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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`
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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
`
`
`
`
`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
`
`
`
`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. T hese
`
`
`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:
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`
`expert reviews
`in motecular medicine
`
`
`! 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
`erythematosus (SLE) (tab001dil)
`
`
`
`
`
`Not studied
`55
`21
`Not studied
`
`5 2
`
`5 (for IgG); 35 (for IgM)
`
`
`
`inSLE
`lesIn
`
`
`
`Anti-DNAantibod
`
`
`
`
`
`Frequencyofpatients studied who have detectable antibodies (%)
`Published studies
`Series at the Bloomsbury
`Antigens the Igs bind to
`
`
`(Ab type)? (worldwide)
`Rheumatology Unit®
`
`
` Cardiolipin
` 34 (for IgG);12 (for IgM)
`20-50
`20-45
`Ctq
`
`
`40-90
`dsDNA
`
`
`~25
`Fc IgG (RF)
`30-80
`Histone
`5-40
`hsp 70
`
`
`5-50
`hsp 90
`10-15
`La
`10-20
`LAC
`
`
`>90
`‘Nucleus’ (ANA)
`20-35
`RNP
`
`
`30-40
`Ro
`
`
`5-30
`sm
`
`
`Not studied
`<70
`ssDNA
`
`
`
`20
`$35
`Thyroid
`
`
`
`
`@ For further details about the antigens used, see Table 13 in Ref. 106.
`
`
`> The Bloomsbury Rheumatology Unit is at University College, London, UK.
`
`Abbreviations used: Ab = antibody; ANA = anti-nuclear antibody; C1q = a complementprotein; dsDNA =
`
`double-stranded DNA; hsp 70 and hsp 90 = two types of human heat shock protein; histone = the protein
`associated with DNA; Ig = immunoglobulin (antibody); LAC = lupus anti-coagulant; RF = rheumatoid factors,
`antibodies (usually IgM) that bind to Ig and are produced by patients with rheumatoid arthritis but also with
`
`
`other diseases; ssDNA = single-stranded DNA; Sm, RNP, Ro and La = ribonuclear proteins; thyroid = an
`
`
`extract of human thyroid.
`
`Ref. 4) have concluded that levels of anti-
`patients with SLE over 40 years ago in four
`different laboratories (reviewed in Ref. 3). The
`dsDNAantibodies [usually measured using
`likely involvement of these antibodies in the
`enzyme-linked immunosorbent assays (ELISAs)
`pathogenesis of humanSLE, and in animal models
`or radioimmunoassays (RIAs)|} generally reflect
`of SLE,is indicated by (1) the close links between
`clinical diseaseactivity. This observation appears
`to be particularly true of renal (kidney) disease,
`disease ‘activity’ and levels of anti-dsDNA
`antibodies in the serum of many, though notall,
`and most of the evidence that anti-dsDNA
`patients; (2) the elution (removal and collection)
`antibodies are pathogenic has been collected from
`studies of the kidney. Thus, high levels of anti-
`of these antibodies from the kidneys of patients
`with SLE and lupus-prone mice; (3) direct
`dsDNAantibodies, as measured using the Farr
`evidenceofthese antibodies being associated with
`assay, which detects high-avidity antibodies
`(those that bind strongly to their target antigens),
`pathogenicity in isolated rat perfusion systems
`in which kidneys are dissected from the rat and
`and low values of the complement enzyme
`their function is maintained artificially for a
`marker CH50 were found predominantly in
`patients with lupus nephritis (i.e. who have
`few hours) and mice with severe combined
`immunodeficiency (SCID); and (4) the fact that
`kidney inflammation associated with SLE;Ref. 5).
`although antibodies to single-stranded DNA
`In contrast, antibodies to ssDNAare notspecific
`for patients with SLE, being present, for example,
`(ssDNA)are frequently found in therelatives of
`patients with SLE, those that bind to dsDNA are
`in many individuals with infectious diseases.
