Manual of Biological Markers of Disease including Sections C

Manual of Biological Markers of Disease Supplement 2, June 1996

INSTRUCTIONS FOR SUPPLEMENT 2: Preliminary pages Replace: page III/ Title page - page IV / Copyright page (addition ISBN) Add after Table of Contents Section A: New table of Contents Section B Table of Contents Section C Section B Add after Chapter B1.4: Chapter B1.5 Section C Add after Chapter B1.8 Insert tab page Section C Introduction to Section C Chapters C1-C8

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MANUAL OF BIOLOGICAL MARKERS OF DISEASE Edited by

W.J.VAN VENROOIJ University of Nijmegen The Netherlands

R.N. MAINI The Mathilda and Terence Kennedy Institute of Rheumatology London, UK

KLUWER ACADEMIC PUBLISHERS NEW YORK / BOSTON / DORDRECHT / LONDON / MOSCOW

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0-306-46852-2 0-792-33808-1

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Contents

Section C: Clinical Significance of Autoantibodies Introduction 1.

Autoantibodies in Rheumatoid Arthritis: 1.1 Rheumatoïd factor 1.2 Antiperinuclear factor and anti-keratin antibodies 1.3 Anti-RA33 antibodies 1.4 Anti-collagen antibodies

2.

J.S. Smolen

Autoantibodies in SLE 2.1 Autoantibodies to double-stranded DNA 2.2 Autoantibodies to histones, Sm and ubiquitins 2.3 Autoantibodies to Ku and related antigens 2.4 Autoantibodies to PCNA 2.5 Autoantibodies to phospholipids

3.

The Editors

A.J.G. Swaak & R.J.T. Smeenk R.M. Bernstein W.H. Reeves, M. Satoh & J J. Langdon Y. Takasaki & E.M. Tan V. Morris & C. Mackworth-Young

Autoantibodies in SLE-overlap syndromes: 3.1 Autoantibodies to UlsnRNP

F.H.J. van den Hoogen & L.B.A. van de Putte

4.

Autoantibodies in Sjögren’s syndrome: A.G. Tzioufas & H.M. Moutsopoulos Anti-Ro/SSA and anti-La/SSB antibodies

5.

Autoantibodies in Scleroderma: 5.1 Anti-centromere antibodies N. F. Rothfield 5.2 Anti-DNA topoisomerase I antibodies 5.3 Anti-RNA polymerase antibodies 5.4 Anti-nucleolar and other autoantibodies

6.

Autoantibodies in Myositis: 6.1 Transfer RNA synthetases

7.

Autoantibodies in Vasculitis: 7.1 Anti-neutrophil cytoplasmic antibody

8.

I. N. Targoff & P.H. Plotz

C.G.M. Kallenberg

Autoantibodies in other diseases: 8.1 Anti-mitochondrial antibody in primary biliary cirrhosis 8.2 Autoantibodies to the coiled body and other nuclear bodies

I.R.Mackay & M. E. Gershwin

L. E. C. Andrade

Introduction to Section C This, the final section of the Manual, is in a sense the raison d’etre for the existence of the entire Manual. In a series of chapters by clinical specialists it examines the significance of autoantibodies which occur ubiquitously in the sera of patients with rheumatic disease. It has been argued that the value of measuring autoantibodies in clinical practice is predicated by the underlying assumption that the presence of a specific autoantibody equals a distinct disease or syndrome. The extent to which this is true or false is critically examined in this section. We have chosen the major immunologically-mediated disorders as the basis of chapter headings in this section. Within each chapter will be found a personal statement by an expert on the frequency of occurrence and the diagnostic specificity of specific autoantibodies. The emphasis is on a practical approach to how the information may be used in a clinical setting. As Editors, we have sought to impose a template of questions which the authors must address. In some instances this has worked splendidly, but in other chapters, sometimes the know-how/data were not sufficient to adhere to this formula, reflecting the diversity of personal opinions and interpretation. However, we are delighted with the overall result and expect that at the very least, this section will provide a mine of useful, up-to-date information. It will also provide a state-of-the-art approach to the use of laboratory tests as an integral part of clinical evaluation of patients with arthritic disorders and multi-system rheumatic diseases. It will become apparent to the reader and user of this Manual that the apparently simple concept enunciated above is subject to a number of conditions. Not only is there a constellation of autoantigens but there is also a heterogeneity of autoantibodies occurring in a serum sample. For example, a serum may contain an autoantibody of many types of isotypes of immunoglobulin and, as a rule, multiple binding specificities to a welldefined macromolecule and its several epitopes (covered in Section B of the Manual). The sensitivity of detection (a positive test) is determined by the amount, the quality, and the characteristics of the method chosen, be it immunofluorescent microscopy, immunodiffusion, counter-immunoelectrophoresis, ELISA, Western blotting, or immunoprecipitation (covered in Section A of the Manual). More importantly. tricks of the trade in laboratory methods, sharpened by exchange of information on the quality and accuracy of results, help to develop a consensus view of the clinical usefulness of the test. This collective wisdom is especially developed by cooperation at an international level and has led to reference standards being agreed, under the aegis of the World Health Organization, the

International League Against Rheumatism, and the International Union of Immunological Societies. In Europe it has found its focal point in annual workshops which we help to convene. These are based on analysis of clinical samples and have evidently led to a raising of standards in detecting autoantibodies for research and application in clinical practice, by involving opinion formers in these areas. Many of these leaders in the various fields have contributed to one or another part of this Manual. Finally, we again want to thank all contributors to this and other sections. Their labour of love can at last be viewed in the context of the whole Manual. It completes the task we had set ourselves two years ago. We believe all our joint endeavours have resulted in the production of a unique compendium of papers. Our original intension was to produce the three sections as a loose-leaf single Manual, which could be updated in parts. Updates to Section B have already started to be incorporated into the Manual this year, and will continue in the forthcoming years for the rest of the Manual, as originally envisaged. However, it has become evident that putting all three parts together is costly and the resulting Manual may be accessible only to a more restricted readership than we wished. For this reason each section may also be published in due course as a freestanding, more economical, paperback. We are in discussion with the publishers about the possibility of making both formats available; the loose-leaf, mainly as a reference manual held by departmental and institutional libraries, and the single sections, to be more widely used by a nonoverlapping readership as personal copies. R.N. MAINI AND WALTHER VAN VENROOIJ LONDON AND NIJMEGEN JANUARY 1996

Autoantibody Manual C1.1, 1–18. 1996 © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

Autoantibodies in rheumatoid arthritis JOSEF S. SMOLEN Klin. Abteilung für Rheumatologie, Univ.-Klinik für Innerr Medizin III AKH, 1090 Wien, Austria

1. Introduction

Rheumatoid arthritis (RA) is the most frequent chronic inflammatory joint disease. Its prevalence is approximately 1% of populations in the western world. RA is a systemic autoimmune disease characterized primarily by polyarticular, symmetric synovitis. It commonly has a relentlessly progressive course, although exacerbations can often interchange with periods of improvement. Rarely, spontaneous remission may occur. RA joint inflammation leads to cartilage degradation, bone erosions and subsequently to extensive joint destruction and functional impairment which can often result in a bed-ridden state. In addition to joint disease, extrarticular disease is seen in up to 40% of the patients; this can be mild (e.g. rheumatoid nodules) or severe (e.g. vasculitis of internal organs) and includes Felty’s syndrome, in which severe RA is associated with splenomegaly and neutropenia. The diagnosis of RA is based primarily on clinical grounds, however the revised criteria for the classification of RA [1] as formulated by the American Rheumatism Associations (today: American College of Rheumatology. ACR) also include a laboratory measure and one determined by joint imaging. These criteria have a specificity and sensitivity of around 90% in distinguishing RA from other disorders. The pathogenesis of RA is still not fully elucidated. There is increasing evidence that activation of cell-mediated immune reactions with extensive involvement of cells belonging to the macrophage and fibroblast lineages lead to massive secretion of cytokines and consequently of metalloproteinases, these are responsible for the unique, destructive and ‘malignant’ properties of synovial membrane and ‘pannus’ [2]. Moreover, RA is linked to HLA-DR4 or, better, to a sequence in the third hypervariable region of most HLA-DR4 (and DRI) 1ß chains [3], further supporting the concept of the significant involvement of the immune system in RA pathogenesis. The etiology of RA is unknown, however RA is regarded an autoimmune disease on the basis of no extrinsic agent being implicated to date as well as of extensive humoral and cellular immunologic reactions to autoantigens. The clinical aspects of the anti-autoantigen responses to be discussed here will mainly deal with those autoantigens which have been described in the AMAN-C1.1/1

previous sections, i.e. rheumatoid factor(s), anti-perinuclear factor and antikeratin Antibodies, anti-RA33, and anti-collagen antibodies. However, it is clear that not all of these tests are available in all laboratories – more sophisticated assays being performed in institutions which are interested in (basic) sciences and not necessarily in routine laboratories. Moreover, central laboratories may not have the same interest in some tests as departmental laboratories. Finally, laboratories with large sample numbers may have different equipment (and techniques) available than small laboratories and vice versa.

2. Historical aspects

Together with other disorders, Klemperer regarded RA as one of the ‘collagen diseases’ or ‘connective tissue diseases’ on grounds of the presence of ‘fibrinoid’ degeneration with some histochemical characteristics resembling collagen in the connective tissue(s) of joints and nodules in the absence of known inciting agents [4]. Waaler [5] discovered the first autoantibody ever described in RA. These antiglobulins, which were named rheumatoid factor (RF) according to the disease. RFs clearly have differential diagnostic value and are even used to describe major subsets of RA (‘seropositive’ vs ‘seronegative’) or by their invariable absence, a major group of non-rheumatoid inflammatory spinal and joint diseases (‘seronegative spondylarthropathies’). However, an involvement in the pathogenesis of RA is unproven, although according to some authors there may be evidence supporting IP’s on the basis of several indirect indications (see later). At a time when the differences between the multiple subtypes of collagen had not yet been recognized and characterized, Carl Steffen demonstrated the presence of anti-collagen antibodies in patients with RA and put forward the hypothesis that autoimmunity to collagen may be a clue in the pathogenesis of RA [6]. This hypothesis proved to be of lasting importance, since humoral and cellular autoimmune responses to collagen can not only be shown in man 17-91, but also in adjuvant arthritis [10], and animal experiments using collagen type II have resulted in a model of chronic, erosive joint inflammation [ 11]. Steffen speculated that anticollagen-collagen immune complexes, on the basis of their sessile nature in vivo in tissue, could even stimulate rheumatoid factor production. However, it is not clear whether the anti-collagen immune response in RA is a primary one or secondary to the extensive tissue degradation observed in the disease. At about the same time as anti-collagen antibodies were described, the anti-perinuclear factor was detected [12]. Three decades later its antigenic component has come close to characterization [13]. It correlates well with anti-keratin antibodies which were described several years later [14] and whose target antigen is currently unknown and is unlikely to be AMAN-C1 .1/2

cytokeratin but appears to be filaggrin (see section B1.5) (see respective chapter). Both types of autoantibodies have some diagnostic value, but their pathogenetic implications are unknown. Anti-RA33 antibodies have been detected in search for RA-specific antinuclear antibody responses [ 15]. The antigen is a heterogeneous nuclear (hn) RNP (16 and Chapter B1.3). Among the many antibodies to nuclear antigens, including those to Epstein-Barr-Virus related antigens (e.g. EBNA) and histones, it is the most RA-specific one on grounds of its low frequency in healthy and EBV-infected individuals and its prevalence in different rheumatic diseases (see below). None of these autoantibodies is entirely RA-specific and thus none is pathognomonic for RA. In many patients they can occur before disease onset [17]. With the exception of APF and AKA which occur together there is no association between these autoantibodies, and therefore they are complementary in their diagnostic value [13]. Moreover, it is of importance to state here that, although these various autoantibodies can occur also in other inflammatory rheumatic diseases and, for example, RF is a particularly ‘promiscuous’ autoantibody, autoantibodies typical of other rheumatic disorders are never or only rarely observed in RA (other than in the context of a clearly identifiable overlap between RA and another disease).

