http://www-ermm.cbcu.cam.ac.uk
`
`expert reviews
`
`on molecular medicine
`
`w
`.J
`Structure-function analysis and the molecular
`
`
`UJ
`C:
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`origins of anti-DNA antibodies in systemic lupus
`erythematosus
`fl>
`(1)
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`·-
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`·-"'C
`0
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`Jatinderpal Kalsi, Chelliah T. Ravirajan, Anisur Rahman and
`.c
`David A. Isenberg
`
`
`
`
`
`Patients with the autoimmune rheumatic disease systemic lupus erythematosus
`�
`
`
`
`(SLE or 'lupus') develop a wide variety of clinical and serological manifestations
`
`
`
`
`
`including the presence of antibodies to double-stranded DNA (dsDNA), which
`<C
`
`
`
`
`
`
`are often diagnostic and potentially pathogenic. In this review, we have examined Z
`
`
`
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`the links between the structure and function of anti-dsDNA antibodies, 't
`
`
`
`
`
`their clinical associations. We have also reviewed studies involving ;:;
`emphasising
`
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`
`
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`animal models, the analysis of human antibody sequences and studies of, and C:
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`
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`using, computer modelling and crystal structure.
`<C
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`....
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`·-
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`Systemic lupus erythematosus (SLE or 'lupus') is
`
`
`
`characterised by thickening of the skin), patients
`
`
`
`
`a major autoimmune rheumatic disease that is
`
`
`with SLE do not develop antibodies to the
`
`
`
`characterised clinically by photosensitive rashes
`DNA-binding enzyme Sci 70 (also known as
`
`and arthritis; in most cases the kidneys, lungs and
`
`
`topoisomerase 1). Furthermore, the relatively
`
`
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`central nervous system are affected by the disease.
`
`
`
`
`restricted autoantibody profile that is typical of
`
`
`
`
`Its clinical diversity is, apparently, matched by its
`
`
`patients with SLE leads to the conclusion that
`
`
`serological diversity (see Table l; tabOOldil).
`
`random polyclonal activation of B cells cannot be
`
`
`
`Antibodies [immunoglobulins (Igs) produced by
`
`
`
`solely responsible for the production of anti­
`
`
`
`B lymphocytes (B cells)] that are found in patients
`
`
`DNA antibodies (Ref. 2). Precisely which of
`
`
`with SLE appear to target a range of 'self antigens'
`
`
`
`these SLE-associated autoantibodies are likely to
`
`
`
`(host-derived antigens). However, the diversity
`
`
`
`be pathogenic remains to be determined, and
`
`
`of the antibodies found in patients with SLE
`
`
`represents a considerable challenge.
`
`is actually rather narrow, given that a
`
`Anti-dsDNA antibodies
`
`
`
`typical mammalian cell contains physiologically
`
`
`significant quantities of as many as 2000 different
`
`
`Among the autoantibodies that are present in the
`
`
`proteins, which are all potential self antigens
`
`serum of patients with SLE, those that bind to
`
`
`(Ref. 1). For example, unlike patients with
`
`
`dsDNA remain of paramount interest. These
`
`
`scleroderma (an autoimmune disease that is
`
`
`antibodies were first identified in the serum of
`
`David A. Isenberg (Corresponding author)
`
`
`ARC Diamond Jubilee Professor of Rheumatology at University College London, Centre for
`
`
`
`
`
`
`
`
`
`
`Rheumatology /Bloomsbury Rheumatology Unit, Department of Medicine, University College
`
`
`
`
`
`London, Arthur Stanley House, 50 Tottenham Street, London WlP 9PG, UK, Tel: +44 (0)171 380
`
`
`9230; Fax: +44 (0)171 380 9278; E-mail: d.isenberg@ucl.ac.uk;
`
`
`
`
`
`Departmental website: http://www.ucl.ac.uk/ medicine /bloomsbury-rheumatology /
`
`Jatinderpal Kalsi, Chelliah T. Ravirajan and Anisur Rahman
`
`
`
`
`Centre for Rheumatology /Bloomsbury Rheumatology Unit, Department of Medicine, University
`
`
`
`
`
`
`
`
`
`
`College London, Arthur Stanley House, 50 Tottenham Street, London WlP 9PG, UK
`
`1
`{99)00042-3a.pdf (short code: txt001 dil); 16 February 1999
`
`Accession information:
`ISSN 1462-3994 ©1999 Cambridge University Press
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`IPR2017-01488
`
`Genentech Exhibit 2023
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`

