathogenicity tend to have multiple somatic mutations in their variable regions. The distribution of these mutations suggests that they have been selected by antigen. This leads to accumulation of certain residues at the antigen-binding sites of these antibodies. Arginine residues are especially important. A computer-generated model of the pathogenic human monoclonal anti-DNA antibody B3 predicted that arginines in the heavy and light chain complementarity-determining regions (CDRs) would interact with dsDNA. We expressed cloned sequences encoding the B3 heavy and light chains in vitro to produce whole IgG. The cloned sequences of the heavy and light chains were manipulated to express a range of variant IgG antibodies. Binding assays on the expressed antibodies showed that altering specific arginine residues reduced binding to dsDNA in a way consistent with computer generated structural models. Changing the pattern of somatic mutations in the light chain altered binding to both dsDNA and histones, but in different ways. A single arginine-to-serine mutation in light-chain CDR1 of B3 reduced binding to both those antigens and may also have reduced the pathogenicity of the expressed antibodies in severe combined immunodeficiency (SCID) mice. Monoclonal human APL were expressed using the same system. Nineteen different heavylight combinations were expressed. The ability to bind cardiolipin correlated well with the presence of exposed arginine residues in the heavy- and light-chain CDRs. The heavy chain of the pathogenic APL antibody IS4 contains four exposed arginines in CDR3. The results of mutagenesis studies suggested that two of these promote binding to cardiolipin whereas the other two have no such effect.
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Introduction
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Imagine a walled city of the Middle Ages. The conquest of such cities was one of the great challenges of mediaeval warfare. Besides the thick walls and fortified gates, any attackers would have to contend with heavily armed defending forces. Nevertheless, such cities could be conquered, and the prizes for such success were great. Amongst the primary methods used to accomplish these conquests were frontal attack, siege and sabotage.
In this essay I propose to consider the body as a walled city, and to show how autoantibody-mediated disease can be considered a form of conquest by sabotage. In particular, I will consider the case of systemic lupus erythematosus (SLE), and show how the autoantibody saboteurs in this disease can be identified and distinguished from other antibody molecules.
If the body were a city, the external defences would be represented by the skin and mucosa, whereas the armed defenders would be the effector cells and molecules of the immune system, both innate and acquired. In major illness, these defences fail and sickness ensues. An overwhelming sepsis is an example of frontal attack, whereas starvation and malnutrition are clearly akin to siege. Sabotage is harder to define.
The secret of a successful saboteur is to be able to pass unnoticed through the city until he reaches the site of his act of sabotage. In the human body, therefore, diseases resembling sabotage would be those in which the agent of damage is not recognizably different from normal constituents of the body. Evading the defence mechanisms that would stop recognizably foreign pathogens, such as bacteria or viruses, these agents of damage can reach vital organs and disable them.
Autoantibodies fit this description. Structurally, they appear very similar to other antibody molecules but differ from them in their ability to bind to antigens within the body itself. Although not all autoantibodies cause diseases, some of them are clearly associated with tissue damage in specific conditions.
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Anti-DNA antibodies in SLE
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The presence of autoantibodies is a cardinal feature of SLE, and two of the 11 criteria for classification of the disease refer to the detection of such antibodies [1]. Although a wide range of different antibodies have been reported to occur in the blood of patients with SLE, very few of these occur in more than 10% of patients with the disease [2]. In even fewer cases is there good evidence that particular autoantibodies are actually involved in causing clinical effects. The work described in this essay concentrates on two types of autoantibody, commonly found in SLE, for which pathogenetic roles are suggested by the available evidence. These are antibodies to double-stranded DNA (anti-dsDNA) and phospholipid (antiphospholipid antibodies, APL).
Anti-dsDNA antibodies occur in 5070% of patients with SLE and are almost specific for this disease [3]. They are very rarely found in healthy people, in patients with other diseases, or in the relatives of patients with SLE [4]. A number of studies have shown that levels of anti-dsDNA antibodies tend to rise during flares of disease activity in SLE, particularly in lupus nephritis [5, 6]. Renal biopsies from patients with SLE contain deposits of anti-dsDNA antibodies [7, 8]. Furthermore, in vivo studies have shown that some anti-dsDNA antibodies can be deposited in the kidneys of mice or rats to cause proteinuria and, in some cases, histological changes very similar to those of lupus nephritis [912].
However, not all anti-dsDNA antibodies are pathogenic. Some patients with SLE have persistently high levels of anti-dsDNA in their blood and yet remain well, with no evidence of disease activity. It was recently reported that stored blood from American soldiers with SLE contained anti-dsDNA and other autoantibodies up to 9.4 yr before they developed any symptoms of the disease [13]. In animal studies, not all the infused anti-dsDNA antibodies cause pathological effects. Some have no effect on the animals at all, even though their ability to bind dsDNA is equal to that of the more pathogenic antibodies [912].