3. Indications for requesting a test

As stated above. diagnosis of RA is based primarily on clinical grounds and the ACR criteria mentioned contain a laboratory test as only one of seven criteria, four of which must be positive [1]. However, once RA is classifiable according to the ACR criteria, we (i) are commonly already dealing with a clinically diagnosable disease, but (ii) are as yet unable to predict the future fate of the disease in an individual patient, particularly if dealing with patients early in their disease course. In very early stages RA may not be unequivocally recognizable (e.g. asymmetric, monoarticular, oligoarticular, lack of hand joint involvement, lack of erosions despite many months duration). Thus, the use of laboratory tests is of particular importance in such early stages. This is considered especially important because early therapy with disease modifying agents (DMARDs) is almost mandatory today, knowing that the fastest progression of joint destruction occurs in the first few years of the disease [19]. Thus, a main indication for requesting one of the tests commonly positive in RA is differential diagnosis, particularly in early disease stages. It is important to note that, aside from other connective tissue diseases (which are accompanied by characteristic autoantibodies) or a diagnosis of virally induced polyarthritis or reactive arthritis are part of the differential diagnosis of early RA. Reactive arthritis, however, is classified as a ‘seronegative’ arthritis, which indicates that these patients are (virtually always!) AMAN-C1.1/3

negative for rheumatoid factor. In fact, they are also negative for APF, AKA and anti-RA33 (1 5, 17, 18). Therefore, not only positive test results, but also negative ones may be diagnostically helpful, since the vast majority (70–80%) of RA patients are positive for at least one of the above tests even in the early stage [17, 18, 20–22) and a negative test, therefore, speaks against RA. Interestingly, whereas the prevalence of RF is lower in early than in established disease (about 50% in early disease, reaching up to 80% at some time during the course of RA) (20, 21, 23, 24), APF, AKA and anti-RA33 occur in similar frequencies both in early as well as in established disease and are present also in RF-negative RA (14–18, 22); thus these newer autoantibodies increase the sensitivity and specificity of laboratory tests for the diagnosis of RA particularly in its early stages [18, 22]. However, the prevalence of some of these autoantibodies may vary between different ethnic populations [25] and their diagnostic value requires evaluation in its context. Anti-collagen antibodies (ACA) have not been studied extensively in early RA patients. It should also be mentioned, that high titer RF is associated with HLA-DR4 according to some authors [26], whereas other autoantibodies have not yet been clearly shown to be immunogenetically linked.

4. The autoantibodies

4.1 Rheumatoid factor 4.1.1 Assays Among other methods (see chapter B1. 1), RF can be measured by a variety of agglutination techniques. The preferred test for large scale clinical use in many hospitals is nephelometry [27, 28]. Nephelometry is a semi-automated method based upon the principle of increasing light scatter intensity in the nephelometer with increasing agglutination, usually of soluble immune complexes. If the antigen-antibody reaction does not permit the formation of large immune complexes, polystyrene (Latex) particle agglutination intensify the reaction. In nephelometry, polystyrene particles coated with human IgG are used for RF determination. This agglutination depends on the RF content of the serum sample (similar to conventional Latex agglutination tests). The signal of scattered light is proportional to the concentration. The RF level is determined by comparison to standards of known RF concentration. This test is probably the best standardised one today, allows expression of results in IU easily and thus good comparability between different centers. Moreover, rheumatoid factor values obtained by nephelometry agree well with conventional Latex fixation tests [28a]. A test result >40 IU/ml is regarded unequivocally positive in our laboratory on the basis of analyses of control sera (unpublished observations). In larger clinical AMAN-C1.1/4

laboratories in central Europe, nephelometry has more or less replaced other agglutination techniques, including the latex test originally described by Singer and Plotz (29) and, today, is the preferred assay for serial routine clinical use on the basis of its simplicity, accuracy, reproducibility, availability and cost. For smaller series of tests, some laboratories use the latex agglutination test (on a slide) as a screening method, since it is simple and fast to perform. If positive, it can then be followed by the classic Waaler-Rose test (see below). As an alternative to nephelometry and latex agglutination, red cell (RBC) agglutination can be regarded as another assay of choice, e.g. by using human Rh positive RBC coated with anti-Rh serum [30, 31] or according to the classic Waaler-Rose method by coating sheep RBC with rabbit antisheep RBC serum [5, 32]. A positive Rose-Waaler test is more specific for RA than the tests using human IgG, including the latex and similar tests, since anti-allotypic antibodies elicited during pregnancy or by transfusion do not react with rabbit IgG [31, 33]; however, to prevent false positive reactions due to heterophilic antibodies, test sera have to be preabsorbed with sheep RBC for the Wader-Rose test. The result is regarded positive beyond a titer of 1:32, a cutoff point clearly above the results obtained in >95%, of healthy control sera. Some laboratories use a latex tests for screening and then follow with a classic Waaler-Rose test. All these tests usually detect RF of the IgM class (IgM-RF). The sensitivity of the mentioned RF tests is in the order of 50–80% (depending upon disease duration. disease manifestations and test employed, see above). The specificity, when compared to healthy controls, exceeds 95%, but when compared to other connective tissue or chronic infectious diseases is significantly lower. Another very important issue is that of standardisation. In order to optimize a test system it is mandatory to use both clearly positive as well as negative controls and to distinguish as correctly as possible between positive and negative in sera containing concentrations of autoantibodies. here RF. at the border between positive and negative. For this purpose, the WHO Standardisation Committee has provided an international reference preparation of rheumatoid arthritis serum which. by definition, contained 100 international units (IU) per ml (33a). The use of this serum has significantly improved performance of and comparability between laboratories (33b) and is now widely established. 4.1.2 Prevalence Rheumatoid factor production can be induced by either antigenic stimulation (e.g. IgG of immune-complexes) or by polyclonal activation [34, 35]. Therefore RF may have important physiologic functions [35]. The term ‘rheumatoid factor’ was coined for the first time [36] several years after its initial detection in RA [5]. RF can occur in 3% of the general AM AN-C1.1/5

population and about 10–15% of healthy elderly (>60 years of age) individuals (20, 37, 38, Table 1). The prevalence of RF in healthy individuals may differ with ethnic background [39]. Although present in up to 80% of RA-patients, RF can also commonly occur in other rheumatologic and nonrheumatic disorders [20, 38,40–53, Table 1). In lymphoproliferative diseases, but occasionally also in RA and in Sjögren’s syndrome, RF can be monoclonal or oligoclonal[53]. 4.1.3 Clinical and immunogenetic associations As always with laboratory tests, πp clinical context and evaluations of patients is of extreme importance for their interpretation. Given the fact that several diseases, such as SLE, Sjögren’s syndrome, polymyositis, viral infections, etc can be accompanied by a symmetric polyarthritis, it is Table 1. Prevalence of RF (by latex agglutination) in different diseases Disease

Approximate prevalence

RA

80%

Sjögren’s syndrome Mixed eryoglobulinemia Systemic lupus erythematosus Mixed connective tissue disease Polymyositis Systemic sclerosis Juvenile chronic arthritis

70%) 70% 30% 25% 20%) 20%) 15%

Subacute bacterial endocarditis Infectious hepatitis EBV, CMV infections Leprosy Tuberculosis Trypanosomiasis Syphilis Sarcoidosis

40% 25% 20% 25% 15%) 15% 10% 10%

Waldenström’s macroglobulinemia Liver cirrhosis Pulmonary interstitial disease

30% 25% 25%

Healthy controls Elderly (>70 years)

<5% 15%

RF is usually of low titer in most of these diseases except for Sjögren’s syndrome and mixed eryoglobulinemia where it can reach titers as high as in RA patients. The prevalences are compounded from the literature quoted and refer to patients with established disease. Rates may fluctuate when considering patients with early disease or after successful therapy.

AMAN-C1.1/6

mandatory to obtain additional clinical, laboratory and serologic information if there are any doubts regarding the diagnosis of RA. Moreover, as stated above, RF-testing is helpful even when providing a negative result, particularly in case of undifferentiated arthritis, Finally, although RF levels (titers) are usually lower in healthy individuals and in patients with other diseases when compared to RA. a positive RF-test, even if high titers are present, is of little help in the absence of clinical information and particularly in the absence of inflammatory joint disease. However, in (younger) healthy individuals the presence of RF may be regarded as a risk factor for the development of RA, since its presence may antedate clinically manifest disease by several years [54, 55]. (Similar risks can also be found for other autoantibodies, see later.) Seropositive RA is a more aggressive joint disease and is more commonly accompanied by extraarticular manifestations than seronegative RA [20, 21, 24, 25, 56, 57]. In particular, rheumatoid nodules and vasculitis occur almost exclusively in seropositive patients [24, 25, 56-58]. Moreover, seropositive RA is associated with increased mortality [59–621. The predisposition to seropositive RA is associated with a shared epitope on HLA-DR4 and DR 1 [26, 56, 63]. HLA-DR4 may generally predispose to RF production as seen in a study of healthy relatives of RA patients [64]. The association of HLA-DR4, (particularly B1*0401-) homozygosity with increased severity of RA [63, 65], is consistent with the association of severe RA with RF positive disease. When looking at the different published studies, one can find the associated HLA molecules (DR4 and/or DRI) or the associated sequence of the third hypervariable region of the HLA-DR ß chain in >80% of RF-positive patients (reaching up to 100% with B1*0401homozygosity), but only <60% of RF-negative patients (with homozygosity seen very rarely). The status of RF can change with disease activity [24, 66, 67], therapy [67] and disease duration, and ‘seroconversion’ in both directions can occur during follow up. Conversion from seropositivity to seronegativity is rare and is usually only observed in the course of successful therapy with disease modifying drugs (DMARDs). Conversion from seronegative to seropositive is more common, since – as already stated – early RA is less commonly RF+ than later RA.The titer of RF in synovial fluids [69–70] is usually similar to that observed in serum, however, occasionally RF can be negative in serum and positive in the SF. The disease should ben be regarded as RF-positive. However, even in ‘seronegative’ patients according to the above definition, there may be ‘hidden’ (masked) RF [71, 72] or RF of other subclasses [70, 73–77]. With regard to IgG-RF it is of interest that it can selfassociate and thereby form (pathogenic) immune complexes [78, 78a]. IgGRF has been found associated with RA-vasculitis and the hyperviscosity syndrome in RA [78b]. RF subclasses are best determined using ELISA [79]. Although their clinical value seems to be limited. there is a suggestion in the literature that patients with IgA-RF have a more severe disease course [79a]. AMAN-C1.1/7