`http://www-ermm.cbcu.cam.ac.uk
`
`expert reviews
`in molecular medicine
`
`-— LIJ
`
`Table 1. Summary of the frequency of serum antibodies (and the antigens they are
`
`detected with) that are commonly detected in patients with systemic lupus
`
`
`emthematosus (SLE) (tab001dil)
`
`
`Frequency ofpatients studied who have detectable antibodies (“/o)
`
`
`Published studies
`Series at the Bloomsbury
`Antigens the lgs bind to
`
`Rheumatology Unitb
`
`(worldwide) (Ab type)a
`
`
` 34 (for lgG);12 (for lgM)
`20—50
` Cardiolipin
`Not studied
`C1q
`20—45
`
`55
`dsDNA
`40—90
`21
`
`
`Fc IgG (RF)
`~25
`Not studied
`Histone
`30—80
`hsp 70
`5—40
`
`
`hsp 90
`5—50
`5 (for IgG); 35 (for lgM)
`La
`10—15
`13
`
`
`14
`LAC
`10—20
`97
`‘Nucleus’ (ANA)
`>90
`
`
`20
`RNP
`20—35
`32
`Ro
`30—40
`
`
`10
`Sm
`5—30
`Not studied
`ssDNA
`s70
`
`
`20
`Thyroid
`S35
`
`
`
`a For further details about the antigens used, see Table 13 in Ref. 106.
`
`
`b The Bloomsbury Rheumatology Unit is at University College, London, UK.
`
`Abbreviations used: Ab = antibody; ANA = anti-nuclear antibody; C1q = a complement protein; dsDNA =
`
`double—stranded DNA; hsp 70 and hsp 90 = two types of human heat shock protein; histone = the protein
`associated with DNA; lg = immunoglobulin (antibody); LAC = lupus anti-coagulant; RF = rheumatoid factors,
`antibodies (usually lgM) that bind to lg and are produced by patients with rheumatoid arthritis but also with
`
`
`other diseases; ssDNA= single—stranded DNA; Sm, RNP, R0 and La = ribonuclear proteins; thyroid = an
`
`
`extract of human thyroid.
`
`
`nSL
`
`
`
`Anti-DNAantibodies
`
`
`
`5 2
`
`patients with SLE over 40 years ago in four
`different laboratories (reviewed in Ref. 3). The
`likely involvement of these antibodies in the
`pathogenesis of human SLE, and in animal models
`of SLE, is indicated by (1) the close links between
`disease ’activity’ and levels of anti—dsDNA
`antibodies in the serum of many, though not all,
`patients; (2) the elution (removal and collection)
`of these antibodies from the kidneys of patients
`with SLE and lupus-prone mice; (3) direct
`evidence of these antibodies being associated with
`pathogenicity in isolated rat perfusion systems
`in which kidneys are dissected from the rat and
`their function is maintained artificially for a
`few hours) and mice with severe combined
`immunodeficiency (SCID); and (4) the fact that
`although antibodies to single—stranded DNA
`(ssDNA) are frequently found in the relatives of
`patients with SLE, those that bind to dsDNA are
`virtually never detected.
`
`Clinical studies
`
`Many papers published during the past 20
`years (reviewed by Spronk and colleagues in
`
`Ref. 4) have concluded that levels of anti-
`
`dsDNA antibodies [usually measured using
`enzyme-linked immunosorbent assays (ELISAs)
`or radioimmunoassays (RIAs)] generally reflect
`Clinical disease activity. This observation appears
`to be particularly true of renal (kidney) disease,
`and most of the evidence that anti—dsDNA
`
`antibodies are pathogenic has been collected from
`studies of the kidney. Thus, high levels of anti-
`dsDNA antibodies, as measured using the Farr
`assay, which detects high-avidity antibodies
`(those that bind strongly to their target antigens),
`and low values of the complement enzyme
`marker CHSO were found predominantly in
`patients with lupus nephritis (i.e. who have
`kidney inflammation associated with SLE; Ref. 5).
`In contrast, antibodies to ssDNA are not specific
`for patients with SLE, being present, for example,
`in many individuals with infectious diseases.
`Lloyd and Schur (Ref. 6) found that complement
`depletion and raised titres of anti-dsDNA
`antibodies were associated more closely with
`renal than non-renal exacerbations in SLE. Ter
`
`Borg and colleagues (Ref. 7), in a prospective
`
`Accession information: (99)OOO42-3a.pdf (Short code: txtOO1dil); 16 February 1999
`ISSN 1462-3994 ©1999 Cambridge University Press
`
`