`Lloyd and Schur (Ref. 6) found that complement
`virtually never detected.
`depletion and raised titres of anti-dsDNA
`Clinical studies
`antibodies were associated more closely with
`Many papers published during the past 20
`renal than non-renal exacerbations in SLE. Ter
`Borg and colleagues (Ref. 7), in a prospective
`years (reviewed by Spronk and colleagues in
`aSSSee
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`
`expert reviews
`in molecular medicine
`
`Anti-DNAantibod
`
`at ‘the scene ofthe crime’. Koffler and colleagues
`study of 72 patients, showed that active lupus
`nephritis was usually associated with high
`(Ref. 12) eluted IgG, complement and IgM from
`the kidneys of patients who had died from lupus
`titres of anti-dsDNA antibodies. More
`recently, Okamura and colleagues (Ref. 8) have
`nephritis. These eluates possessed anti-nuclear-
`demonstratedaclose relationship between renal
`bindingactivity; although a specific test for anti-
`disease activity (assessed by biopsy) and the
`DNAbinding was not performed, the anti-
`isotype of Ig (e.g. IgG compared with IgM)
`nuclear-binding activity of the eluted antibodies
`could be partially inhibited by the addition of
`produced that bound DNA:activity correlated
`with IgG reactive against dsDNAbutnot with IgG
`dsDNA,suggestingthat at least some anti-dsDNA
`reactive against ssDNA or with IgM reactive
`antibodies were present.
`In a study (Ref. 13) of over 40 families affected
`against either dsDNA or ssDNA. Bootsma and
`colleagues (Ref. 9), using the conceptof a rise in
`by SLE (where 21% of 147relatives had antibodies
`levels of anti-dsDNA antibodies as a means of
`to ssDNA), only tworelatives had antibodies
`predictingaclinical relapse, showedthattreating
`to dsDNA,strongly implicating anti-dsDNA
`such patients with highlevels of prednisolone (30
`antibodies, but not the anti-ssDNA antibodies, in
`the disease process (Ref. 14).
`mg/day) reduced the relapse rate, compared with
`a control group, who were treated with either
`lower doses of prednisolone or no steroids.
`
`Spontaneous disease modeis of SLE;
`pathogenicity of anti-DNA antibodies
`Assessment of disease activity in SLE
`Somestrains of mice spontaneously develop
`A major problem with many of these earlier
`autoimmune disorders that have many of the
`features that are typical of human SLE (Ref. 15).
`studies was the unsatisfactory nature of the
`The most commonly studiedstrains of mice with
`indices of disease activity that were usedto assess
`murine lupusare: (1) New Zealand black (NZB),
`SLE. Validated and reliable global-score indices
`(i.e. score systems in which activity in each system
`(2) BWF1 [NZB x New Zealand white (NZW) F1],
`(3) MRL/++, (4) MRL-Ipr/Ipr, (5) MRL/Ipr, (6)
`is ‘lumped together’) have been developed
`only in the past decade. However, with a
`BXSB, and (7) SNF1 [(NZB) x (SWR) F1] (for
`disease as diverse as SLE, an activity index that
`a comparison of their features see Table 2;
`tab002dil). These mice produceelevatedlevels of
`demonstrates ‘at a glance’ the degree of disease
`activity in each of the major organs or systems
`total Igs and autoantibodies (such as anti-dsDNA)
`and are thought to develop nephritis and arteritis
`has obvious advantages. The British Isles Lupus
`Assessment Group (BILAG) has described and
`as a result of deposition of immune complexesin
`the kidneys or arteries. Like patients with SLE,
`validated such a system, based on the ‘physician’s
`these mice presenta diversity of disease patterns.