This raises an important question. How can we distinguish potentially pathogenic autoantibodies from antibodies that are likely to be harmless? In other words, how does one recognize a potential saboteur from the innocent bystanders in the vicinity? Clearly, saboteurs must be recognizable if you know what you are looking for. A large spanner or a quantity of high explosive would be important clues. In the case of pathogenic autoantibodies the clues are more subtle, but it is equally important to discover them. This is because if we know what distinguishes pathogenic autoantibodies from other antibody molecules we may be able to deduce two important things.
Firstly, what caused these antibodies to develop in such a way that they became pathogenic? Secondly, what properties of these antibodies enable them to cause tissue damage in a way that other antibodies cannot emulate? If we can answer the first question, we may be able to develop ways of preventing the production of the pathogenic autoantibodies. If we can answer the second, we may be able to find ways of preventing the antibodies from causing disease.
Fortunately, there are some isotype and binding properties that have proved useful in distinguishing pathogenic from non-pathogenic anti-dsDNA antibodies. Both clinical and murine studies show that levels of immunoglobulin (Ig) G antibodies that bind dsDNA are more closely related to disease activity and tissue damage than IgM or anti-single-stranded DNA (ssDNA) antibodies [3, 14]. Two separate groups have shown that pathogenic murine anti-dsDNA antibodies can be distinguished from non-pathogenic antibodies by their ability to bind a 100 kDa protein in glomerular lysates [10, 15]. This protein is
-actinin, which plays an important role in maintaining cytoskeletal structure and function in renal podocytes [16]. We have recently shown that affinity-purified polyclonal anti-DNA antibodies from patients with active renal lupus had higher anti-
-actinin activity than similar anti-dsDNA samples from patients with lupus, but no renal activity [17].
The identification of isotype and binding properties which correlate with pathogenicity enables us to select monoclonal antibodies with these properties for further study. This allows us to identify the molecular features which are characteristic of those antibodies, and which may therefore be important in determining the potential of a particular antibody molecule to become a pathogenic saboteur.
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APL in the antiphospholipid antibody syndrome
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The same line of reasoning can be applied to the study of pathogenic APL. APL occur in 15% of healthy people, and can occur in certain infections or malignancies, or in association with certain drugs [18]. In these people, however, the APL do not cause the features of arterial or venous thrombosis, recurrent fetal loss and thrombocytopenia which are characteristic of the antiphospholipid antibody syndrome (APS). APS can occur in the absence of any other autoimmune condition (primary APS) or in the presence of another syndrome, such as SLE (secondary APS). Approximately 2530% of patients with SLE have blood APL or lupus anticoagulant and the occurrence of thrombosis in such patients is well documented [19, 20].
As in the case of anti-dsDNA antibodies in SLE, there is compelling evidence to support the contention that APL play a role in causing the clinical effects seen in APS. High levels of APL, especially IgG APL, are associated with increased risk of developing thrombosis [21]. In murine models, the administration of polyclonal or monoclonal APL has been shown to cause increased and prolonged thrombus formation after pinching the femoral vein [22], increased fetal loss [23], and activation of endothelial cells [24].
Just as for anti-DNA antibodies, however, not all APL are equally pathogenic and those of greater pathogenicity can be distinguished by particular isotype and binding properties. APL found in people without APS bind neutral and anionic phospholipids in the absence of any serum-derived cofactors. Conversely, APL from people who have APS usually bind only anionic phospholipids and require the presence of protein cofactors [25]. A number of phospholipid-binding proteins can act as cofactors, but the one for which the most evidence of clinical importance has accrued is ß2-glycoprotein I (ß2GPI) [26, 27]. In fact, it has been shown that APL from patients with APS will bind ß2GPI in the absence of phospholipids, under appropriate conditions [28].
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The mark of a saboteur: which molecular features might distinguish pathogenic from non-pathogenic autoantibodies?
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The molecular properties of an antibody molecule are implicit in its amino acid sequence. However, only a part of the antibody sequence varies between one antibody molecule and another. An Ig molecule is composed of two heavy chains and two light chains. Only the N-terminal half of the light chain and the N-terminal quarter of the heavy chain vary in sequence between different antibody molecules. These regions are therefore called the heavy-chain variable region (VH) and the light-chain variable region (VL). It is the sequences of the variable regions that encode the antibody-binding arms of the antibody molecule, so it is in these sequences that we may find the structural features that distinguish pathogenic from non-pathogenic antibodies.