`

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`
`expert reviews
`in molecular medicine
`
`study of 72 patients, showed that active lupus
`nephritis was usually associated with high
`titres of
`anti—dsDNA antibodies. More
`
`recently, Okamura and colleagues (Ref. 8) have
`demonstrated a close relationship between renal
`disease activity (assessed by biopsy) and the
`isotype of Ig (e.g. IgG compared with IgM)
`produced that bound DNA: activity correlated
`with IgG reactive against dsDNAbut not with IgG
`reactive against ssDNA or with IgM reactive
`against either dsDNA or ssDNA. Bootsma and
`colleagues (Ref. 9), using the concept of a rise in
`levels of anti-dsDNA antibodies as a means of
`
`predicting a clinical relapse, showed that treating
`such patients with high levels of prednisolone (30
`mg / day) reduced the relapse rate, compared with
`a control group, who were treated with either
`lower doses of prednisolone or no steroids.
`
`Assessment of disease activity in SLE
`A major problem with many of these earlier
`studies was the unsatisfactory nature of the
`indices of disease activity that were used to assess
`SLE. Validated and reliable global-score indices
`(i.e. score systems in which activity in each system
`is ’lumped together’) have been developed
`only in the past decade. However, with a
`disease as diverse as SLE, an activity index that
`demonstrates ’at a glance’ the degree of disease
`activity in each of the major organs or systems
`has obvious advantages. The British Isles Lupus
`Assessment Group (BILAG) has described and
`validated such a system, based on the ’physician’s
`intention to treat’ principle (Ref. 10). Thus, eight
`organs or systems are distinguished, allowing the
`easy correlation of activity in a particular organ
`or system with any serological marker. Using this
`system in a serial, longitudinal study of 14 Afro-
`Caribbean patients with SLE, antibodies to
`dsDNA were shown to correlate with renal
`
`disease, cardiopulmonary disease and global
`scores, but not with musculoskeletal, central
`nervous system or haematological involvement.
`Blood samples taken over periods of follow—up
`in the range of 3—15 years were analysed in this
`study (Ref. 11).
`
`Historical evidence that anti-dsDNA
`
`antibodies can be pathogenic in SLE
`Detection alone of anti-DNA antibodies in the
`
`lesinSLE
`
`Anti-DNAantibod
`
`at ’the scene of the crime’. Koffler and colleagues
`(Ref. 12) eluted IgG, complement and IgM from
`the kidneys of patients who had died from lupus
`nephritis. These eluates possessed anti-nuclear-
`binding activity; although a specific test for anti-
`DNA binding was not performed, the anti-
`nuclear-binding activity of the eluted antibodies
`could be partially inhibited by the addition of
`dsDNA, suggesting that at least some anti-dsDNA
`antibodies were present.
`In a study (Ref. 13) of over 40 families affected
`by SLE (where 21% of 147 relatives had antibodies
`to ssDNA), only two relatives had antibodies
`to dsDNA, strongly implicating anti-dsDNA
`antibodies, but not the anti-ssDNA antibodies, in
`the disease process (Ref. 14).
`
`Spontaneous disease models of SLE;
`pathogenicity of anti-DNA antibodies
`Some strains of mice spontaneously develop
`autoimmune disorders that have many of the
`features that are typical of human SLE (Ref. 15).
`The most commonly studied strains of mice with
`murine lupus are: (1) New Zealand black (NZB),
`(2) BWF1 [NZB x New Zealand white (NZW) F1],
`(3) MRL/++, (4) MRL-lpr/lpr, (5) MRL/lpr, (6)
`BXSB, and (7) SNFl [(NZB) x (SWR) F1] (for
`a comparison of their features see Table 2;
`tabOOZdil). These mice produce elevated levels of
`total Igs and autoantibodies (such as anti-dsDNA)
`and are thought to develop nephritis and arteritis
`as a result of deposition of immune complexes in
`the kidneys or arteries. Like patients with SLE,
`these mice present a diversity of disease patterns.
`In NZB and BWF1 mice, disease occurs primarily
`in the females. The disease in NZB mice is
`
`characterised mostly by a type of haemolytic
`anaemia that is Coombs positive (with anti-red-
`cell antibodies produced), and this can be
`associated with variable production of anti-
`nuclear antibodies (ANAs). The disease in
`BWF1 mice is characterised by the production
`of ANAs and immune-complex-mediated
`glomerulonephritis (in their kidneys). In MRL/
`lpr mice, the lupus disease more equally affects
`male and female mice, whereas in BXSB mice, the
`disease affects males, because of the influence of
`
`a Y—linked gene (Ref. 15).
`
`kidneys of patients with SLE does not prove such
`MRL/ ++ and MRL/ lpr mice are used as models
`antibodies were responsible for the development
`of this complication; however, it does place them
`of SLE, and genetically differ at the lpr locus,
`—3
`Accession information: (99)OOO42—3a.pdf (short code: txtOO‘idii); 16 February 1999
`ISSN 1462—3994 ©1999 Cambridge University Press
`
`The role of the lpr gene in mouse models
`of SLE
`
`