`intention to treat’ principle (Ref. 10). Thus, eight
`organsor systemsare distinguished, allowing the
`In NZB and BWF1 mice, disease occurs primarily
`in the females. The disease in NZB mice is
`easy correlation of activity in a particular organ
`or system with any serological marker. Using this
`characterised mostly by a type of haemolytic
`anaemia that is Coombspositive (with anti-red-
`system inaserial, longitudinal study of 14 Afro-
`Caribbean patients with SLE, antibodies to
`cell antibodies produced), and this can be
`associated with variable production of anti-
`dsDNA were shownto correlate with renal
`nuclear antibodies (ANAs). The disease in
`disease, cardiopulmonary disease and global
`BWF1 mice is characterised by the production
`scores, but not with musculoskeletal, central
`of ANAs and immune-complex-mediated
`nervous system or haematological involvement.
`glomerulonephritis (in their kidneys). In MRL/
`Blood samples taken over periods of follow-up
`in the range of 3-15 years were analysed in this
`Ipr mice, the lupus disease more equally affects
`study (Ref. 11).
`male and female mice, whereas in BXSB mice, the
`disease affects males, because of the influence of
`a Y-linked gene (Ref. 15).
`
`Historical evidence that anti-dsDNA
`antibodies can be pathogenic in SLE
`Therole of the [pr gene in mouse models
`Detection alone of anti-DNA antibodies in the
`of SLE
`kidneys of patients with SLE does not prove such
`MRL/++ and MRL/Ipr 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 Ipr locus,
`—————SSSSSa ee
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`
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`expert reviews
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`eeeSSSe
`Table 2. Features of disease in lupus-prone mouse models (tab002dil)
`
`
`
`Anti-DNAantibodiesinSLE
`
`Major immunological
`features
`
`Production of anti-erythrocyte
`antibodies, hyperproduction of
`IgM, generalised lymphocyte
`dysfunction.
`
`Production of anti-nuclear and
`anti-DNAantibodies,
`generalised lymphocyte
`dysfunction.
`
`Production of anti-nuclear
`antibodies and rheumatoid
`factors, proliferation of Ly1*
`cells?, generalised lymphocyte
`dysfunction.
`
`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.
`
`
`
`Mousestrain;
`MHC haplotype
`
`New Zealand black
`(NZB); H-29
`
`Majorclinical features
`
`Haemolytic anaemia,
`glomerulonephritis,
`lymphomas.
`
`Survival time for 50%
`of animals in typical
`group; sex of animal
`mostaffected
`
`18 months; both sexes
`affected equally.
`
`Severe immune-complex-
`mediated nephritis.
`
`7-8 months; females.
`
`Lymphoproliferation,
`immune-complex-mediated
`nephritis, rheumatoid
`arthritis, vasculitis.
`
`2—4 months; females.
`
`BWF1 [F1 of (NZB
`x New Zealand white
`(NZW)]; H-2%
`
`MRL-/pr/ipr, H-2*
`
`MRL**; H-2*
`
`BXSB; H-2°
`
`As for MRL-/pr/pr but less
`severe.
`
`18 months; females.
`
`As for MRL-/pr/pr but less
`severe.
`
`Haemolytic anaemia,
`lymphadenopathy,
`glomerulonephritis.
`
`2-4 months; males.
`
`Moth-eaten; H-2°
`
`Hair loss, glomerulonephritis,
`increased susceptibility
`to infections.
`
`4 month; both sexes
`affected equally.
`
`Palmerston—North;
`H-29
`
`Polyarteritis nodosa,
`immune-complex-mediated
`nephritis.
`
`Swan; H-2*
`
`Mild glomerulonephritis.
`
`11 months; females.
`
`18 months; both sexes
`affected equally.
`
`Production of anti-DNA
`antibodies, thymic atrophy
`occurs earlier than normal.
`
`SNF 1; H-2:
`
`Severe glomerulonephritis.
`
`4-8 months; females.
`
`Production of anti-DNA and
`anti-nucleosome antibodies.
`
`@Ly1* cells = mouse T lymphocytes(Tcells) that have Ly1 antigens ontheir cell surface, a T-helper subset.
`Abbreviations used: H-2 = mouse major histocompatibility complex (MHC) class I; IgM = isotype of
`immunoglobulin (Ig); NTA = natural thymocytotoxic antibody.