The VH region is encoded by a composite sequence made up of three gene segments, VH, DH and JH. These segments are separate in most cells of the body. The human genome contains about 50 functional VH genes, 30 DH genes and six JH genes [29]. In an antibody-producing B lymphocyte, one VH gene, one DH gene and one JH gene are brought together to produce the sequence encoding the variable region of the heavy chain secreted by the cell. In a large population of human B cells, however, not all VH genes are equally likely to be expressed. Some are far more popular than others, in that they are much more likely to be rearranged next to DH and JH genes in a way that allows the production of a functional heavy chain [30, 31]. This means that, at the level of the whole immune system, there are more antibodies encoded by some heavy-chain genes than by others.
There are two different types of light chain, kappa and lambda, and the variable regions of these chains are designated V
and V
respectively. Just as in the heavy chains, V
and V
are encoded by composite sequences. These are produced by bringing together just two segments, V
and J
(or V
and J
), and some V
and V
genes are used more frequently than others [32, 33].
Consequently, one possible explanation for the fact that pathogenic antibodies are made in patients with conditions such as SLE, but not in other people, could be that these patients have different patterns for preferential use of the heavy- and light-chain genes. By rearranging and expressing genes that are rarely expressed in healthy people, these patients might be able to produce antibodies with unusual binding and pathogenic properties.
After the heavy- and light-chain genes have been rearranged, the B cell begins to produce antibody, and a process of somatic hypermutation is triggered [34]. This leads to a high frequency of somatic mutations in the part of the B cell DNA that encodes the expressed heavy- and light-chain variable regions, but not in any other genes [34]. This is an important process, because it enables focusing and fine-tuning of an antibody response so that it becomes more specifically targeted to recognize a particular antigen.
As a B cell divides, its descendants accumulate a range of different somatic mutations such that, after several generations, a number of different (though closely related) variable-region sequences are present within the clone. Since the variable regions are responsible for binding to antigen, antibodies with different variable region sequences are likely to differ in their antigen-binding properties. As long as a particular antigen is present, those antibodies which bind the antigen most strongly will be at an advantage. B cells bearing such antibodies will bind more antigen, divide more prolifically and come to dominate the clone. Somatic mutations that enhance binding to antigens will therefore tend to accumulate [35].
This antigen-driven accumulation of somatic mutations is particularly characteristic of IgG antibodies, because class switching and somatic hypermutation tend to occur at similar time periods in B-cell development. Since IgG anti-dsDNA antibodies and IgG APL are particularly closely related to disease activity in SLE and APS respectively, it is plausible that particular patterns of mutation in these IgG antibodies are responsible for their pathogenic effects and arise due to the presence of particular antigens. Under this paradigm, pathogenic antibodies would arise in patients with SLE not because of unusual patterns of gene usage but because the process of antigen-driven somatic hypermutation acts upon the rearranged heavy- and light-chain genes to produce unusual sequence motifs within their variable regions.
To decide whether gene usage or somatic hypermutation plays the major role in the production of human anti-dsDNA antibodies and APL, it is necessary to analyse the sequences of large numbers of such antibodies. Monoclonal antibodies are particularly useful for this, since both their sequences and functional properties may be readily determined. The initial focus of my research was on the sequence analysis of monoclonal human anti-DNA antibodies and APL.
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The systematic analysis of human autoantibody sequences
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I began my research at the Centre for Rheumatology, University College London, at a time when several human monoclonal anti-DNA and APL antibodies had been produced there. These had been produced by making hybridomas from the peripheral blood lymphocytes or splenocytes of patients with SLE. Due to the work of a number of people, especially Richard Watts [36], Michael Ehrenstein [37, 38], Chelliah Ravirajan [12] and Sanj Menon [39], the isotype and binding properties of these antibodies had been characterized, and some of them had been administered to severe combined immunodeficiency (SCID) mice in order to determine their potential for causing pathogenic effects [11, 12].
The analysis of the VH and VL sequences of these antibodies by Celia Longhurst and myself showed that the IgG antibodies tended to carry more somatic mutations than IgM and that the patterns of these mutations were suggestive of antigen-driven somatic mutation [12, 36, 38, 39]. In particular, replacement mutationswhich alter amino acid sequencetended to accumulate in the complementarity-determining regions (CDRs), but not in the intervening framework regions. Silent mutations, which do not alter the amino acid sequence, did not accumulate in the CDRs. This pattern of asymmetrical distribution of replacement mutations is exactly that which had been proposed as being strongly suggestive of an antigen-driven process [35]. Crystallographic studies have shown that the CDRs encode the bulk of the antigen-binding sites of antibody molecules [40], so it is logical that replacement mutations in the CDRs should particularly affect binding to antigens.