`

`
`
`Anti-DNAantibodiesinSLE
`
`http://www—ermm.cbcu.cam.ac.uk
`
`expert reviews
`in molecular medicine
`
`§ T
`
`able 2. Features of disease in lupus-prone mouse models (tab002dil)
`
`Mouse strain;
`MHC haplotype
`
`New Zealand black
`(NZB); H-2d
`
`Major clinical features
`
`Haemolytic anaemia,
`glomerulonephritis,
`lymphomas.
`
`Survival time for 50%
`of animals in typical
`group; sex of animal
`most affected
`
`18 months; both sexes
`affected equally.
`
`BWF1 [F1 of (NZB
`x New Zealand white
`(NZW)]§ H-2d’Z
`
`MRL—lpr/Ipr, H-2k
`
`Severe immune-complex-
`mediated nephritis.
`
`7—8 months; females.
`
`Lymphoproliferation,
`immune-complex-mediated
`nephritis, rheumatoid
`arthritis, vasculitis.
`
`2—4 months; females.
`
`Major immunological
`features
`
`Production of anti-erythrocyte
`antibodies, hyperproduction of
`lgM, generalised lymphocyte
`dysfunction.
`
`Production of anti-nuclear and
`anti-DNA antibodies,
`generalised lymphocyte
`dysfunction.
`
`Production of anti-nuclear
`antibodies and rheumatoid
`factors, proliferation of Ly1+
`cells“, generalised lymphocyte
`dysfunction.
`
`MRL‘“; H—2k
`
`As for MRL-lpr/lpr but less
`severe.
`
`18 months; females.
`
`As for M RL-lpr/Ipr but less
`severe.
`
`
`
`BXSB; H-2h
`
`Haemolytic anaemia,
`lymphadenopathy,
`glomerulonephritis.
`
`2—4 months; males.
`
`Moth-eaten; H-2b
`
`Hair loss, glomerulonephritis,
`increased susceptibility
`to infections.
`
`1 month; both sexes
`affected equally.
`
`Palmerston—North;
`H—2q
`
`Polyarteritis nodosa,
`immune-complex-mediated
`nephritis.
`
`Swan; H-2k
`
`Mild glomerulonephritis.
`
`11 months; females.
`
`Production of anti-DNA
`antibodies, anti-NTA and
`anti-erythrocyte antibodies,
`thymic atrophy occurs earlier
`than normal.
`
`Production of anti-DNA
`antibodies, anti-NTA and anti-
`erythrocyte antibodies,
`immunosuppression.
`
`Production of anti-DNA
`antibodies, hyperactivity of B
`lymphocytes.
`
`18 months; both sexes
`affected equally.
`
`Production of anti-DNA
`antibodies, thymic atrophy
`occurs earlier than normal.
`
`SNF1; H-ZL"d
`
`Severe glomerulonephritis.
`
`4—8 months; females.
`
`Production of anti-DNA and
`anti-nucleosome antibodies.
`
`a Ly1+ cells = mouse T lymphocytes (T cells) that have Ly1 antigens on their cell surface, a T-helper subset.
`Abbreviations used: H-2 = mouse major histocompatibility complex (MHC) class I; lgM = isotype of
`immunoglobulin (lg); NTA = natural thymocytotoxic antibody.
`
`which is mutated in lpr mice and is an autosomal
`recessive gene (Ref. 16). In the homozygous state,
`lpr causes an immunological
`illness that
`is characterised by lymphoproliferation
`and accelerated autoimmunity. MRL/ ++ mice,
`which have two copies the wild-type (unmutated)
`gene at the lpr locus, develop an autoimmune
`
`syndrome with a much longer survival time of
`the mice. Congeneic mice on various genetic
`backgrounds that have two copies of the mutated
`lpr gene (such as C57BL/ 6-lpr/lpr, Bé-lpr/Zpr,
`C3I-I/HeI-lpr/lpr and AKR-lpr/ lpr) all develop
`’spontaneous’ lymphoproliferation and produce
`autoantibodies. Despite the fact that anti-DNA
`
`—A
`
`ccession information: (99)OOO42-3a.pdf (short code: txtOO1dil); 16 February 1999
`ISSN 1462—3994 ©1999 Cambridge University Press
`
`