In clinical practice, RF commonly occurs together with other autoantibodies: in RA particularly with anti-collagen, anti-RA33, APF and antikeratin. However, these latter autoantibodies are also found in RF-negative RA, usually in a similar frequency as in seropositive RA (see respective descriptions). Antinuclear antibodies, as analysed by conventional indirect immunofluorescence techniques, also occur in RA, and more commonly in seropositive than seronegative cases, however antibodies to nuclear subsets characteristic of other connective tissue diseases are only rarely observed in RA. In contrast, when RF is found in Sjögren’s syndrome it is usually accompanied by anti-Ro (and anti-La), in SLE by anti-dsDNA, anti Sm, anti-Ro, anti-RNP, in mixed connective tissue disease by anti-RNP, in scleroderma by anti-Sc170, etc. In all these other connective tissue diseases RF is almost never found as the only autoantibody. RF has not been shown to be linked to clinical disease activity as clearly as, the acute phase response [80, 81]. However, there is some evidence for a correlation of RF titers to disease activity [24, 66, 67, 82], and there is indirect linkage to disease activity since RF-titers decrease with successful therapy, particularly methotrexate and parenteral gold [67, 79, 83, 83a]. Should RF be determined more than once during follow up? Clearly, in early RA a single determination with a negative result is not sufficient to rule out seropositive RA with all its implications. Therefore, when RA is suspected (or diagnosed) and RF is negative, follow up determinations every six to twelve months should be performed. Once a positive test has been obtained (and confirmed), one does not need to follow RF throughout the disease course. However, in addition to the clinical status and the acute phase response, successful therapy is often accompanied by a fall in RF titre, and, therefore, some centers follow RF levels every six to twelve months during therapy. Particularly with respect to such follow up. nephelometry is an assay of choice because it allows easy quantitation in international units (IU).

4.2 Antiperinuclear factor and anti-keratin antibodies Assays Antiperinuclear and anti-keratin antibodies are detected by indirect immunofluorescence (see this Manual, chapters A.10., B.1.2 and B.1.5), since no other detection systems are currently available. The assays are usually only offered by some specialty and not by routine laboratories: currently no cell line containing the antiperinuclear antigen (which is associated with the keratohyaline granule) is available, and only 10% of donors, who also have to be easily available (!), have sufficient antigen in their buccal mucosal cells. Also, no cell line containing the target antigen of antikeratin antibodies (AKA), which is present in the lower middle third of the rat oesophagus, is available. As long as the antigens are not fully defined (perinuclear factor AMAN-C 1.1/8

Table 2. Prevalences of antiperinuclear factor Disease

Approximate prevalence of APF

AKA

Rheumatoid arthritis Ankylosing spondylitis Psoriatic arthritis SLE Primary Sjögren’s syndrome Scleroderma

75% 5% 5% 10% 30% 10%

50% 2% 2% 3% 10% 10%

Autoimmune thyroiditis Infectious mononucleosis Fibrosing alveolitis

20% 30% n.t.

n.t. 10% 70%

1%

<5%

Healthy individuals

may be similar to the AKA-target), the limitations of the assay systems will preclude widespread use. In addition to the Chapters in this Manual, a recent review has dealt extensively with these two antigen-antibody systems [97]. 4.2.2 Prevalence, sensitivity and specificity The specificity of APF for RA has been described to be between 73 and 99'21 [97, 98, 12], the sensitivity between 49 and 87% [12, 97, 99]. A median estimate of published work would bring specificity to approximately 90% and sensitivity to about 70% The specificity of AKA is generally above 90%, the sensitivity ranges between 40 and 60% in published work [13, 97]. 40–90% of AKA-positive sera are also APF positive [13, 97, Chapter B. 1.2]. The specificity of APF and AKA has also to be viewed vis-a-vis different specific diseases (Table 2). Thus, in patients with primary Sjögren’s syndrome APF has been described in up to 30% [13, 100] and in SLE up to 50% [98]. Patients with infectious mononucleosis and autoimmune thyroiditis have repeatedly been found APF-positive in significant (20–50%) proportions [97, 101, 102]. Similarly (Table 2), AKA have been observed in significant (10 to >50%) proportions of scleroderma and primary Sjögren’s syndrome patients as well as in fibrosing alveolitis [97, 103, 104]. In healthy controls, however, AKA and APF occurred in <5% in most of the studies. Most APF-positive sera are also RF-positive, although up to 20% APFpositive specimens are RF-negative [ 105]. An association between HLADR4 and APF or AKA has not been established [106–108], but APF may be DR4-associated in RF-seronegative patients [ 106]. 4.2.3 Clinical findings Although early studies have indicated that the presence of APF or AKA may be related to certain disease manifestations, such as rheumatoid nodules [12], AMAN-C1.1/9

neither APF nor AKA have been consistently shown to correlate to clinical disease expression, be it activity, severity or outcome. On the other hand, APF, like AKA, may precede the disease onset [109], but the proportion of respective patients is low. APF and AKA can also be found in synovial fluids, but their prevalence there does not exceed the one observed in serum [12, 110]. Serial studies of APF and AKA have only rarely been performed in RA patients [109]. There is currently no indication that the assay needs to be repeated during follow up. However, confirmation of its presence or lack about six months after the first determination is recommended. APF and AKA expand the diagnostic armamentarium, since they are also positive in RF-negative RA patients. However, they do not predict disease severity nor therapeutic responsiveness.

4.3 Anti-RA33 antibodies 4.3.1 Assays Anti-RA33 is detected by immunoblot technique using nuclear extracts (see chapter B1 .3). In contrast to most other anti-nuclear antibodies detectable by immunoblot, the concentration of anti-RA33 is usually low and, therefore, a low dilution (approx. 1:100) is recommended for testing. In addition, an ELISA has been developed (see chapter B1.3), but as long as its specificity and reproducibility is not fully confirmed and the necessity for quantitation of the antibody not established, the preferred assay for clinical use is the immunoblot. The antigen, RA33, has been defined as the A2 protein of hnRNPs, the heterogeneous nuclear ribonucleoproteins [ 16 and Chapter B1.3]. 4.3.2 Prevalence, sensitivity and specifcity Anti-RA33 is the most recently described antibody occurring in RA [15]. However, the antibody is also found in MCTD and SLE patients [16, 111]: its prevalence in all these three disorders is between 25 and 40%. In SLE and MCTD, however, it is always associated with at least one other characteristic autoantibody, such as anti-RNP, anti-Sm, or anti-DNA. It is found in <5% of other disease controls and healthy individuals [15, 16, 18, 111] (Table 3). Its sensitivity for RA is about 35%, its specificity, excluding MCTD and SLE, over 95%. Thus, its sensitivity is lower than that of RF, APF and AKA, but its specificity may be somewhat higher than that of APF and AKA, particularly since it is not found in infectious mononucleosis [15]. Anti-RA33 is present in a similar proportion of seropositive and seronegative RA [15], and it can also be found in APF/AKA-negative patients [18]. The antibody may precede the onset of disease by a shorter period than other markers and may then be a marker for imminent RA [18, 25]. However, anti-RA33 may differ in prevalence in different ethnic groups (it AMAN-C1.1/10

is rare in Greek and Finnish RA patients) [25 and unpublished observations]. This finding is surprising since such differences are usually not observed for the other autoantibodies detected in RA. The anti-RA33 autoantibody may occur together with antibodies to the A1 protein of hnRNPs [111, 11] which is of lesser sensitivity and specificity for RA. There is no known HLA association. 4.3.3 Clinical associations The antibody is not associated with certain disease subsets or clinical manifestations in RA, but in contrast to RF it is usually present in similar frequencies in early as well as late RA [112]. In SLE, however, anti-RA33 may be associated with more severe (erosive) forms of arthritis [111, 113]. Anti-RA33 can also be found in synovial fluids, its presence there does not extend its diagnostic potential. In contrast to RF, ACA, APF and AKA, for which epitope mapping is either not helpful, has not been performed yet or the antigen has not been characterized, epitope mapping of RA33 may be helpful in differentiating Table 3. Prevalences of anti-RA33 Diseases

Approxim. prevalence

Rheumatoid arthritis SLE Mixed connect. tissue disease Primary Sjögren’s syndrome Scleroderma Poly/Dermatomyositis Psoriatic arthritis Ankylosing spondylitis Reactive arthritis Osteoarthritis

35% 25% 40% <5% 7% <5% <5% <5% <5% <5%

Infectious mononucleosis

<5%

Healthy controls

3%

epitopes recognized by RA from those recognized by MCTD and SLE sera [114, 115 and chapter B1.3]. Although according to current knowledge the antibody does not reflect disease activity, it has been seen to become negative with successful therapy (unpublished observations). Nevertheless, except for a single confirmatory assay repeated three to six months after an original negative result there is currently no indication that the test should be redone regularly. Moreover, the autoantibody does not predict responsiveness to therapy.

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4.4 Anti-collagen antibodies 4.4.1 Assays Anti-collagen antibodies (ACA) can be measured by passive hemagglutination (PH), indirect immunofluorescence, radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) [6, 7, 86, and chapter B1.4]. Passive hemagglutination, in which f.i. tanned RBC are coated with collagen, was the first technique used to reliably show the presence of anticollagen antibodies in synovial fluids of RA patients [6]. Correlation between RIA and hemagglutination is good [7]. However, the sensitivity of RIA and ELISA is greater than that of passive hemagglutination and indirect immunofluorescence and the sensitivity of RIA is even greater than that of ELISA [7, 86]; therefore RIA is the preferred assay for clinical use. Although autoantibodies to many types of collagen, including the major types I and III [6, 7, 86], are found in RA, autoimmunity to type II fits the collagen hypothesis best, since type II is the major cartilage collagen and has been shown to induce chronic arthritis [11]. Therefore assays for anti-collagen II are the most important ones for RA. The availability of the ACA-RIA in routine clinical settings is rare, and usually only few specialty laboratories have this and other techniques established.