`
`whichis mutated in lpr mice and is an autosomal
`recessive gene(Ref. 16). In the homozygousstate,
`lpr causes an immunological
`illness that
`is characterised by lymphoproliferation
`and accelerated autoimmunity. MRL/++ mice,
`which havetwocopiesthe 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
`backgroundsthat have twocopiesof the mutated
`Ipr gene (such as C57BL/6-Ipr/Ipr, B6-Ipr/Ipr,
`C3H/HeJ-lpr/lpr and AKR-Ipr/Ipr) all develop
`‘spontaneous’ lymphoproliferation and produce
`autoantibodies. Despite the fact that anti-DNA
`
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`

`expert reviews
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`
`
`
`Anti-DNAantibodiesinSLE
`
`http://www-ermm.cbcu.cam.ac.uk
`___EeeeEee
`of CD4* T cells and CD8* T cells appeared to
`antibodies are produced by, and foundin,all of
`be normal (Ref. 22). Failure of apoptosis of
`the strains of mice, only MRL/Ipr mice (with one
`self-reactive CD8* T cells can then lead to
`wild-type and one mutant lpr gene) develop
`downregulation of CD8 and persistence as
`severe immune-complex renal disease, which is
`CD4- CD8- T cells, which contribute to the
`responsible for the death of these mice by 6-9
`lymphadenopathy. It should be noted that in the
`monthsof age. Therefore, the underlying or
`MRL/lIpr mice, the size of the thymus increased
`primary mechanism of autoimmunity in the MRL
`in parallel with the augmentedactivity of the Th
`modellies in the MRL/++ background, and the
`T cells (Ref. 23).
`Ipr gene appears to act by accelerating and
`increasing the severity of the disease.
`The Ipr gene is now knownto encode a
`defective configuration of the Fas receptor
`(Ref. 17), the normal form of which mediates a
`signalling pathway that initiates apoptosis
`(programmedcell death; Ref. 18). Fas is a cell-
`surface protein that plays a majorrole in the
`induction of apoptosis in lymphoid cells. Mice
`with mutations in Ipr and/or Ipr®, the gene(s)
`encoding Fasin mice (Ref. 19), are characterised
`by prolongedsurvivalof B cells and otherfeatures
`that are similar to those found in patients with
`SLE, suggestingthat in these animals interference
`with apoptosis might be important in the
`pathogenesis of their autoimmune disease (see
`below). Because of the involvement of Ipr in
`apoptosis, which is important to T-cell function,
`werecently investigated whether MRL/Ipr mice
`hadincreasedactivity 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,
`cause the accumulation of large numbers of
`lymphocytes in the spleen and lymph nodes.
`TheseT cells are either conventional CD4* T cells
`or CD4 CD8T cells (also known as ‘double
`negative’ T cells). In MRL/Ipr mice, a failure of
`apoptosis of CD4' T cells in the periphery of the
`thymusallowsself-reactive T cells to persist and
`ultimately to drive autoantibody production
`by B cells (Ref. 21). Our studies revealed no
`significant difference in the extent of apoptosis in
`the organs of MRL/Ipr mice compared with
`normal (non-autoimmune) BALB/c mice at one
`monthof age. At this age, the MRL/Ipr mice had
`low levels of serum anti-ssDNAantibodies and
`anti-dsDNAantibodies, and showed nodetectable
`impairment in renal functions. This observation
`confirms that in MRL/Ipr mice, at one month,
`when the distribution of apoptotic cells was
`normal, there was no detectable lupus disease.
`Initially, in the MRL/Ipr mice, there were no global
`defects in the negative selection of the T-cell
`repertoire, and the positive selection of subsets
`
`Anti-DNAantibodies in mice
`In mice, the genetic basis of the ability to produce
`anti-dsDNAantibodies has not been completely
`defined. In most strains, nephritis is associated
`with the co-inheritance of several genes including
`major histocompatibility complex (MHC)genes.