To some extent, the conclusions derived from our antibody sequences were in agreement with those previously published by other groups. Over the last few years, the entire human variable region gene repertoire has been defined [29, 41, 42] and accurate statistical methods for analysing patterns of mutations have been developed [43], so that systematic analysis of the sequences of all published human monoclonal anti-DNA and APL antibodies has become possible. We have recently completed these analyses [44, 45].
We found 66 published human anti-DNA and 36 published human APL sequences in the literature. We matched each sequence to the germline gene used to encode it and thus identified all the sites of somatic mutation within these sequences. We determined the distribution of replacement and silent mutations within each sequence. We used the multinomial method of Lossos et al. [43] to analyse these distributions statistically and computed P values denoting the probability that each sequence arose as a result of antigen-driven selection of mutations.
By comparing the distribution of heavy- and light-chain gene usage amongst these monoclonal antibodies with the expected gene usage in healthy adults [3033] and in patients with SLE [31, 46, 47], we showed that there was no good evidence for preferential utilization of any genes to encode anti-DNA antibodies or APL in humans [44, 45]. This was a very different conclusion from that previously reported for monoclonal anti-DNA antibodies derived from various murine models of SLE. Radic and Weigert [48] showed that these murine antibodies tend to be encoded by particular mouse VH and V
genes and postulated that these genes might encode structures particularly suited to the formation of a DNA binding site. We found no strong evidence that the same could be said for any human VH, V
or V
genes.
Radic and Weigert also showed that murine IgG anti-DNA antibodies tended to carry more mutations than IgM anti-DNA and that antigen-driven selection of these mutations was partially responsible for an observed high frequency of the amino acids arginine (Arg), asparagine (Asn) and lysine (Lys) in the CDRs [48]. It was postulated that these amino acids, when present at the antigen-binding site, enhance binding to DNA. Arg and Lys are positively charged, and might therefore form charge interactions with the negatively charged DNA molecule. Asn is uncharged but could interact with DNA by either donating or accepting hydrogen bonds. Although there has been no large scale analysis of murine monoclonal APL sequences, some authors have suggested that Arg residues might also play a role in binding to negatively charged phospholipids [49].
Our analysis of human monoclonal anti-DNA and APL antibodies showed that IgG or IgA antibodies were far more likely to carry multiple somatic mutations than IgM antibodies. Statistical analysis suggested that antigen-driven somatic mutation had occurred in 13/20 IgG and IgA anti-dsDNA antibodies [44] and 9/14 IgG APL antibodies [45].
There was accumulation of Arg, Lys and Asn residues at the contact sites of IgG and IgA anti-dsDNA antibodies and IgG APL and many of these residues were created by somatic mutation away from the germline gene sequence. The contact sites are those positions in the sequence predicted to be most likely to make contact with antigens on the basis of information from known crystal structures [50]. Most contact sites are in the CDRs.
The systematic analysis of human anti-DNA and APL sequences therefore suggests that somatic hypermutation, rather than altered gene usage, is responsible for the production of pathogenic IgG antibodies in SLE and APS. To an extent, these antibodies may be distinguished from non-pathogenic antibodies by their high content of Arg, Lys and Asn residues at contact sites.
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The delineation of antibody binding sites by computer modelling
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The analysis and comparison of multiple antibody sequences was useful in deriving a general hypothesis that Arg, Lys and Asn residues may be important in binding to DNA or phospholipids. The mere presence of these amino acids at contact sites, however, is not enough to confer DNA or phospholipid binding potential upon an antibody molecule. The actual positions occupied by these residues are critical.
Radic and Weigert [48] had used computer modelling programs to show how Arg, Asn and Lys residues at particular points in the sequences of murine anti-DNA antibodies might interact with the DNA double helix. Kalsi et al. [51] published computer models of two human anti-DNA antibodies that had been produced in our unit. The most interesting model was of the antibody B3. This is an IgG antibody, derived from a patient with SLE, which binds to ssDNA and dsDNA [37, 38]. When administered to SCID mice, B3 bound to a number of organs, including the kidney, and these mice developed proteinuria [11]. More recently, we found that B3 also binds
-actinin [17].
The light and heavy chains of B3 are encoded by genes which are among the most commonly expressed VH and V
genes in healthy people and in patients with SLE [30, 31, 33, 47]. This suggests that the molecular properties of B3 may be relevant to those of antibodies produced in many patients with this disease. Both the VH and V
sequences of B3 are heavily mutated and statistical analysis suggests that these mutations have been selected by antigen [38]. Mutations at a number of sites, particularly in V
CDR1, create new Arg residues.