`

`http://www—ermm.cbcu.cam.ac.uk
`
`expert reviews
`in molecular medicine
`
`— a
`
`ntibodies are produced by, and found in, all of
`the strains of mice, only MRL / lpr mice (with one
`wild-type and one mutant lpr gene) develop
`severe immune-complex renal disease, which is
`responsible for the death of these mice by 6—9
`months of age. Therefore, the underlying or
`primary mechanism of autoimmunity in the MRL
`model lies in the MRL/ ++ background, and the
`lpr gene appears to act by accelerating and
`increasing the severity of the disease.
`The lpr gene is now known to encode a
`defective configuration of the Fas receptor
`(Ref. 17), the normal form of which mediates a
`signalling pathway that initiates apoptosis
`(programmed cell death; Ref. 18). Fas is a cell-
`surface protein that plays a major role in the
`induction of apoptosis in lymphoid cells. Mice
`with mutations in lpr and / or lprcg, the gene(s)
`encoding Fas in mice (Ref. 19), are characterised
`by prolonged survival of B cells and other features
`that are similar to those found in patients with
`SLE, suggesting that in these animals interference
`with apoptosis might be important in the
`pathogenesis of their autoimmune disease (see
`below). Because of the involvement of lpr in
`apoptosis, which is important to T—cell function,
`we recently investigated whether MRL / lpr mice
`had increased activity in the T—helper (Th) subset
`of T cells (Ref. 20). T cells in the lymph nodes (of
`both mice and humans) produce soluble factors
`that enhance B—cell activation and, in turn,
`
`Control of anti-DNA antibodies in mice
`
`cause the accumulation of large numbers of
`lymphocytes in the spleen and lymph nodes.
`These T cells are either conventional CD4+ T cells
`or CD4‘ CD8‘ T cells (also known as ‘double
`negative’ T cells). In MRL/ lpr mice, a failure of
`apoptosis of CD4+ T cells in the periphery of the
`thymus allows self-reactive T cells to persist and
`by T cells and cytokines
`ultimately to drive autoantibody production
`T cells probably play an important role in these
`by B cells (Ref. 21). Our studies revealed no
`mouse models of SLE because experimental
`significant difference in the extent of apoptosis in
`elimination of Th T cells in SNF1 mice virtually
`the organs of MRL/lpr mice compared with
`prevented disease, and the removal of Th cells in
`normal (non-autoimmune) BALB/ c mice at one
`BWF1 mice also improved disease outcome (Ref.
`month of age. At this age, the MRL/ lpr mice had
`25). CD4+ T cells, which can induce B cells to
`low levels of serum anti—ssDNA antibodies and
`secrete cationic IgG anti-dsDNA antibodies, have
`anti-dsDNA antibodies, and showed no detectable
`been cloned from SNF1 mice, which develop
`impairment in renal functions. This observation
`an SLE—like autoimmunity (Ref. 26). Studies on
`confirms that in MRL/ lpr mice, at one month,
`the role of
`those T cells
`that express
`when the distribution of apoptotic cells was
`CD40 ligand (CD4OL) in the development of
`normal, there was no detectable lupus disease.
`glomerulonephritis in SNF1 mice have shown that
`Initially, in the MRL / lpr mice, there were no global
`blocking the interaction between CD4OL on the
`defects in the negative selection of the T—cell
`pathogenic Th cells and CD40 on the lupus B cells,
`repertoire, and the positive selection of subsets
`— 5
`Accession information: (99)OOO42-3a.pdf (short code: txtOOi dil); 16 February 1999
`ISSN 1462—3994 ©1999 Cambridge University Press
`
`
`
`Anti-DNAantibodiesinSLE
`
`of CD4+ T cells and CD8+ T cells appeared to
`be normal (Ref. 22). Failure of apoptosis of
`self-reactive CD8+ T cells can then lead to
`
`downregulation of CD8 and persistence as
`CD4“ CD8‘ T cells, which contribute to the
`lymphadenopathy. It should be noted that in the
`MRL/ lpr mice, the size of the thymus increased
`in parallel with the augmented activity of the Th
`T cells (Ref. 23).
`
`Anti-DNA antibodies in mice
`
`In mice, the genetic basis of the ability to produce
`anti—dsDNA antibodies has not been completely
`defined. In most strains, nephritis is associated
`with the co—inheritance of several genes including
`major histocompatibility complex (MI 1C) genes.
`In the SNF1 mouse, nephritis is linked to the I—Acl
`locus of the normal SWR parent, whereas in BWF1
`mice, it is linked to the I-Eb chain from the NZW
`parent, a different MHC gene.
`Each of these autoimmune mouse strains
`
`has a polyclonal B-cell hyperactivity (many
`clones of B cells are affected) that causes
`hypergammaglobulinaemia (high levels of IgG
`antibodies) and an increased number of antibody-
`forming cells, including those that produce
`antibodies that bind to nuclear antigens. Although
`the B cells that are capable of producing the high-
`affinity anti-dsDNA antibodies are present in
`normal mice, they do not proliferate and actually
`produce such antibodies. Experimentally,
`to investigate the relationship between
`autoimmunity and Fas-mediated apoptosis (see
`below), using appropriate fusion—partner cells
`(Ref. 24), autoantibody—producing cells can be
`’immortalised’ to produce Igs in culture.
`
`