`In the SNF1 mouse, nephritis is linked to the I-A‘
`locus of the normal SWRparent, whereas in BWF1
`mice, it is linked to the I-E® 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 numberof antibody-
`forming cells, including those that produce
`antibodiesthat bindto nuclear antigens. Although
`the B cells that are capable of producing the high-
`affinity anti-dsDNA antibodies are present in
`normal mice, they do notproliferate and actually
`produce such antibodies. Experimentally,
`to investigate the relationship between
`autoimmunity and Fas-mediated apoptosis(see
`below), using appropriate fusion-partnercells
`(Ref. 24), autoantibody-producingcells can be
`‘immortalised’ to produce Igs in culture.
`
`Control of anti-DNA antibodies in mice
`by T cells and cytokines
`T cells probably play an importantrole in these
`mouse models of SLE because experimental
`elimination of Th T cells in SNF1 mice virtually
`prevented disease, and the removalof Th cells in
`BWF1 micealso improved disease outcome(Ref.
`25). CD4* T cells, which can induce B cells to
`secrete cationic IgG anti-dsDNAantibodies, have
`been cloned from SNF1 mice, which develop
`an SLE-like autoimmunity (Ref. 26). Studies on
`the role of
`those T cells
`that express
`CD40 ligand (CD40L) in the developmentof
`glomerulonephritis in SNF1 mice have shownthat
`blocking the interaction between CD40L on the
`pathogenic Th cells and CD40 on the lupusB cells,
`
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`

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`
`expert reviews
`in molecular medicine
`
`cardiolipin and to nucleic acids (Fig. 1; fig001dil;
`at a particular window of time, delayed the
`Ref. 32). Together, these data suggest that DNA
`expansion of autoimmune memoryBcells, and
`”/)
`itself might not be the immunogenthat is
`resulted in long-term therapeutic benefits in the
`responsible for inducing what are commonly
`mice (Ref. 27). Thus,it is theoretically possible that
`knownas anti-DNA antibodies.
`patients with SLE might benefit from treatment
`with monoclonal antibodiesthatare reactive with
`CD4 or CD40.
`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 BWF1 mice with an anti-IL-6-receptor antibody
`loweredthe production of IgG autoantibodies and
`suppressed the development of autoimmune
`disease (Ref. 29). Again, these data suggest that
`the use of appropriate monoclonalantibodies in
`patients with SLE mightbe beneficial (Ref. 30).
`
`
`
`cAnti-DNAantibodies
`
`Non-mammalian DNAasaneliciting
`agent
`As reviewed elsewhere (Ref. 33), mammalian
`DNAis poorly immunogenic in mammals. One
`of the key questions, therefore, concerns the
`identity of the agent(s) responsible for eliciting
`the anti-DNA-antibody responsein patients with
`SLE.Several candidates have been suggested, and
`have been reviewed below.
`Experiments have shownthat bacterial DNA
`is able to induce strong antibody responses in
`mice. Gilkeson and colleagues (Refs 34, 35) have
`proposed that bacterial DNA could be the
`immunogen that induces the production of
`antibodies that are reactive to DNAin patients
`with SLE. When bacterial DNA wasinjected into
`healthy mice, antibodies that lacked specificity for
`mammalian DNA were mostly found; these
`antibodies lacked the molecular characteristics
`(notably arginine residues in the V,, 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,
`polyomavirus, have produceddata that are more
`promising. Hyperimmunisation of BALB/c mice
`(Ref. 36) with this virus has been shownto 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
`characteristic of lupus in mice. Rekvig and
`colleagues (Ref. 37) have shownthatthe injection
`of BK into young lupus-prone mice (NZB x NZW
`F1) before the natural onsetof the disease induced
`a strong and persistent anti-dsDNAresponse,
`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 andanti-
`histone antibodies (histones are the protein core
`
`In vivo stimulation of anti-DNA antibody
`production
`The anti-DNAantibody 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 separatedby three adjacent carbon atoms,
`see Fig. 1; fig001dil) on apparently diverse
`molecules such as phospholipids, proteins,
`polysaccharides and cell-membranestructures
`(reviewedin 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
`polynucleotidesthat 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-DNAantibodies.