4.4.2 Prevalence, sensitivity and specificity of ACA The value of the antibody determination is limited: the prevalence of ACA is relatively low in RA-sera [9, 86, 87] and ACA are not uncommonly found in many other diseases [87–89a] (Table 4). Thus, specificity and sensitivity of type II ACA for RA are low. Anti-collagen antibodies occur with different prevalence rates depending upon the type of collagen used, but usually do not exceed 30%) in serum [9, 86, 88]. Neither the occurrence of serum autoantibodies to native or denatured type I nor type II collagen is specific for RA [88–90], but synovial fluids of RA patients, compared to controls, contain anti-collagen antibodies in higher frequencies and amounts [7]. This can be demonstrated by RIA, passive haemagglutination and other techniques. (The potential interaction of fibronectin or glycosaminoglycans in PH is of little relevance, since at the titers of anti-collagen antibodies observed by PH fibronectin and other substances become too highly diluted to be still reactive [90]). Anti-collagen antibodies (ACA) are also contained in synovial fluid immune complexes and have also been eluted off rheumatoid cartilage [91, 92]. Thus, ACA-determination in SF is of greater value than in serum. Regarding the immune response to collagen it should be borne in mind that the first complement component, C1q, has a collagen-like tail [93] and that there is both humoral and cellular cross-reactivity between collagen and AMAN-C1.1/12

Clq [94]. Moreover, Clq is partly degraded by granulocyte collagenase [95], and fibronectin also binds to the collagen-like fragment of Clq [96]. In particular the immunological cross-reactivities have to be taken into account whenever antibodies to collagen are found, but also when immune complexes are determined using Clq. Moreover, antibodies directed against Clq which Table 4. Prevalences of anti-collagen II antibodies Disease

Approximate prevalence

Rheumatoid arthritis SLE Scleroderma Relapsing polychondritis Osteoarthritis

30% 20% 15% 50% 10%

Leprosy

50%

Healthy controls

5%

Antibodies to other types and to denatured collagens may be more prevalent in RA. but also in other diseases. including chronic liver diseases

have been described in rheumatic diseases may well be cross-reactive anticollagen antibodies, at least in part. In addition, the presence of anti-DNA antibodies in patient sera may give false positive reactions due to binding of DNA (similar to other polyanions) to collagen, and such sera may have to be DNAse-treated before testing. All these issues, which are currently not well resolved, decrease the value of ACA-determination unless performed in truly specialised laboratories. In any case. since type II collagen is the predominant cartilage collagen and since type II collagen can induce an experimental arthritis in mice and rats [II], autoimmunity to type II collagen is of special interest in RA. However. neither the predictive nor the diagnostic value of ACA exceed that of the other tests discussed here. Hence, since also specificity and sensitivity are low, determination of ACA is currently more important for scientific than for diagnostic purposes. 4.2.3 Clinical associations There are no particular associations to subtypes of RA or clinical manifestations. ACA occur not only in RF-positive, but also in seronegative RA. Correlations to other autoantibodies have not been analysed yet. Anticollagen II antibodies detected by an ELISPOT assay on B-cells from joints showed a further prevalence in HLA-DR 4 patients with RA (Klareskog).

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5. General recommendations

In patients with clinically undifferentiated arthritis RF, APF, AKA, and anti-RA33 can be helpful for differential diagnosis. If negative and with continuing clinical activity, tests should be repeated 3–6 months later. The positivity of 1 of these tests indicates the possibility of RA; the positivity of 2 or more of these tests (in the absence of other known autoantibodies, particularly to nuclear antigens) makes RA highly likely [18], since in combination these autoantibodies they occur only very rarely in other arthritic disorders. In practice, only RF is established for routine clinical use. An international standard is available for rheumatoid factor [33a, b], and should be used for expression of results. Standards are lacking for all the other serological markers, and consensus finding study groups have only recently started to improve techniques in the field [116] and standard serum preparations do not exist. Moreover the substrates and antigens for APF and AKA are not available in a stable or consistent form. The determination of anti-RA33, currently depends upon Western blotting, a technique which has not been established in routine laboratories.

6. Future trends and prospects

RF remains an important autoantibody in evaluating the diagnosis and prognosis of RA. Future detailed investigations into RF subclasses [76] may prove to be of importance in monitoring response to therapy. Moreover, determination of several of the autoantibodies discussed here and HLA-DR4 subtyping may lead to a better definition of outcome of RA and of increasing accuracy of the diagnosis of early disease. Finally, quantitation of APF, AKA and anti-RA33 may reveal a potential value in the follow up of RA. The search for disease specific autoantibodies in RA is continuing. New candidate autoantigens appear in the literature but have not yet been clearly characterized [117, 118]. It will, also be important to characterize the target antigens of APF and AKA and to determine the epitopes relevant for RA. Such epitope mapping, which has already been successfully started for antiRA33, may reveal subpopulations of autoantibodies that give further insight into pathogenesis, if not further assistance to the clinician in diagnosis and management of RA.

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Autoantibody Manual C2.1. 1-17. 1996 © 1996 Kluwer. Academic Publishers Printed in The Netherlands.

Clinical aspects of antibodies to double-stranded DNA A.J.G. SWAAK' and R.J.T. SMEENK² ¹ Department of Rheumatology, Dr. Daniel den Hoed Clinic, Rotterdam, The Netherlands ² Department of Autoimmune Diseuses, Central Lab. Netherl. Red Cross Bloodtr. Serv. ( CLB), Amsterdam, The Netherlands

1. Introduction

A broad spectrum of autoantibodies can be demonstrated in the serum of SLE patients. It was the observation by several investigators back in 1957 that there may exist a reaction between DNA and SLE sera [1–4] that started the serological studies of this disease. The earliest techniques used for detection of anti-dsDNA were relatively insensitive precipitation methods such as complement fixation and hemagglutination. At this moment specific and sensitive assays like radio immunoassay (RIA), immunofluorescence test on Crithidia luciliae (IFT) and enzyme linked immunosorbent assay (ELISA) are available for detecting and quantitating antibodies to DNA. As was discussed by Stollar [5] the measurement of anti-dsDNA antibodies depends on numerous variables, amongst which are included the purity of the test antigen, the distinction between binding by antibodies and non-immunoglobulin proteins, the isotype of the antibody and its functional properties such as complement fixing ability and avidity. Nowadays the presence of antibodies to double-stranded DNA (dsDNA) is regarded as highly specific for SLE. Of the few patients with other connective tissue diseases found to have elevated anti-dsDNA, most have been found to have SLE on reevaluation or on follow-up [6, 7]. Because of the fact that no absolute definition exists for SLE, an existing concept of the disease relies on a description a group of symptom complexes without any obvious cause and lacking a pathognomonic feature, the diagnosis is often difficult to make. The widely used definitions are the criteria for classification of SLE, before 1982 the preliminary criteria of 1971 [8], and since 1982, the modified criteria [9]. These are intended for use in classification rather than diagnosis of SLE. The revision of the criteria in 1982 was considered necessary in order to add to a positive LE cell preparation, other laboratory tests such as antiDNA, which had proven to be SLE specific. In this chapter, focused on antibodies to DNA in patients with SLE. special attention will be paid to their role in the diagnosis, the follow-up, prognosis and pathogenesis of the disease. AMAN-C2.1/1

2. Detection methods

Caused by the high specificity of anti-dsDNA for SLE, a lot of effort has been put into proper detection of these antibodies. This has resulted in the development of quite a number of anti-dsDNA assays. Out of these the anti-dsDNA ELISA, the PEG assay, the indirect immuno-fluorescence technique with Crithidia luciliae and the Farr assay are the most widely used assays.

3. Assays to detect antibodies to DNA

3.1 Farr assay The Farr assay employs ammonium sulphate precipitation to separate DNA/Anti-dsDNA complexes from free (radiolabeled) DNA [10]. This radio immunoassay (RIA) was shown to be specific for SLE provided pure double stranded DNA is used as the antigen [11]. The use of iodine-labelled calfthymus DNA makes the assay prone to non-specific DNA binding [12]. Also the choice of assay conditions employed will influence the assay [11].

3.2 PEG assay The PEG assay is a RIA in which radiolabeled (PM2) DNA is used as antigen. DNA/anti-DNA complexes are separated from free radiolabeled DNA by means of a 3,5% polyethylene glycol precipitation [13, 14]. This assay is very sensitive to non-specific DNA binding by serum proteins; the non-specific DNA binding can easily be eliminated by the addition of an excess of the polyanion dextran sulphate to the incubation mixture.

3.3 Crithidia test Crithidia luciliae, haemoflagellates containing a giant mitochondrion which consists of pure dsDNA, are used as substrate [15]. Immunofluorescence of this so-called kinetoplast therefore indicates the unequivocal presence of antibodies to dsDNA.

3.4 ELISA Double-stranded DNA binds very poorly to the plastic, because both DNA and plastic are negatively charged. Therefore a precoat of the plastic is generally employed, either using poly-L-lysine or protamine sulphate [16]. AMAN-C2.1/2

More recently, streptavidin precoated plates have been applied in combination with photobiotinylated DNA [17, 18]. DNA is coated overnight to such a precoated plate. In between, plates are washed with PBS. Dilutions of sera are incubated with the plates for 2 h and bound antibodies are detected by means of a horse-radish peroxidase conjugated anti-immunoglobulin antiserum. The bound enzyme catalyses the oxidation of a suitable substrate by H2O2 to a coloured reaction product. In most cases, the reaction is stopped by the addition of an equal volume of 2N H2SO4, and the plates are read in a Titertek Multiskan at 450 nm.

3.5 Comparison of anti-dsDNA assays General comparison of the four anti-dsDNA assays using sera of patients with SLE leads to high levels of correlations between the various assays. Calculated statistical correlations are summarized in Table 1. Table 1. Correlation between four anti-dsDNA assays Anti-dsDNA assay

PEG assay

Crithidia test

ELISA

Farr assay

r = 0.67" t = 0.63b

r = 0.85 t = 0.69

r = 0.67 t = 0.53

PEG assay

–

r = 0.83 t = 0.69

r = 0.62 t = 0.63

Crithidia test

–

–

r = 0.70 t = 0.66

Anti-dsDNA levels of sera of defined SLE patients were determined by the Farr assay. PEG assay. Crithidia test and ELISA a coefficient of correlation by linear regression analysis b Kendall’s rank correlation coefficient Taken from: Smeenk (1991) Rheumatol Int 11:101–107

3.6 Discrepancies between the assays Because anti-dsDNA assays are often routinely used to screen sera for the presence of anti-dsDNA to provide evidence for, or to refute the diagnosis SLE. another approach is to compare the behaviour of sera which are sent for routine anti-DNA determination in these four assays. In general, as shown in Table 1, a comparison using sera of patients with defined SLE led to high levels of correlation between these assays [ 19, 20]. However, discrepancies were also found, as illustrated in Table 2, when the results are compared obtained in well defined sera of patients with a variety of autoimmune diseases. The specificity further decreased by AMAN-C2.1/3

comparing the results obtained in the different assays in sera with a possible diagnosis of SLE (Table 3). Table 2. Comparison of the disease specificity of 4 anti-dsDNA assays Percentage of sera positive in assay

Active SLE Rheumatoid arthritis Sjögren’s syndrome Scleroderma Autoimmune liver disease Autoimmune thyreoiditis

Farr

I FT

PEG

ELISA

98 1 0 0 0 0

96 0 0 0 0 0

96 5 7 0 20 7

100 3 20 30 15 13

Taken from: Smeenk (1992) Mol Biol Rep 17:71–79

Table 3. Screening of 14,417 sera sent for routine anti-dsDNA determination by 3 antidsDNA assays Anti-dsDNA assay

Number of sera positive/negative

Crithidia-test Farr assay PEG assay

470 + 535+

790– 255–

Taken from: Smeenk (1982) Clin Exp Immunol 50:603–610

A number of these discrepancies can be explained on the basis of differences in avidity of anti-dsDNA found in patients. The Farr assay is strictly selective for antibodies of high avidity, whereas the other assays are not. Furthermore, DNAianti-dsDNA complexes have to be quite large to be precipitated in the PEG assay, which explains why some sera react positive in the IFT on Crithidia luciliae but negative in the PEG assay. Finally the ELISA may non specifically detect IgM antibodies, irrespective of their specificity. Using the Crithidia test or the ELISA as screening assay for routine anti-dsDNA determination high as well as low avidity antibodies are detected. A second advantage of these techniques is the intrinsic check for detecting antibodies only. However, a positive assay result is not always indicative that the patient or will develop SLE: anti-dsDNA of lower avidity may occur in other diseases as well. Additional problems encountered when using an anti-DNA ELISA include the use of precoats such as poly-L-lysin or protamin, to which complexes containing DNA readily bind. AMAN-C2.1/4

Caused by the fact that measurement of high avidity anti-dsDNA antibodies has the highest specificity for SLE, an assay which preferentially detects high avidity antibodies, such as the Farr assay, is preferable for diagnostic reasons. Therefore, in our view screening for the presence of anti-DNA is best performed using either the Crithidia test or the ELISA, but positive results should always be confirmed using a Farr assay. In case an in-house Farr assay is not available, commercial kits such produced by Amersham-Kodak (U.K.) or Diagnostics Products Corporation (DPC; Los Angeles, U.S.A.) provide excellent alternatives.