`

`http://www—ermm.cbcu.cam.ac.uk
`#m
`cardiolipin and to nucleic acids (Fig. 1; fig001dil; —l
`at a particular window of time, delayed the
`Ref. 32). Together, these data suggest that DNA w
`expansion of autoimmune memory B cells, and
`itself might not be the immunogen that is
`resulted in long—term therapeutic benefits in the
`responsible for inducing what are commonly :
`mice (Ref. 27). Thus, it is theoretically possible that
`known as anti-DNA antibodies.
`I—
`patients with SLE might benefit from treatment
`with monoclonal antibodies that are reactive with
`CD4 or CD40.
`
`expert reviews
`In molecular medicine
`
`tibodies
`
`Non-mammalian DNA as an eliciting
`agent
`As reviewed elsewhere (Ref. 33), mammalian
`DNA is poorly immunogenic in mammals. One
`of the key questions, therefore, concerns the
`identity of the agent(s) responsible for eliciting
`the anti—DNA-antibody response in patients with C
`SLE. Several candidates have been suggested, and ‘5
`have been reviewed below.
`Experiments have shown that bacterial DNA <
`is able to induce strong antibody responses in 2
`mice. Gilkeson and colleagues (Refs 34, 35) have 0
`proposed that bacterial DNA could be the
`I
`immunogen that induces the production of "I':
`antibodies that are reactive to DNA in patients :
`with SLE. When bacterial DNA was injected into <
`healthy mice, antibodies that lacked specificity for
`mammalian DNA were mostly found; these
`antibodies lacked the molecular characteristics
`
`Studies of mouse models of SLE have suggested
`a role for two cytokines, interleukin 6 (IL-6) and
`interleukin 10 (IL-10), in the pathophysiology of
`SLE. Treatment of BWF1 mice with an anti-IL-10
`monoclonal antibody delayed the onset of
`autoimmune manifestations and the production
`of autoantibodies (Ref. 28). Similarly, treatment
`of BWFl mice with an anti-IL—6-receptor antibody
`lowered the production of IgG autoantibodies and
`suppressed the development of autoimmune
`disease (Ref. 29). Again, these data suggest that
`the use of appropriate monoclonal antibodies in
`patients with SLE might be beneficial (Ref. 30).
`
`In vivo stimulation of anti-DNA antibody
`production
`The anti-DNA antibody response in SLE has been
`shown to be heterogeneous (Ref. 31). Serum-
`derived antibodies and monoclonal anti—DNA
`antibodies recognised epitopes that occur in
`different nucleic acids (such as phosphodiester
`groups separated by three adjacent carbon atoms;
`see Fig. 1; fig001dil) on apparently diverse
`molecules such as phospholipids, proteins,
`polysaccharides and cell—membrane structures
`(reviewed in Ref. 31). These observations were not
`due either to non—specific binding of low-affinity
`antibodies or merely to a charge interaction,
`because it has
`also been shown that
`anti—’DNA’ antibodies do not necessarily bind
`polynucleotides that have similar patterns of ionic
`charge (Ref. 31). Furthermore, IgG antibodies that
`have high affinity for DNA (Ref. 31) can show the
`same degree of polyreactivity (i.e. ability to react
`with different epitopes) as some low-affinity IgM
`anti—DNA antibodies.
`
`(notably arginine residues in the VH CDR3
`region) that are typical of the anti-dsDNA
`antibodies that are found in patients with SLE
`(Ref. 35). Autoantibodies in the serum of
`patients with SLE can bind to a widely shared
`bacterial epitope (on DNA), though these
`antibodies were mainly directed against ssDNA
`rather than dsDNA.
`
`a human dsDNA
`Studies using BK,
`polyoma virus, have produced data that are more
`promising. Hyperimmunisation of BALB/ c mice
`(Ref. 36) with this virus has been shown to induce
`the production of anti-DNA antibodies, and
`analysis of the variable-region genes of the
`antibodies revealed that the induced antibodies
`resembled the anti—DNA antibodies that are
`
`This polyreactivity of DNA antibodies could
`be due to multiple binding sites in variable regions
`in individual Igs or to the same (or similar)
`epitopes in different antigens. Furthermore,
`hybridoma cells (a fusion of antibody-producing
`cells and a transformed cell line) that were
`prepared from mice that had been immunised
`with conjugates of cardiolipin (a phospholipid)
`and protein have been shown to produce
`monoclonal antibodies that bound both to
`—6
`Accession information: (99)OOO42-3a.pdf (short code: txtOO1diI); 16 February 1999
`ISSN 1462-3994 ©1999 Cambridge University Press
`
`characteristic of lupus in mice. Rekvig and
`colleagues (Ref. 37) have shown that the injection
`of BK into young lupus—prone mice (NZB x NZW
`F1)before the natural onset of the disease induced
`a strong and persistent anti-dsDNA response,
`which is more typical of the response found in
`patients with SLE. In a subsequent report
`(Ref. 38), the same group showed that in vivo
`expression of a single DNA-binding protein,
`the polyoma virus T antigen, was sufficient to
`initiate the production of anti-dsDNA and anti-
`histone antibodies (histones are the protein core
`
`