`This polyreactivity of DNA antibodies could
`be due to multiple bindingsites in variable regions
`in individual Igs or to the same(or similar)
`epitopes in different antigens. Furthermore,
`hybridomacells (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
`
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`
`

`

`
`
`Anti-DNAantibodiesinSLE
`
`http://www-ermm.cbcu.cam.ac.uk
`
`expert reviews
`in molecular medicine
`
`Phosphodiester
`group
`
`Three adjacent
`carbon atoms
`
`Putative
`shared epitope
`for anti-DNA-
`antibody binding
`
`Expert Reviews in Molecular Medicine ©1999 Cambridge University Press
`
`C,
`
`]Ho” O. BASE
`His Cc’
`H
`Oo He
`R
`
`Gh
`
`u
`
`H2C\ 9_¢G-R
`
`Single strand of DNA
`
`Cardiolipin (phospholipid)
`
`Potential shared epitope between DNAandcardiolipin
`
`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 shownto 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 epitopein their primary sequence, consisting
`of one or more phosphodiester groups (Ref. 107) separated by three adjacent carbon atoms (shownherefor
`ssDNAandcardiolipin). Abbreviation used: R = side chain. Modified from Ref. 107 (fig001dil).
`
`around which dsDNA ‘wraps’). They also
`provided further evidence that DNAaloneis
`not immunogenic in vivo, thus adding support
`to the increasingly popular theory that DNA
`that is complexed to histones in the form of
`nucleosomesis the key immunogenicparticle
`
`responsible for the production of anti-DNA
`antibodies.
`
`Nucleic-acid—protein conjugates as
`eliciting agents
`Nucleosomesare the fundamental repeating units
`
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`
`

`

`
`
`Anti-DNAantibodiesinSLE
`
`expert reviews
`in molecular medicine
`
`http://www-ermm.cbcu.cam.ac.uk
`EESeeeSS
`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 autoimmunediseases, such as
`(Ref. 39). Bell and colleagues (Ref. 40) have
`Sm-D antigens, ubiquitinated H2A antigen and
`demonstrated that B-cell hybridomacells from
`poly(ADP-ribose)antigens (Ref. 48). Recently, we
`patients with SLEin culture released nucleosomes
`have shownthat, in the mouse strain MRL/Ipr,
`that stimulated proliferation and Ig synthesis in
`whichis genetically susceptible to lupus, the
`normalB cells of mice.
`injection of aslittle as 1 1g of mouse histone-RNA
`In mouse models of SLE, the onset of the
`complex induced increased antibody responsesto
`autoimmune response has been shown to be
`murine histones, dsDNAand ubiquitinated H2A,
`characterised by the early emergence of antibodies
`and exaggeratedthe disease in vivo in young mice
`that recognise conformational epitopes of the
`(Ref. 49).
`nucleosomeparticles (Refs 41, 42). As the immune
`Antibodies to dsDNA were also induced in
`response progressed, autoantibodies that reacted
`non-autoimmune mice by immunisation with
`to dsDNA or histones became apparent. In
`complexes formed from mammalian DNA andthe
`addition, in mice with lupus, nucleosomes were
`Fus-1 protein from the parasite Trypanosoma cruzi
`a major immunogenfor inducing ThTcells that
`(Ref. 50). However, the significance of these
`could stimulate pathogenic autoantibodies
`observations to the aetiology of human SLE
`(Ref. 43).
`remains uncertain.