4. Diagnostic significance of anti-dsDNA antibodies

Early methods for detection of anti-dsDNA antibodies showed that the presence of these antibodies was strongly correlated with SLE. However with the development of more methods for detection, these antibodies were found to occur in numerous clinical syndromes like uveitis, discoid lupus erythematosus, rheumatoid arthritis, juvenile rheumatoid arthritis and further in a wide variety of patients [21–24]. From the beginning it became clear that from a diagnostic point of view using 2 assays was superior in terms of sensitivity and specificity the Farr assay using well defined radioactive dsDNA and the immunofluorescence technique (IFT) using the kinetoplast of Crithidia luciliae as substrate [25]. The most frequently used method for testing the sensitivity and specificity of a newly developed assay is by testing panels of serum samples from selected patients with well defined clinical diseases. In this way the specificity for SLE of anti-dsDNA measured by the Farr assay and by the immunofluorescence test (IFT) on Crithidia luciliae was confirmed (Table 2). However when sera for routine anti-dsDNA determination were tested, the specificity for SLE of both methods was completely lost [6]: only 33% and 46% of the patients positive in the IFT, and the Farr assay respectively fulfilled the ARA criteria. One should consider that sera which are tested for routine examination will come from patients suspected of having SLE, and may, therefore, include many patients with incomplete expression of its disease. The only way to prove the specificity of anti-dsDNA for the diagnosis SLE, was to follow the non-SLE patients with anti-dsDNA in their circulation, to see whether these patients would develop SLE or an SLE-like syndrome [7] at a future time point. Overall, the results we obtained show that nearly 15% of the patients positive in the Farr assay may never develop SLE. In our view the diagnostic significance of anti-dsDNA can only be (and is) proven in this prospective way. So far, only the diagnostic significance of the Farr assay has been evaluated in this way. This would imply that the diagnostic significance of results obtained in other assays are still questionable.

AMAN-C2.1/5

5. The prognostic significance of anti-dsDNA antibodies

As described earlier, SLE patients are characterized by a variety of autoantibodies, of which anti-dsDNA antibodies are the most specific for SLE and are detected in almost all patients with active disease [26]. Several studies have claimed a relation between disease course and the profiles of these antibodies [26–29], but others did not find such a correlation [30]. In asymptomatic patients very high levels could also be observed [31]. Major drawbacks of the mentioned studies are that they were mostly of retrospective character, without quantitation of the amount of antibodies. In a longstanding prospective study of 130 SLE patients [32, 33] which were followed monthly. a firm and clear correlation between anti-dsDNA antibody levels and disease activity could be observed. In these 130 SLE patients, thirteen patients developed fifteen periods of an exacerbation during the study period. All exacerbations were preceded by a continuous increase of anti-dsDNA and followed by a sharp drop of the levels, mostly at the period

Fig. 1. Anti-dsDNA profile and clinical course of patient Bu. Patient Bu. female, born 1944. was first admitted to the hospital in 1971 for vague complaints of the intestine. slight exanthema. a positive LE cell phenomenon and arthralgia. In April 1975 complaints of her intestine increased. in June followed by skin lesions. Early in July she was readmitted to the hospital for a pleural rub was heard. combined with an active arthritis. At that occasion. also for the first time, she developed a proteinuria. (Taken from: Swaak AJG (1979) Arthritis Rheum 22:1414–1415.) AMAN-C2.1/6

the disease manifestations became manifest. Fig. 1 illustrates the need for quantitation (in units/ml) but also the clear correlation between anti-dsDNA profiles and disease course. Results comparable to our data have more recently be obtained by Ter Borg et al. [34, 35]. An important proviso is that the claimed correlation is only proven for high avidity anti-dsDNA antibodies, using the Farr assay. Looking specifically at low avidity anti-dsDNA antibodies, contrasting results were obtained. In a longitudinal study of the relevance of low avidity anti-dsDNA measured by the PEG assay [36] showed that these patients had a rather mild course of their SLE, with absence of renal involvement. Furthermore, antidsDNA detected by the PEG assay had little predictive value in respect to clinical exacerbations. In patients defined by having only anti-dsDNA detectable in the PEG assay, a change in anti-dsDNA avidity (i.e. antidsDNA becoming Farr positive) was rarely observed. These observations illustrate that the prognostic significance of anti-dsDNA is strongly influenced by its avidity c.q. the assay which will be used: Farr versus PEG assay.

6. Prevalence of anti-dsDNA antibodies in SLE and other diseases

Following the initial observations of the occurrence of antibodies to dsDNA in patients with SLE over the years much research has been devoted to the uniqueness of these antibodies for SLE patients. The initially developed methods showed that the presence of anti-dsDNA was strongly associated with SLE, later more sensitive methods were developed and anti-dsDNA was also found to occur in other clinical syndromes (Table 4). Most of the work on the specificity of anti-dsDNA assay has been carried out by testing panels of serum samples from selected patients with well defined clinical manifestations as shown in Tables 2 and 4. Testing panels of sera from selected patients with well defined clinical manifestations will exclude analysis of sera from those patients for which the use of anti-dsDNA as a diagnostic tool is most relevant, namely Table 4. Incidence of anti-dsDNA in serum of patients with various autoimmune diseases Author

Koffler (1969) Kredich (1 973) Aarden (1975) Fritzler (1980) Sarai (1980)

Ref.

SLE

41 42 43 44 45

PSS**

%

+

%

+

%

+

50 33 48 80 56

60 88 98 28 95

32 67 150 30 10

9 0 0 7 0

– – 17 8 30

– – 0 0 0

–

* Primary Sjögren‘s syndrome

AMAN-C2.1 /7

Prim. SS*

+

** Progressive systemic sclerosis

+ Positive findings

RA

70 17 26 15

%

0 0 0

patients with more or less SLE-like syndromes, not meeting the actual diagnosis SLE. By screening 4431 sera sent to our laboratory for diagnostic reasons, anti-dsDNA antibodies were found in 66 patients (Table 5) who did not fulfil the ARA criteria for SLE and did not diagnostically have SLE [6]. By selecting patients sera in this way the specificity of the detection was 46%. In order to evaluate the relevance of the presence of anti-dsDNA in nonSLE patients, a prospective study was performed from 1974 until 1982, in which all non-SLE patients that once had anti-dsDNA in their serum, were followed to see whether they would develop SLE. Of a group of 441 non-SLE patients 70% developed SLE within the first year after anti-dsDNA had been Table 5. Diagnosis of 66 non-SLE sera positive for anti-dsDNA in the IFT Anti-dsDNA in the Farr assay Neg. (<10 units/ml)

Pos. (≥10 units/ml)

Arthritis unclassified Renal disease Liver disease Drug induced LE Diseases of the respiratory tract Diseases of the digestive tract Myasthenia gravis Other diagnosis

16 6 5 4 2 2 1 6

17 0 0 0 0 0 0 7

Total

42

24

Table 4 shows the clinical diagnosis of the non-SLE patients with a positive kinetoplast fluorescence (IFT). The diagnosis arthritis represents a diversity of diagnoses. mainly RA [21]. polysynovitis [2] or arthralgia [10]. The following diagnoses represent renal diseases: glomerulonephritis [1]. focal glomerulonephritis [1]. deterioration of the renal function without a clinical-pathohistological diagnosis [4]. for liver disease, autoimmune hepatitis [1]. chronic hepatitis [1]. cirrhosis [2], liver carcinoma [1], for diseases of the digestive tract, colitis [1]. malabsorption syndrome [1] and for diseases of the respiratory tract. pneumonitis [I]. pleuritis [1]. Other diagnosis: unclassified diseases [10], Dühring’s disease [1], Hodgkin’s disease [1], Raynaud’s syndrome [1] Taken from: Swaak AJG (1982) Ann Rheum Dis 41:388–395

detected by means of the Farr assay. Eighty two of the remaining 137 nonSLE patients were studied for a very long period of time, depending on their year of entry [7]. From the data presented in Table 6 it can be seen that there is a tendency to either develop SLE in the following three years or not at all. From these findings it can be concluded that of the non-SLE patients with anti-dsDNA in the circulation nearly 85% will develop SLE within a few years. Only 15% of the patients did not fulfil the ARA criteria after 5 years. The above findings clearly show that prevalence, but also the incidence of anti-dsDNA antibodies, is strongly influenced by the assay used. AMAN-C2. 1/8

Table 6. Follow-up data of the anti-dsDNA positive non-SLE patients (1974–1982) Follow-up years

0–1 1–2 2–3 3–4 4–5 5–6 6–7 7–8 8–9

Number of followed patients in relation to the duration of the follow-up

386 82 81 60 41 32 20 6 1

Patients that developed SLE

No.

%

304 1 10 15 2 3 – – –

69 1 12 25 5 9 – – –

137 non-SLE patients were evaluated which were upon screening positive for anti-dsDNA antibodies measured with the Farr assay. During the follow-up 55 patients were lost. Ten of these died of non-SLE related causes and most of the other 45 visited physicians who did not participate in the study Taken from: Swaak AJG (1985) Ann Rheum Dis 44:245–251

7. Genetic aspects of anti-dsDNA antibodies

A number of studies has suggested an association of both HLA-DR2 and DR3 antigens with SLE [37, 38], or an association with only HLA-DR3 [39, 40]. Other studies failed to show any correlation of SLE with a particular major histocompatibility (MHC) product [41, 42]. It would appear that ethnic and/or racial backgrounds influence the HLA association with SLE. Restriction fragment length polymorphism (RFLP) analysis of HLADR, DQ, DP and C4 null alleles in Caucasian patients with SLE revealed an increase in DR3(DRw17) and DQw2.1 in association with the presence of C4 nullgenes [43]. The authors observed no association of DR or C4 null alleles with clinical features of the patients studied, nor was there any association with anti-dsDNA or levels thereof.