The molecular model of the B3dsDNA complex, originally developed by Kalsi et al. [51] and modified by Sylvia Nagl, is shown in Fig. 1. The DNA double helix binds in a cleft on the surface of the molecule. Three Arg residues at the margins of the cleft stabilize this binding. These Arg residues (shown in yellow in Fig. 1) are at position 27a in V
CDR1, position 54 in V
CDR2 and position 53 in VHCDR2. Two of these arginines (V
27a and VH53) are derived from somatic mutations.
This model gives us an image of the fine structure of a human antibodydsDNA interaction. This image is much more exact than the general observation, based on sequence analysis, that some Arg, Asn and Lys residues are likely to be important. In order to test this model of the B3dsDNA interaction experimentally, it was necessary to establish a system for expression of cloned antibody sequences in vitro. The cloned sequences could then be modified so that the effects of point mutations or alterations in the pattern of somatic mutations in B3 could be determined in assays of binding to antigen.
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The expression of whole human IgG anti-DNA antibodies in vitro
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The expression system that we used was initially developed by the Medical Research Council Collaborative Unit, Mill Hill, London [52]. The expression vectors enable the cloned antibody VH and VL sequences to be ligated 3' to an immunoglobulin leader sequence and 5' to a block of cDNA encoding the entire human C
, C
or C
sequence. There are three separate vectors for heavy, kappa and lambda sequences respectively.
In the initial experiment, the VH and VL sequences of B3 and of another human monoclonal anti-DNA antibody, WRI176, were cloned into these expression vectors [53]. Simultaneous transfection of a heavy-chain expression vector and a light-chain expression vector into COS-7 monkey kidney cells caused these cells to secrete whole IgG antibody. The supernatants of the transfected cells were harvested and tested for antigen-binding activity using enzyme-linked immunosorbent assays (ELISA).
Using the four cloned sequences, four different heavylight chain combinations were expressed. The combination B3VHB3V
bound to both ssDNA and dsDNA in ELISA. However, replacement of either the heavy chain with WRI176VH or the light chain with WRI176VL completely removed the ability to bind DNA [53]. Thus, both the heavy and light chains of B3 are important in binding to DNA, as suggested by the model [51].
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Importance of somatic mutations and arginine residues in the B3 lambda and heavy chains in binding to dsDNA
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In order to investigate the roles played by somatic mutations and specific arginine residues in binding to dsDNA, we then used the COS-7 cell expression system to pair the B3 heavy chain with five different light chains, which differed only in their patterns of somatic mutations.
Three of these light chains were B3V
, 33H11V
and UK-4V
. B3, 33H11 and UK-4 are all human IgG
monoclonal antibodies, produced from peripheral blood lymphocytes of three different patients [37, 39, 54]. 33H11, kindly donated by Thomas Winkler and Joachim Kalden (Erlangen, Germany), is an anti-DNA antibody, whereas UK4 is an APL, which does not bind DNA. B3V
, 33H11V
and UK-4V
are very similar in sequence. They are all encoded by the human V
gene 2a2 and differ only in the sites and nature of the somatic mutations. Since they were all paired with the same heavy chain (B3VH), any difference in the DNA binding properties of these heavylight chain combinations must arise from the different patterns of somatic mutation in the light chains.
The other two light chains paired with B3VH in this experiment were produced by site-directed point mutagenesis of the cloned B3V
sequence. Light-chain B3V
a was exactly the same as B3V
except that the arginine at position 27a in CDR1 had been reverted to a serine (Ser). Serine was chosen because it is uncharged and because position 27a of the unmutated germline gene 2a2 encodes serine. Light-chain B3V
b also had this Arg-to-Ser mutation, but also had a glycine (Gly)-to-serine mutation at position 29, a little further on in CDR1.
Figure 2 shows the results of this experiment [55]. The graph plots binding to dsDNA (measured by ELISA) against concentration of antibody. The wild-type combination B3VHB3V
showed the strongest binding to dsDNA. The single point mutation in B3VHB3V
a reduced binding to dsDNA significantly, and the second mutation Gly29Ser in B3VHB3V
b reduced it even further.

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FIG. 2. Results of anti-dsDNA ELISA for B3 heavy chain paired with five different light chains derived from lambda gene 2a2. Binding to dsDNA is plotted as optical density (measured by ELISA) against concentration of whole IgG antibody in the supernatant tested. In each experiment, a negative control sample of supernatant from COS-7 cells, which had been electroporated in the absence of plasmid DNA, was also tested and was found to contain no IgG and no anti-dsDNA activity. Reprinted from Rahman et al. [55], © 2001, with permission from Elsevier Science.