`Ourresearch group has produced from one
`patient with SLE a paneloffive IgG (andfive IgM)
`human hybridoma-derived monoclonal anti-
`DNAantibodies; two of these IgG antibodies
`bound to both dsDNAandhistones, whereas none
`of the IgM antibodiesdid (Ref. 44). Moreover, the
`binding of these human monoclonal antibodies
`to histones was enhancedbythe presence of DNA,
`which wasalso detectable in the supernatants of
`the hybridomas. In a subsequent study, we
`analysedthe pathogenic properties of monoclonal
`human hybridoma anti-DNAIgG antibodies by
`implanting and growing the hybridoma-secreting
`cells in the peritoneal cavity of SCID mice. Only
`the IgG antibodies that boundto both dsDNAand
`histones (or nucleosomes) deposited in the renal
`glomeruli of the mice, producing renal damage
`(Refs 45, 46). These data imply that some anti-
`dsDNAantibodies have the capacity to bind
`directly to the renal tissue and cause damage,
`perhaps withoutthe help of complement.
`Conjugatesof nucleic acid andproteins, where
`the nucleic acids can be DNAorother nucleic
`acids, and the proteins can be histone or other
`proteins, can also induce anti-DNAantibodies. For
`example, in normal, non-SLE-susceptible mice,
`bacterial DNA that is complexed to methylated
`bovine serum albumin (BSA) in a suitable
`adjuvant can inducethe production of hightitres
`of IgG anti-DNAantibodies (Ref. 47). However,
`anti-DNA antibodies induced in this way do not
`bind mammalian dsDNA,despite their reactivity
`with mammalian histone proteins that are
`complexed with mammalian RNA. These IgG
`antibodies react with mammalian histones,
`
`Molecular mimicry and cross-reactivity
`A microorganism that possesses both foreign
`epitopes and epitopes that mimic autoantigens
`has the potentialto elicit an autoimmune response
`in.a humanor animal that is exposedto it. Indeed,
`Diamondandcolleagues(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, whichis a
`componentofthe bacterial cell wall. Although the
`antibody 99D.7E provided partial protection
`against the microorganism, it was pathogenicto
`the kidney. It is also possible that immune
`responses might arise against a particular
`componentbecauseofits close association with
`another component. Rabbits that were immunised
`with purified rabbit RNA polymerase 1 (Ref. 53)
`producedantibodies that werereactive against the
`RNApolymerase 1 enzymeand alsorabbit 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 polymerase1
`and nucleic acids rendered the nucleic acids
`immunogenic. A recent experimental model of
`lupus (Ref. 54) that used peptide immunisation
`of normal rabbits extols the concept of epitope
`spreading as one of the mechanisms that might
`generate anti-DNA antibodies. Our own work,
`however, does not substantiate this model
`(Ref. 55).
`
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`ISSN 1462-3994 ©1999 Cambridge University Press
`
`

`

`Lu
`and
`
`ins
`lies
`
`
`
`Anti-DNAantibod
`
`codingfor Fasor the Fas ligands. However, levels
`of soluble Fas are elevated ina minority of patients
`whohave SLEonly (Ref. 57).
`Bcl-2 is one of a family of proto-oncogene
`products; the principal Bcl-2 protein is unusual
`among such products in that it enhances the
`survival of lymphoid cells by interfering
`with apoptosis, rather than by promoting cell
`proliferation. Much fundamental research work
`is in progress to explore how apoptosis is
`Nuclear DNA damage induced by phagocytes
`regulated; some members of the Bcl-2 family
`Activated
`phagocytic
`cells
`(such
`as
`enhance apoptosis whereasothers blockit (Ref.
`polymorphonuclear leucocytes and macrophages)
`57). Althoughnotall studies agree, it is probable
`are capable of releasing highly reactive oxygen
`that in patients with SLE, T cells, but notBcells,
`species (ROS), which might be able to penetrate
`overexpress Bcl-2, and that this overexpression
`cell membranes, and thereby interact with, and
`correlates with SLE disease activity (Ref. 57).
`damage, nuclear DNA. Subsequentrelease of this
`‘altered’ DNA might, in turn, stimula