8. Anti-dsDNA and disease spectra

Antibodies to DNA differ widely in their avidity towards DNA. This is reflected in their assay behaviour. Based on this distinct behaviour. patients can be defined and evaluated to see whether differences in anti-dsDNA avidity are reflected in the spectrum of disease features shown by the patient. Farr assay and PEG assay are both RIAs in which the same radio-

AMAN-C2.1/9

labeled DNA is used, but which differ with respect to the way DNA/antidsDNA complexes are precipitated. As stated before, the Farr assay is strictly selective for high avidity anti-dsDNA whereas the PEG assay is not. This opens a possibility to obtain a measure of anti-dsDNA avidity if anti-dsDNA is detectable by both Farr assay and PEG assay: a relative index of anti-dsDNA avidity can be acquired by calculating an index between results of both assays, expressed in terms of Units/ml. We found that optimal discrimination between high and low avidity DNA was obtained using a Farr/PEG-ratio of 5 as a cut-off value [44]. With the use of this approach we compared the sera of 17 SLE patients with nephritis with the sera of 17 patients with central nervous system (CNS) involvement [45]. To permit an optimal comparison the sera were selected in pairs matched for anti-dsDNA titre measured with the Crithidia test. It was found that the anti-dsDNA avidity of patients with nephritis was significantly higher than that of patients with CNS involvement: 11/17 patients with renal involvement had a Farr/PEG-ratio of >5, opposed to 5/17 patients with CNS involvement (Table 7). In a series of studies on the physicochemical nature of the interaction between DNA and anti-dsDNA [46–48] it was shown that this interaction is primarily of an electrostatic nature, with the possible involvement of secondary hydrogen bonds in the interaction between DNA and high avidity antibodies. On this basis, an increase in ionic strength or pH will tend to dissociate DNA/anti-dsDNA complexes, starting with those composed of low avidity antibodies and DNA. Therefore, discrimination between anti-dsDNA of low and high avidity can also be obtained by means of elution studies employing buffers of either increased ionic strength or increased pH. Using the Crithidia test and defining a titre drop of two or more steps as significant, indeed it was found that we could discriminate between renal and CNS involvement (Table 7). Autoantibodies different from anti-DNA that have been described to occur in SLE include anti-cardiolipin, anti-histone, anti-nucleosome, antirRNP, anti-Sm, anti-nRNP, anti-Ro/SS-A and anti-La/SS-B. Incidences of these antibodies (and the levels thereof) vary, sometimes in relation to

Table 7. Comparison of patients with renal involvement with patients with CNS involvement Farr/PEG-ratio Patients with Renal involvement CNS involvement

Titredrops at pH 10

n

5

<5

>2

<2

17 17

11 5

6 12*

4 10

13 7+'

* p < 0.05 (Wilcoxon rank correlation test on individual serum pairs) + p < 0.05 by chi-square analysis with Yates correction Taken from: Smeenk (1988) J Immunol Meth 109:27–35 AMAN-C2.1/10

certain clinical features. For instance, patients with central nervous system involvement have the lowest frequency of anti-dsDNA (32% vs. 91% in the total SLE population) but a significantly increased incidence of antinRNP, anti-Sm and anti-La/SS-B [49]. Longitudinal studies of anti-Sm [50], anti-Ro/SS-A and anti-La/SS-B (Veldhoven et al., submitted) and anti-rRNP [51] generally failed to show a correlation between antibody level and disease activity (exacerbation), in sharp contrast to the findings for anti-dsDNA. Levels of soluble interleukin 2 receptors (sIL2R) have also been studied longitudinally in patients with SLE, in relation with anti-DNA levels. Ward et al. observed that in three out of ten patients, sIL2R fluctuated in parallel with anti-DNA, but independently from anti-DNA in the other seven patients [52]. In a more recent longitudinal study, no correlation at all was observed between sIL2R and anti-DNA levels, although sIL2R levels were increased during exacerbations of the disease[53].

9. Complement fixation by anti-dsDNA and nephritis

To study the complement fixing ability of anti-dsDNA, most people have used a modification of the Crithidia test. Generally, the conclusion was reached that presence of complement fixing anti-dsDNA was closely related to the occurrence of nephritis in the patient. On the other hand, it was also observed that patients with nephritis generally had higher anti-dsDNA titres. From comparable studies, Beaulieu et al. [54] and Ballou and Kushner [55] both reported a highly significant degree of association (r = 0.73; p < 0.001) between complement fixing anti-dsDNA and anti-dsDNA titres. Yet, the former concluded upon a casual relationship between nephritis and complement fixing anti-dsDNA, whereas the latter decided upon dependence of renal disease and complement fixing anti-dsDNA titre. These contradictions prompted us to study the relation between antidsDNA and complement fixing anti-dsDNA in our patients [56]. If patients were selected on the basis of presence or absence of nephritis, a relation between the presence of complement fixing anti-dsDNA and nephritis was found (Table 8). However, mean anti-dsDNA titres of both groups of patients were also found to differ widely. Therefore, sera were studied from patients with nephritis which had been matched according to titre in the Crithidia test with sera from patients without nephritis. In this way a comparable incidence and mean titre of complement fixing anti-dsDNA in both groups of sera were found (Table 8). On the basis of these results it was concluded that complement fixing titres are a direct reflection of antidsDNA titres. Patients with nephritis generally will have higher titres of antidsDNA than patients without nephritis, and, therefore more complement fixing anti-dsDNA will be found in sera of patients with nephritis.

AMAN-C2.1/11.

Table 8. Correlations between nephritis and complement fixing anti-DNA Patients

n

Mean titre anti-DNA titre

CF anti-DNA present yes

no

a) selected clinically with nephritis without nephritis

12 12

1/400 1/75

10 3

2 9*

b) titre-matched with nephritis without nephritis

12 12

1/1175 1/1175

11 9

1 3+

*

p > 0.01. chi-square analysis + Not significant Taken from: Swaak AJG (1985) In: Peeters H (Ed) Protides of the Biological Fluids, 33th Ed. pp. 317–320

10. Considerations on pathogenicity of anti-dsDNA antibodies

Antibodies to DNA have always been claimed to play an important role in the pathogenesis of SLE. Traditionally, SLE is considered an immune complex disease [57]. In this concept, anti-DNA binds DNA and the resulting immune complexes are deposited in the tissues. This binding of DNA by antibodies may occur in the circulation, but it may also happen in situ [58]. At the site of deposition, subsequent complement activation then leads to inflammation and the characteristic disease features of SLE. An alternative to this model presented in the eighties was based on studies that showed that anti-DNA could bind directly to tissue structures on the basis of charge interactions or crossreactions with such structures [59–62]. Studies using monoclonal antibodies to DNA have lent support to such theories, as these monoclonals were often found to crossreact with structures like phospholipids, proteoglycans or other polynegative molecules [63–66]. In more recent years, we carefully evaluated anti-DNA binding to heparan sulphate (HS), the negatively charged side chain of HS-proteoglycan. HSPG is an intrinsic constituent of the glomerular basement membrane (GBM). Initially, it seemed that anti-DNA could crossreact (directly) with HS [66], but when these crossreactive anti-dsDNA antibodies were purified under dissociating high salt conditions (3M NaCl) on a protein A-sepharose column, they completely lost their ability to bind HS and HSPG. Reconstitution of the purified anti-dsDNA antibodies with the column effluent restored the HS and HSPG binding [67,68]. In the protein A column effluent histones and DNA were identified. Further experiments showed that the cross reactivity of anti-DNA with HS was mediated via a ligand consisting of histones and DNA, i.e. nucleosome particles [69]. Therefore, it can be concluded that binding of anti-DNA to the GBM is AMAN-C2.1/12

mediated through nucleosomes, composed of histones and DNA [70, 71]. Interestingly, reactivity with HS in ELISA is correlated with the presence of renal disease in SLE patients [67, 72]. These findings are of special interest, since recent data have shown that nucleosomes may form the immune target for T cells of SLE patients [73]. Furthermore, antibodies with specificity for nucleosomes (not reactive with DNA or histones alone) have been identified in murine models of SLE [74. 75] as well as in human SLE (Koutouzov, personal communication). In the MRL/lpr mouse model, the anti-nucleosome antibody response was reported to precede the anti-DNA and anti-histone response in time [74, 75].

11. Anti-dsDNA antibodies in relation to therapeutic management

The prognosis of SLE has considerably improved during the past twenty years [76, 77]. This is partly based on the introduction of more sophisticated treatment protocols. Conventional treatment strategies consist mainly of non-steroidal anti-inflammatory and anti-malarial drugs, During periods of more serious disease, corticosteroids with or without immunosuppressive drugs are used [78, 79]. The possibility of predicting impending disease exacerbations based on changes in the levels of anti-DNA, complement factors or sIL2R [32–361 has opened a way to a more dedicated therapeutic approach. Using high daily doses of prednisone, this approach has been applied by Appel et al. and Jarrett et al. [80, 81]. More recently, Spronk et al. demonstrated that treatment with corticosteroids as soon as a significant rise in anti-DNA occurred, resulted in the prevention of relapses in most instances [82]. These authors treated the patients with an additional dose of 30 mg prednosilone, without increasing the cumulative dose of corticosteroids.

12. Conclusions

In this chapter, an overview is given of the present knowledge on one particular antinuclear antibody: the one against DNA. Detection of this antibody in the circulation of a patient by an anti-DNA assay selective for high avidity anti-DNA (Farr assay) is highly diagnostic for SLE. Anti-DNA of lower avidity (Crithidia test, ELISA, PEG assay) would seem to occur in rheumatic diseases other than SLE as well, making detection of such antibodies of less diagnostic value. Not only do disease features of SLE vary from patient to patient, but anti-DNA avidity does so too. By studying the possible relationship between anti-DNA avidity and disease features, it was found that SLE patients with nephritis tend to have anti-DNA of a high avidity, whereas CNS involvement in patients with low avidity anti-DNA is more proAMAN-C2.1/13

minent. Also, it was observed that the avidity of anti-DNA remained rather constant in time. Patients with low avidity anti-DNA mostly present with a milder form of the disease and continue in this way. Longitudinal studies with frequent serum sampling showed that exacerbations tend to be preceded by an increase in anti-DNA level in the serum of the patient, and followed by a sharp drop of this level. This makes frequent measurement of anti-DNA (every 4–6 weeks) with the use of a truly quantitative assay very valuable in the monitoring of individual SLE patients. Treatment of patients on the basis of increasing anti-DNA levels might prevent upcoming exacerbations. With respect to the pathogenesis of SLE, it is generally believed that anti-DNA is not an epiphenomenon, but plays an active role in the induction of disease phenomena. The mechanism through which anti-DNA elicits disease features is a current subject of debate. Traditionally, it was thought that DNA/antiDNA complexes mediate tissue damage through local deposition. Later, the possibility of in situ formation of DNA/anti-DNA complexes was added to this concept, and also the hypothesis that anti-DNA does not mediate disease phenomena through the formation of DNA/anti-DNA complexes but through direct binding to crossreactive structures such as phospholipids and proteoglycans. More recent data suggest that nucleosomes play a central role in the initiation of the glomerular inflammation of SLE. In this concept antidsDNA antibodies are complexed to nucleosomes either in circulation or in situ in the GBM. These nucleosomes are, via their histone part, bound to the GBM through charge interactions with anionic sites. Once these deposits are formed their presence might enhance further deposition of anti-dsDNA antibodies, via binding to the DNA part of the nucleosome.