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The combination B3VHUK-4V
did not bind dsDNA at all, even when tested at antibody concentrations much higher than those which gave detectable binding for all the other combinations. Conversely, B3VH33H11V
bound dsDNA well. Only the wild-type B3 molecule was a better binder.
These results showed that Arg27a in B3V
does play a role in binding to dsDNA and that B3VH will interact with some, but not all, patterns of somatic mutation in the V
gene 2a2 to produce a DNA binding site. To explain the results in more detail, we modelled these heavylight chain combinations (Figs. 35
).
Figure 3 shows the model of B3VHUK-4V
. The antigen-binding cleft characteristic of wild-type B3 has been blocked by a bulky structure arising from UK-4V
CDR3. An arginine residue (Arg 94) is at the centre of this blocking structure. This shows that the actual site occupied by Arg residues is critical to whether they aid or inhibit binding to dsDNA. This message is underlined by the model of B3VH33H11V
(Fig. 4). Binding to dsDNA is enhanced by an interaction with an arginine at position 92 in 33H11V
CDR3, which compensates for the lack of an Arg 27a.
Figure 5 shows a magnified view of the interaction between position 27a and dsDNA in B3V
and B3V
a. Arg27a, shown in blue in the upper part of the figure, can form a charge interaction with the DNA molecule. Ser27a, shown in green in the lower part of the figure, cannot form this interaction. This explains the significant reduction in binding to dsDNA caused by this single amino acid change. The additional change, Gly29Ser in B3V
b, causes an additional adverse steric interaction between the antigen and antibody.
When the same system was used to express a variant of B3VH in which Arg53 was mutated to serine, this single change abolished binding to dsDNA, regardless of which light chain was paired with the mutagenized heavy chain. Like Arg 27a in the light chain, Arg53 in B3VH was predicted by the computer model [51] to make important contacts with dsDNA. The results of the expression experiments were consistent with these predictions.
These expression and modelling experiments [53, 55] had enabled us to localize particular regions of sequence as being markers for the ability to bind DNA. This ability was critically sensitive to the positions of certain arginine residues.
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The differential effects of somatic mutations and arginine residues on binding to DNA and histones
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Binding to DNA is not an absolute predictor of pathogenicity [56]. On the basis of data from experiments in murine models, a number of authors have suggested that the pathogenicity of anti-DNA antibodies depends not only on binding to DNA but also on the ability to cross-react with other antigens [10, 15, 57]. A particularly interesting hypothesis is that binding to nucleosomes is crucial [57, 58]. Nucleosomes are complexes of DNA and histones, which are released from apoptotic cells. Clearance of this apoptotic material is retarded in patients with SLE [59], suggesting that nucleosomes may be more readily available as antigens in these people than in others. Nucleosomes are therefore potentially important both as the antigens to drive the production of pathogenic antibodies in SLE and as antigens in the target tissues. In these tissues, notably the kidney, the antibodynucleosome interaction could be involved in causing tissue damage [57, 58].
On this basis, pathogenic anti-DNA antibodies might be distinguished from other antibodies by their content of some somatic mutations which enhance binding to DNA and others which favour binding to histones. In the next series of experiments, we used the expression system to try to distinguish these features.
Ten different light chains were paired with the B3 heavy chain. The light chains included those tested in the previous experiment, as well as chimeric V
sequences that were generated from them by genetic manipulation. Using appropriate restriction digests, these chimeric sequences were designed to contain CDR1 from one sequence with CDR2 and 3 from a different V
sequence. The purpose of this was to isolate the effects of particular CDRs. The results of these experiments [60] are summarized in Table 1.
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TABLE 1. Differential effects of CDR exchange and mutagenesis in the light chain on binding to dsDNA and histones
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The presence of UK-4 V
CDR3 always blocked binding to either dsDNA or histones. This was consistent with the previous model (Fig. 3), which suggested a dominant blocking effect exerted by this CDR. Conversely, when separated from UK-4V
CDR3, the CDR1 region of UK-4V
does not block binding to either antigen.
If Arg27a in B3V
CDR1 and Arg 92 in 33H11V
CDR3 both enhance binding to DNA, as predicted by the models (Figs 1 and 4), one would expect the chimeric light chain containing both these CDRs to be a particularly good binder to DNA. This was indeed the case.
Table 1 shows differences between the way in which these heavylight chain combinations bound to dsDNA and the way they bound histones. Arg27a in B3V
does play some role in binding to histones, because point mutation at this site in B3V
a reduced this binding. However, B3V
CDR2 and CDR3 are more important than B3V
CDR1 in binding to histones. Somatic mutations in B3V
CDR2 and CDR3 probably play an important role in binding to histones, because replacement of these CDRs by the corresponding regions from UK-4 or 33H11 has a marked adverse effect on this binding.