`

`http://www—ermm.cbcu.cam.ac.uk
`
`expert reviews
`in molecular medicine
`
`Phosphodiester
`group
`
`Three adjacent
`carbon atoms
`
`Putative
`shared epitope
`for anti-DN'A-I
`antibody binding
`
`
`
`Anti-DNAantibodiesinSLE
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`Single strand of DNA
`
`Cardiolipin (phospholipid)
`
`Potential shared epitope between DNA and cardiolipin
`Expert Reviews in Molecular Medicine @1999 Cambridge University Press
`
`Figure 1. Potential shared epitope between DNA and cardiolipin.Anti-DNA antibodies found in the serum
`of patients with systemic lupus erythematosus (SLE) have been shown to bind to many antigenic targets
`including single- and double-stranded DNA (ssDNA and dsDNA) and phospholipids such as cardiolipin. In
`addition, mouse monoclonal antibodies from mice immunised with cardiolipin can bind to nucleic acids and
`cardiolipin (Ref. 32). This cross-reactivity might be due to a shared epitope in their primary sequence, consisting
`of one or more phosphodiester groups (Ref. 107) separated by three adjacent carbon atoms (shown here for
`ssDNA and cardiolipin). Abbreviation used: R = side chain. Modified from Ref. 107 (figOO1dil).
`
`responsible for the production of anti-DNA
`antibodies.
`
`around which dsDNA ’wraps’). They also
`provided further evidence that DNA alone is
`not immunogenic in Vivo, thus adding support
`to the increasingly popular theory that DNA Nucleic-acid—protein conjugates as
`that is complexed to histones in the form of
`eliciting agents
`nucleosomes is the key immunogenic particle Nucleosomes are the fundamental repeating units
`— 7
`Accession information: (99)OOO42-3a.pdf (short code: tXtOO‘l dil); 16 February 1999
`ISSN 1462—3994 ©1999 Cambridge University Press
`
`