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73. Mohan C. Adams S. Stanik V & Datta SK (1993) J Exp Med 177: 1367–1381 74. Burlingame RW. Boey ML. Starkebaum G & Rubin RL (1994) J Clin Invest 94: 184–194 75. Amoura Z. Chabre H. Koutouzov S. Lotton C. Cabrespines A. Bach JF & Jacob L (1994) Arthritis Rheum 37: 1684–1688 76. Swaak AJG. Nossent JC. Bronsveld W. Van Rooyen A. Nieuuenhuys EJ. Theuns L & Smeenk RJT (1989) Ann Rheum Dis 48: 447–454 77. Swaak AJG. Nossent JC. Bronsveld W. Van Rooyen A. Nieuwenhuys EJ. Theuns L & Smeenk RJT (1989) Ann Rheum Dis 48: 455–460 78. Austin HA 111. Klippel JH & Balow JE (1986) N Engl J Med 314: 614–619 79. Donadio JV Jr & Glassock RJ (1993) Am J Kidney Dis 21: 239–250 80. Appel AE, Sablay LB. Golden RA Grayzel AI & Bank N (1978) Am J Med 64: 274–283 81. Jarrett MP. Sablay LB. Walter L. Barland P & Grayzel AI (1981) Am J Med 70: 1067–1072 82. Bootsma H. Spronk PE. Limburg PC. Gmelig-Meyling FHJ. Kater L. Derksen RHWM & Kallenberg CGM (1995) (submitted for publication)

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Autoantibody Manual C2.2. 1–7. I996 © 1996 Kluw er A1 ademic Publishers Printed in The Netherlands

Autoantibodies to histones, Sm and ubiquitins ROBERT BERNSTEIN Rheumatology Department, Stopford Building, The University of Manchester, M13 9PT. U. K.

1. Antihistones antibodies

There are five histones for packaging DNA: four make a bobbin and the fifth a cleat. Autoantibodies recognising histones are amongst the most abundant in health and disease. They tend to arise with ageing and chronic infection, and as a response to therapy with various drugs. They may occur in rheumathoid arthritis and juvenile chronic arthritis. Titres are almost invariably high in drug induced lupus, and antihistone antibodies are particularly common in systemic lupus erythematosus and autoimmune chronic active (lupoid) hepatitis; they are probably responsible for the LE cell phenomenon. Antibodies to chromatin include antibodies to DNA, poly(ADP)-ribose, HMG proteins and each histone. Further antigens arise from the binding together of these individual components, such as the H2A-HZB-DNA complex. Antibody responses to fragments of histones, such as a hydrophilic region of histone H2A, can be defined, but it is important to remember that the whole response is likely to be more than the sum of its parts. The anti-chromatin response differs in subtle ways between drug induced lupus, human idiopathic SLE, and canine lupus. If we disregard responses to single stranded DNA which are common and generally considered a nuisance in diagnostic tests, then in drug induced lupus we have antibodies to histones, and poly(ADP)-ribose, in human SLE we have antibodies to histones, poly(ADP)-ribose and double stranded DNA, and in canine lupus we have antibodies to histones but rarely to DNA (poly(ADP)-ribose not having been studied).

1.1 Assays Antihistone antibodies have been detected by immunofluorescence on acidextracted histone-reconstituted tissue, by immunoblotting and by radioimmunoassay and ELISA. The antibody populations recognized by these tests are by no means complete or similar, but ELISA is now used most frequently for research [1]. Such tests are not generally part of the routine service, even though many clinicians believe wrongly that antihistone antibodies are a specific test for drug-induced lupus. AMAN-C2.2/1

1.2 Drug induced lupus In drug-induced lupus the antihistone response tends to be larger than in the much more usual situation of drug-induced antibodies without disease, and the response tends to differ according to the drug involved. Thus, procainamide and quinidine induce IgG antibodies reactive with an H2AH2B-DNA complex, while hydralazine and chlorpromazine are more likely to induce antibodies that bind H1, H3-H4 and H2A-H2B [2]. It has been suggested that an immunoglobulin class switch and concomitant increased complement binding ability may herald the development of drug induced lupus. Renal disease is infrequent in drug induced lupus and may be associated with high levels of antibody to myeloperoxidase (one of the ANCA specificities), which also arises frequently in drug induced lupus [3]. On the other hand, antihistone antibodies may well mediate nephritis in canine lupus, so why not in human lupus nephritis? Antibody levels decline gradually over the months after drug withdrawal. Once lupus has begun, antihistone antibody have no known prognostic value, and disease activity is more readily assessed on clinical grounds. Among asymptomatic patients being treated with a suspect drug, those with high titres of ANA and antihistone antibody may be most at risk of developing drug induced lupus, but this cannot be relied on.

1.3 SLE In systemic lupus erythematosus antihistone antibodies are common. They occur in between a half and two-thirds of active cases. Any of the five histones can be the predominant target, with the response to H1 and H2B most often the highest [4]. Epitope mapping of histones by synthesis of candidate peptides suggests that putative hydrophilic regions are antigenic, and immunisation with such peptides induces antihistone responses in certain strains of rabbits and mice. Neither the antihistone antibody response as a whole nor any subpopulation of antibodies has been associated with any clinical manifestation of SLE or lupus subset. In a longitudinal study, disease activity tended to correlate with levels of antibodies to core histones but not anti-H1 [5]. Future clinical interest can be expected because histones may be important to the immunogenicity of chromatin and hence to the production of anti-DNA antibodies, and also because of a possible role in the pathogenesis of lupus nephritis. Thus, heparan sulphate in the kidney basement membrane can bind histones and histone-DNA complexes, hence localising potential targets for autoantibodies [6]. There are no pressing grounds, just yet, to measure antihistone antibodies on a routine basis.

AMAN-C2.2/2

Clinical aspects of' antihistone antibodies Detection by ELISA and Immunoblotting May occur with ageing. chronic infection. RA. JCA and certain drug therapies High levels in SLE, lupoid hepatitis and drug induced lupus Little diagnostic specificity No statistical association with any lupus feature or subset Possible role in pathogenesis of lupus nephritis

2. Antibodies to ubiquitin and uH2A

Ubiquitin and the ubiquitinated form of histone H2A (uH2A) have been identified as major autoantigens in SLE. Anti-ubiquitin antibodies have been detected with a frequency of 79% by ELISA: antibodies to uH2A were detected by immunoblotting [7, 8]. These antibodies were also found in murine lupus, by eight weeks of age in NZBxNZW mice and soon after four weeks of age in MRL – Ipr/lpr mice [9]. Confirmation of these findings was lacking when biochemically isolated uH2A and purified ubiquitin were employed as antigens in immunoblotting experiments with seven lupus sera. Nor were antibodies to purified ubiquitin demonstrated by ELISA in a larger number of lupus sera [10]. Both groups used the same chemical lot of ubiquitin, and it is not clear what minor technical differences, such as in fixation, could explain such different results. With regard to immunoblotting of uH2A, the first group deduced by immunological criteria that bands of 43 kDa and 52 kDa were uH2A [8], whereas the second group showed that uH2A migrates in SDSPAGE at 22 kDa. Thus, it remains to be established whether antibodies to ubiquitin are truly ubiquitous in SLE or as hard to find as that damned elusive Pimpernel. Clinical aspects of antibodies to ubiquitin and uH2A Detection by ELISA (ubiquitin) and immunoblotting (uH2A) Frequency in SLE ?0%. ?79% (controversy)

3. Antibody to Sm

Antibody to the soluble nuclear antigen Sm was described by Tan and Kunkel in 1966 [11], just six years after the first description of antibodies to what we now call the Ro and La antigens by Anderson et al. in the United Kingdom. Anti-Sm antibody is said to be highly specific for SLE. and its presence was incorporated by the American Rheumatism Association as a categorisation criterion for SLE. Nevertheless. the antibody has been found in at least two cases of rheumatoid arthritis. AMAN-C2.2/3

3.1 Racial variation The frequency of anti-Sm antibody in SLE was long given as about 25% on the basis of American studies. The European experience has been that anti-Sm antibody is much less common: with an overall frequency in SLE below 10%), it became clear that this was because the frequency of anti-Sm antibody is nearly ten times greater in Black and Chinese patients (about 30%) than in White patients (4–8%) [12].

3.2 HLA associations Among black patients anti-Sm antibody is associated with an increased frequency of DR2 and increases in DQA1*0102 (p = 0.007, odds ratio 6.7) and DQB1*0602 (p = 0.001, odds ratio 9.1) [13].

3.3 Lupus subsets Within the clinical spectrum of SLE there are suggestions that the antibody is associated with more severe disease and poor outcome [14, 15]. These associations are not strong [16] and are probably explained by the tendency to more severe disease in Black patients. Moreover, there is a dissociation between the presence of anti-Sm antibody on the one hand and antibodies to Ro and La antigens on the other, with the latter antibodies being associated with those rather milder forms of lupus that tend to evolve into Sjögren,s syndrome. Anti-Sm antibody is, of course, not lonely, as it is usually accompanied by anti-(U1) RNP (a more promiscuous antibody).

3.4 Assays The development of ELISA assays for antibodies to Sm and to the B and D polypeptides has shown the antibodies to be rather more common than when detected by immunodiffusion or counterimmunoelectrophoresis, and antibody to B (and its close variant B´) is more common than anti-D. A commercial ELISA using recomblnant antigen was also more sensitive than immunoblotting where native epitopes may have been lost [ 17]. Alternatively, ELISA may give false positive results, and one wonders whether that may be the case in a study finding anti-Sm antibody in SLE (40%, Sjögren’s syndrome (12%), rheumatoid arthritis (6%) and miscellaneous autoimmune conditions (12%) [ 18]. No new clinical associations have emerged with this technology. Measuring fluctuations in antibody level by ELISA may be of use in following disease activity in selected cases [19]. AMAN-C2.2/4

3.5 Origins of anti-Sm response in mice and men The anti-Sm response is thought to be antigen driven. In young MRL-1pr mice injection of a monoclonal anti-Sm antibody induced early production of endogenous anti-Sm of unrelated idiotypes, and immunisation with Sm antigen induced antibodies to Sm and RNP; it may be that the monoclonal antibody worked by delaying the clearance of endogenously released Sm antigen [20]. In MRL-1pr mice only a proportion with lupus develop antiSm antibody, and this may be controlled via the IgH locus; mice bearing IgHb were 78% anti-Sm positive, whereas mice bearing IgHj were only 27% anti-Sm positive [21]. Anti-Sm antibodies derived from MRL-1pr mice are oligoclonal in individual mice yet variable between mice and variable in the extent of somatic mutation – consistent with antigen selection [22]. In human SLE the anti-Sm response is relatively unrestricted with regard to IgG subclass; there is some preference for IgGl, but this is much less marked than the IgGl K restriction of the anti-La response [23, 241. One justification for epitope mapping of the antigens has been the search of databases for homologous sequences in viruses which might then be invoked as possible triggers for the autoimmune response. A sequence at the C terminus of the SmD peptide (residues 95–119) shows strong homology with a sequence of EBNA I (residues 35–58), a nuclear antigen induced by Epstein-Barr virus infection. Antibodies reactive with the D peptide also bound the EBNA I peptide and detected the EBNA I molecule in an extract of EBV-infected cells. Immunisation of mice with the EBNA I peptide induced antibodies which reacted with EBNA and SmD [25]. One cannot help but smell a rat here in the murine immune system, but taken at face value the ubiquity of EBV highlights the importance of rare host factors in the loss of control of autoimmune phenomena. Clinical aspects of anti-Sm antibodies Detection by immunodiffusion, counterimmunoelectropheresis. immunoblotting or ELISA Specificity for SLE (with rare exceptions) Frequency in SLE 4–8% in white patients, 30% in black and chinese patients More severe lupus? Association with anti-(U1)RNP, dissociation from antibodies to Ro and La HLA-DQ associations in black patients