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The effect of a point mutation in B3V on pathogenicity in SCID mice
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The expression experiments had enabled us to delineate sequence features which distinguished antibodies with the ability to bind DNA and histones. The next step was to seek some evidence that these sequence and binding properties were relevant to the pathogenicity of the antibodies.
In order to do this, we used the expression vectors described previously to create two stably transfected Chinese hamster ovary (CHO) cell lines. One line secreted the wild-type B3 antibody whereas the other secreted B3 with the single Arg27aSer point mutation in V
CDR1.
Separate groups of SCID mice were implanted with these lines and control groups were implanted with CHO cells secreting no antibody or with no cells at all. These experiments were carried out twice, and on both occasions the groups of mice containing the secreted antibodies suffered major adverse effects compared with the control groups. Furthermore, there was a difference between the groups which received cells secreting wild-type and mutated antibodies [61].
The mice that received cells secreting wild-type B3 developed significant proteinuria and died about 25 days after the CHO cells were implanted (mean day of death was 26). Mice that received cells with the mutated antibody sequence lived longer (mean day of death was 39) and developed significantly less proteinuria. Mice that received non-secreting CHO cells lived up to 50 days and most of them developed little or no proteinuria [61]. These results suggest that the Arg27aSer mutation may truly have an effect on pathogenicity, but must be interpreted with caution since we were unable to demonstrate deposition of the antibody in the kidneys of any of the mice.
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The expression of monoclonal human APL
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We then applied these techniques to the study of human monoclonal APL. We planned to study APL which were likely to be relevant to pathogenicity in APS. Dr Pojen Chen and Dr Reginald Chukwuocha, from the University of California, Los Angeles, donated the antibodies IS4 and CL24, which are IgG APL derived from patients with APS and which show ß2GPI-dependent binding to cardiolipin (CL) [62]. Both antibodies are thrombogenic in vivo in a murine pinch-induced thrombosis model and both activate endothelial cells in vitro [24]. IS4V
is derived from germline gene 2a2, but has a different pattern of somatic mutations from the light chains studied previously. It is not rich in Arg residues, but IS4VH contains five arginines in CDR3 [63].
Using the COS-7 cell expression system, we expressed a panel of 19 different heavylight chain combinations and tested the supernatants for binding to CL. The results are shown in Table 2. IS4VH exerts a dominant effect on binding to cardiolipin, since it bound this antigen in combination with four out of five light chains tested, three of which were derived from the germline gene 2a2 [64]. Only one heavylight combination lacking IS4VH bound CL in this experiment.
The nature of the light chain paired with IS4VH is important in determining the strength of binding to cardiolipin. The strongest binding was obtained when this heavy chain was combined with B3V
or CL24V
. The computer models shown in Fig. 6a and b show that both these light chains contain multiple exposed arginine residues, whereas this was not the case for the wild-type IS4V
sequence (Fig. 6d). In fact, the combination IS4VH IS4V
binds CL considerably less well than either IS4VH B3V
or IS4VH CL24V
.

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FIG. 6. Computer models of light and heavy chains of expressed antiphospholipid antibodies, showing surface-exposed Arg residues. (a) B3 light chain in spacefill mode. Secondary structure of the heavy chain is shown in light blue. CDR1 is shown in yellow, CDR2 in orange and CDR3 in red. (b) CL24 light chain in secondary structure mode. (c) IS4 heavy chain in spacefill mode. CDR1 is shown in yellow, CDR2 in orange and CDR3 in red. (d) IS4 light chain in secondary structure mode. In all four figures, surface-exposed Arg residues are shown in dark blue. The models were made by Dr Sylvia Nagl. Reprinted from Giles et al. [64], © 2003, with permission from Elsevier.
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The computer model of IS4VH is shown in Fig. 6c. Four of the five arginines in CDR3 are surface-exposed, and might therefore be involved in binding to CL. To test this hypothesis, we used site-directed mutagenesis to produce and express variants of IS4VH in which one or more of these arginines were changed to serine [65]. The results are shown in Fig. 7. In combination with B3V
, wild-type IS4VH binds CL well. This binding is completely abolished when all four arginines at positions 96, 97, 100 and 100 g are changed to serine. These four arginines, however, do not have equal effects. Both Arg96 and Arg97 can be changed to serine without any reduction in binding to CL, but changing either Arg100 or Arg100 g reduces binding by about 50% [65].