`

`non-autoimmune mice by immunisation with
`complexes formed from mammalian DNA and the
`Fus-1 protein from the parasite Trypanosoma cruzi
`(Ref. 50). However, the significance of these
`observations to the aetiology of human SLE
`remains uncertain.
`
`expert reviews
`in molecular medicine
`
`(O
`
`http://www- ermm. cbcu.cam. ac uk
`—m
`several mammalian histone peptides and also
`of chromatin; they are generated during apoptosis
`other components that are common target
`by internucleosomal cleavage of the chromatin
`antigens in autoimmune diseases, such as
`(Ref. 39) Bell and colleagues (Ref. 40) have
`Sm-D antigens, ubiquitinated HZA antigen and
`demonstrated that B-cell hybridoma cells from
`poly(ADP——ribose)antigens (Ref. 48) Recently, we
`patients with SLE in culture released nucleosomes
`have shown that, in the mouse strain MRL/ lpr,
`that stimulated proliferation and Ig synthesis in
`normal B cells of mice.
`which is genetically susceptible to lupus, the
`In mouse models of SLE, the onset of the
`injection of as little as 1 ug of mouse histone—RNA
`complex induced increased antibody responses to
`autoimmune response has been shown to be
`murine histones, dsDNA and ubiquitinated H2A,
`characterised by the early emergence of antibodies
`and exaggerated the disease in vivo in young mice
`that recognise conformational epitopes of the
`(Ref. 49).
`nucleosome particles (Refs 41, 42). As the immune
`Antibodies to dsDNA were also induced in
`response progressed, autoantibodies that reacted
`to dsDNA or histones became apparent. In
`addition, in mice with lupus, nucleosomes were
`a major immunogen for inducing Th T cells that
`could stimulate pathogenic autoantibodies
`(Ref. 43).
`Our research group has produced from one
`patient with SLE a panel of five IgG (and five IgM)
`human hybridoma-derived monoclonal anti-
`DNA antibodies; two of these IgG antibodies
`bound to both dsDNA and histones, whereas none
`of the IgM antibodies did (Ref. 44). Moreover, the
`binding of these human monoclonal antibodies
`to histones was enhanced by the presence of DNA,
`which was also detectable in the supernatants of
`the hybridomas. In a subsequent study, we
`analysed the pathogenic properties of monoclonal
`human hybridoma anti-DNA IgG antibodies by
`implanting and growing the hybridoma-secreting
`cells in the peritoneal cavity of SCID mice. Only
`the IgG antibodies that bound to both dsDNA and
`histones (or nucleosomes) deposited in the renal
`glomeruli of the mice, producing renal damage
`(Refs 45, 46). These data imply that some anti-
`dsDNA antibodies have the capacity to bind
`directly to the renal tissue and cause damage,
`perhaps without the help of complement.
`Conjugates of nucleic acid and proteins, where
`the nucleic acids can be DNA or other nucleic
`acids, and the proteins can be histone or other
`proteins, can also induce anti-DNA antibodies. For
`example, in normal, non-SLE-susceptible mice,
`bacterial DNA that is complexed to methylated
`bovine serum albumin (BSA) in a suitable
`immunogenic. A recent experimental model of
`adjuvant can induce the production of high titres
`lupus (Ref. 54) that used peptide immunisation
`of IgG anti—DNA antibodies (Ref. 47). However,
`of normal rabbits extols the concept of epitope
`anti—DNA antibodies induced1n this way do not
`spreading as one of the mechanisms that might
`bind mammalian dsDNA, despite their reactivity
`generate anti-DNA antibodies. Our own work,
`with mammalian histone proteins that are
`however, does not substantiate this model
`complexed with mammalian RNA. These IgG
`antibodies react with mammalian histones,
`(Ref. 55).
`# 8
`Accession information: (99)OOO42-3a.pdf (short code: txtOO1diI); 16 February 1999
`ISSN 1462—3994 ©1999 Cambridge University Press
`
`Molecular mimicry and cross-reactivity
`A microorganism that possesses both foreign
`epitopes and epitopes that mimic autoantigens
`has the potential to elicit an autoimmune response
`in a human or animal that is exposed to it. Indeed,
`Diamond and colleagues (Refs 51, 52) have shown
`that autoantibodies were generated as part of the
`immune response to a bacterial immunogen.
`This group described 99D.7E, a cross—reactive
`monoclonal mouse IgG antibody that bound to
`both dsDNA and phosphorylcholine, which is a
`component of the bacterial cell wall. Although the
`antibody 99D.7E provided partial protection
`against the microorganism, it was pathogenic to
`the kidney. It is also possible that immune
`responses might arise against a particular
`component because of its close association with
`another component. Rabbits that were immunised
`with purified rabbit RNA polymerase 1 (Ref. 53)
`produced antibodies that were reactive against the
`RNA polymerase 1 enzyme and also rabbit nucleic
`acids. It is possible that these antibodies against
`nucleic acids were induced not by cross-reactive
`epitopes on RNA polymerase 1, but because the
`physical association between RNA polymerase 1
`and nucleic acids rendered the nucleic acids
`
`EA
`
`
`
`nti-DNAantibodiesi
`
`

`

`http://www—ermm.cbc:u.cam.ac.uk
`#m
`coding for Fas or the Fas ligands. However, levels
`Other stimuli of anti-dsDNA-antibody
`_I
`of soluble Fas are elevated in a minority of patients
`production
`who have SLE only (Ref. 57).
`Abnormalities of the immune system in human
`Bcl—2 is one of a family of proto-oncogene
`SLE no doubt serve to enhance or perpetuate
`products; the principal Bcl-2 protein is unusual
`the anti—DNA-antibody response. It has been
`among such products in that it enhances the
`suggested that several other factors might also
`survival of lymphoid cells by interfering
`’assist’ in the generation of the autoantibody
`with apoptosis, rather than by promoting cell
`response.
`proliferation. Much fundamental research work
`is in progress to explore how apoptosis is
`regulated; some members of the Bcl-2 f