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FIG. 7. Effect of point mutations from Arg to Ser in IS4VH CDR3 on binding to cardiolipin. The graph shows cardiolipin binding of IgG in COS-7 cell supernatants containing wild-type or mutant forms of IS4 heavy chain expressed with wild-type B3 or IS4 light chains. Binding to CL was only seen in combination with B3 light chain. The IS4VH mutants VHi, VHii, VHiii and VHiv contain single Arg-to-Ser point mutations at positions 96, 97, 100 and 100 g respectively, whilst VHi and VHii contain Arg-to-Ser at positions 96 and 97, and VHx has an Arg-to-Ser mutation at all four positions. Binding to cardiolipin is plotted as optical density (measured by ELISA) against concentration of whole IgG antibody in the supernatant tested. In each experiment, a negative control sample of supernatant from COS-7 cells, which had been electroporated in the absence of plasmid DNA, was also tested and was found to contain no IgG and no anti-cardiolipin activity.
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We have therefore shown that this expression system provides a powerful method of disentangling the influences of various sequence motifs on binding to three different antigens. The presence of somatic mutations in the light chain is important in binding to dsDNA, histones and cardiolipin. Arginines at specific positions in the heavy and light chains are particularly important in binding to dsDNA or cardiolipin. This overlap between the structural properties of anti-dsDNA antibodies and APL is interesting, since there is evidence that both may arise due to the presence of immunogenic apoptotic debris. Apoptotic blebs, unlike intact cells, may present nuclear antigens and anionic phospholipids on their surfaces. Radic and colleagues have shown that the presence of particular arginine residues in the murine monoclonal antibody 3H9 enhances binding to DNA, CL and apoptotic material [49, 66]. Our studies in human antibodies have led to similar findings concerning the importance of arginines in anti-dsDNA antibodies and APL.
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The science of sabotage
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Saboteurs are the most elusive and audacious of opponents. Moving unobtrusively, they may be unrecognizable until after their crimes have been committed. The current treatments for SLE and APS may be considered imperfect attempts to counter sabotage. In SLE, treatment is often instituted only after the sabotage has begun to cause deleterious effects, such as glomerulonephritis. In APS, we may try to prevent such effects by long-term treatment with warfarin, but this is akin to preventing sabotage by running a police state. We hope that repression of all clotting will prevent pathogenic clotting caused by APL. It would preferable if we could recognize and counter the pathogenic APL specifically.
The best way to defeat saboteurs is to recognize and apprehend them before they can do any harm. In the case of autoantibodies, this is difficult because the pathogenic antibodies resemble all other antibodies in terms of gene usage and basic molecular structure. The research described in this essay is part of an effort by many groups world-wide which aims to find common features between pathogenic antibodies, which are not shared with non-pathogenic antibodies. Such features could be either structural or functional. This essay has concentrated on structural aspects, because a better knowledge of the structure of pathogenic antibodies may one day help in the rational design of drugs which will remove or disable the pathogenic antibodies but not the innocent ones. Alternatively, it may be possible to use antibody structures to deduce the important epitopes on apoptotic debris which play a role in driving formation of these pathogenic antibodies in the first place. By blocking or removing such epitopes, could we prevent or reduce production of the antibodies?
There are major challenges in this work. In particular, since autoantibody responses are polyclonal, how much can we really learn by studying a limited number of antibody structures? One reason to be optimistic about this is that recurrent themes, such as the importance of somatic mutations and arginine residues, arise both from the systematic analysis of large populations of human and murine antibodies and from the detailed study of single antibodies.
This suggests that the molecular properties of pathogenic autoantibodies, while intriguing in themselves, may also illuminate future attempts to treat SLE and APS.
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Acknowledgments
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I would like to thank all my colleagues at the Centre for Rheumatology, Department of Medicine, University College London and at the Medical Molecular Biology Unit, Institute of Child Health, London. I would particularly like to acknowledge Celia Longhurst, Tindie Kalsi, Chelliah Ravirajan, Jo Haley, Ian Giles, Nancy Lambrianides and Lesley Mason, who have all been valued colleagues in these studies over many years. I am very grateful to Professor David Isenberg and Professor David Latchman, who have supported and encouraged me throughout these studies. Much of this work was funded by the Arthritis Research Campaign and the Wellcome Trust. Figures 1 and 36

are printed by kind permission of Dr Sylvia Nagl. Figures 16



are reproduced with permission from Elsevier, the publishers of Journal of Molecular Biology and Molecular Immunology, in which these figures originally appeared [55, 64].
The author has declared no conflicts of interest.
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Submitted 22 June 2004;
revised version accepted 6 July 2004.