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References I. Burlingame RW & Rubin RL (1994) Manual of Biological Markers of Disease B2.2: 1–28, Kluwer Academic Publishers, Dordrecht/Boston/London 2. Burlingame RW & Rubin RL (1991) J Clin Invest 88: 680–690 3. Cambridge G. Wallace H, Bernstein RM & Leaker B (1994) Br J Rheumatol 33: 109–114 4. Bernstein RM, Hobbs RN, Lea DJ, Ward DJ & Hughes GRV (1985) Arthritis Rheum 28: 285–293 5. Muller S. Barakat S, Watts R. Joubaud P, Isenberg D (1990) Clin Exp Rheumatol 8: 445–453 6. Schmiedeke T, Stoechl F, Muller S et al. (1992) Clin Exp Immunol 90: 453–458 7. Muller S. Briand JP & Van Regenmortel MHV (1988) Proc Natl Acad Sci USA 85: 8176–8180 8. Plaue S. Muller S & Van Regenmortel MHV (1989) J Exp Med 169: 1607–1617 9. Elouaai F, Lule J. Benoist H et al. (1994) Nephrol Dial Transplant 9: 362–366 10. Suzuki T, Burlingame RW. Casiano CA, Boey ML & Rubin RL (1994) J Rheumatol 21: 1081–1091 11. Tan EM & Kunkel H (1966) J Immunol 96: 464–471 12. Bernstein RM, Bunn CC, Hughes GRV, Francoeur AM & Mathews MB (1984) Mol Biol Med 2: 105–120 13. Olsen ML, Arnett FC. Reveille JD (1993) Arthritis Rheum 36: 94–104 14. McCarty GA, Harley JB. Reichlin M (1993) Arthritis Rheum 36: 1560–1565 15. Thompson P. Juby A, Davis P (1993) Lupus 2: 15–19 16. Gulko PS, Reveille JD, Koopman WJ et al. (1994) J Rheumatol 21: 224–228 17. Delpech A, Gilbert D, Daliphard S et al. (1993) J Clin Lab Anal 7: 197–202 18. Gripenberg M, Teppo AM. Friman C (1991) Rheumatol Int 11: 209–213 19. Muller S, Barakat S, Watts R. Joubaud P & Isenberg D (1990) Clin Exp Rheumatol 8: 445–453 20. Stocks MR. Williams DG, Maini RN (1991) Eur J Immunol 21: 267–272 21. Halpern MD. Graven SY, Cohen PL, Eisenberg RA (1993) J Immunol 151: 7268–7272 22. Bloom DD, Davignon JL, Retter MW et al. (1993) J Immunol 150: 1591–1610 23. Tokano Y, Yasuma M, Harada S et al. (1991) J Clin Immunol 11: 317–325 24. Meilof JF. Hebeda KM, De Jong J, Smeenk RJ (1992) Res Immunol 143: 711–720 25. Sabbatini A. Bombardieri S, Migliorini P (1993) Eur J Immunol 23: 1146–1152

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Autoantibody Manual C2.3. 1–11. 1996 © 1996 Kluw er Academic Publishers Printed in The Netherlands

Clinical significance of autoantibodies to Ku and related antigens WESTLEY H. REEVES, MINORU SATOH and JENIFER J. LANGDON Division of Rheumatology and Immunology, Departments of Medicine and Microbiology and Immunology. and UNC Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599–7280, U.S. A.

1. Introduction

The Ku antigen was first described by Mimori et al. [1] as a nonhistone nuclear antigen producing a precipitin line with serum from a patient with scleroderma-polymyositis overlap syndrome. Although originally thought to be relatively specific for this unusual subset of autoimmune disease, subsequent studies have shown that autoantibodies to Ku are also found in some sera from patients with systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), scleroderma, polymyositis, Grave’s disease, and primary pulmonary hypertension [2–6]. Thus, serum autoantibodies specific for Ku cannot at present be regarded as a diagnostic marker for any particular autoimmune disease subset.

2. Indications for requesting a test

Serologic testing for autoantibodies to Ku is not routinely available, and should be considered a research test. Indeed, there is no standard clinical test for anti-Ku antibodies at present (Table 1). The “gold standard” for detecting this specificity is either double immunodiffusion [1] or radioimmunoprecipitation [2], although an antigen capture ELISA shows promise for becoming a routine test amenable to large scale screening of serum samples [7]. The ELISA may eventually become the preferred assay for routine clinical use, because of its simplicity, sensitivity and specificity. However, largely due to the unavailability of simple tests for large scale serum testing, the usefulness of anti-Ku antibodies in differential diagnosis, monitoring disease activity or defining prognosis is not known. However, there is limited evidence that the levels of anti-Ku antibodies reflect disease activity in a subset of patients [8]. Although additional studies are needed to verify whether or not anti-Ku titers correlate with disease activity, it may be worthwhile to consider serial determinations of anti-Ku antibody levels in some instances. AMAN-C2.3/1

3. Methods used for detection

3. 1 Immunodiffusion The initial report of anti-Ku autoantibodies employed double immunodiffusion (ID) [1]. Although most high titer anti-Ku sera contain precipitating antibodies, ID is relatively insensitive compared with immunoprecipitation and ELISA. Thus, although the specificity of this assay is high, ID is considerably less sensitive than immunoprecipitation or ELISA. Nevertheless, double immunodiffusion is useful because it is simple and widely available. A detailed procedure is reported in reference [1]. Briefly, Ouchterlony double immunodiffusion is performed in 0.6% SeaKem agarose in PBS-NaN3 using calf thymus extract at a concentration of 100 mg/ml in saline as a source of antigen. Although initial reports utilized calf thymus as a source of antigen, primate cells appear to be a richer source of Ku [9–11], and certain anti-Ku autoantibodies display selective binding to human Ku [12]. Thus, the sensitivity of the assay could probably be enhanced somewhat by using antigenic extracts derived from human cells. Table 1. Methods for detecting autoantibodies to Ku Technique

Advantages

Disadvantages

Availability

Immunodiffusion

High specificity Native antigen used

Low sensitivity

Yes

Immunoprecipitation

High specificity High sensitivity Native antigen used

Use of isotopes Technically demanding

Research only

Ag capture ELISA

High specificity High sensitivity Native antigen used Simplicity

Not generally available

Research only

Fusion protein ELISA

High specificity

Low sensitivity Denatured antigen used

Research only

Western blot

High specificity Simplicity

Low sensitivity Denatured antigen used

Research only

3.2 Immunoblotting Cellular as well as recombinant human p70 and p80 Ku antigens can be used in a western blot assay for detecting autoantibodies to Ku [3, 4, 13]. The western blot assay has the advantage of simplicity, but its usefulness is limited by the fact that most sera containing autoantibodies to Ku AMAN-C2.3/2

recognize the native form of the antigen preferentially [13]. Sera that are reactive by western blot generally contain high titer antibodies to Ku. Many high titer anti-Ku sera contain antibodies reactive with both p70 and p80, but the relative strength of the binding to p70 and p80 is variable, with some sera showing significantly stronger reactivity with one or the other subunit [4, 14].

3.3 Immunoprecipitation In view of the fact that autoantibodies to Ku recognize primarily conformational epitopes that do not survive immunoblotting [ 13], radioimmunoprecipitation assays have become a standard technique for detecting anti-Ku antibodies. For this assay, human cell lines, such as K562 (human erythroleukemia) or HeLa (human cervical carcinoma), are metabolically labeled with 35S-methionine and extracts of these cells are used as a source of Ku antigen. Detailed protocols for immunoprecipitation have been published [2, 4, 15]. Typically, cells are lysed in buffer containing 0.15 M NaCI, 50 mM Tris pH 7.5, 2 mM EDTA, 0.3% Nonidet P-40 and protease inhibitors. The extract is cleared twice by centrifuging at 10,000 × g in a microcentrifuge, and cell extract (typically from 1.5 × 106 cells in a volume of 200 µl) is incubated for 1 h with 15–20 µI of packed protein A-Sepharose beads previously incubated for 1 h with 10 µl of human serum. A variety of washing protocols have been used, but one or more high salt washes (≥ / = 0.5 M NaCl in 50 mM Tris pH 7.5, 2 mM EDTA) may be important for avoiding false positive reactions (M Satoh. unpublished observations, [7]). The beads are then washed in the same buffer containing 0.15 M NaCI, and immunoprecipitated proteins are eluted by boiling in SDS sample buffer followed by analysis on 10%) SDSpolyacrylamide gels. The gels are fluorographed, dried, and exposed to Xray film for 3 days. Positive sera immunoprecipitate a characteristic doublet of 70 and ~ 86 kDa proteins (see Antigen Description, Figure 2).

3.4 ELISA An antigen capture ELISA based on murine anti-Ku mAbs that correlates well with immunoprecipitation and employs stringent washing conditions to dissociate Ku-associated proteins and DNA has been developed [2, 7]. False positive reactions caused by anti-histone, anti-DNA, or anti-DNAPK antibodies (see Antigen Description) can be avoided by prewashing the antigen-coated wells with 1.5 M NaCl before adding antiserum [7]. The procedure for the antigen capture ELISA is summarized in Fig. 1. The sensitivity and specificity of this assay appears comparable to that of the radioimmunoprecipitation assay, and the ELISA is considerably simpler to AMAN-C2.3/3

1.

2.

3.

4.

5.

6.

Fig. 1. Antigen capture ELISA for detecting anti-Ku antibodies. (1) Duplicate wells of a microtiter plate are coated overnight at 4 ºC with murine anti-Ku mAbs (mAbs 162 and 111 [2] are useful for this purpose) at 10 µg/ml. The wells are then blocked with PBS containing 10%) bovine calf serum. (2) Cleared cell extract from the human erythroleukemia cell line K562 (50 µl of extract containing 2 × 106 cell equivalents) is added to the “A” wells for 90 min at 22 ºC. Buffer alone is added to “B”wells. (3) Wells are washed with 1.5 M NaCI. 50 mM Tris pH 7.5, 2 mM EDTA to remove bound DNA and proteins. (4) Human serum (1:100–1:500 dilution) is added for 1 h at 22 ºC, followed by washing. (5) Peroxidaseconjugated goat anti-human immunoglobulin (kappa and lambda light chain specific, 1 :2500 dilution of each) is added for 1 h at 22 ºC, followed by washing. (6) Substrate is added for 45–60 min and optical density at 490 nm is determined. O.D. in “B” (control) wells is subtracted from that in the “A” (antigen coated) wells.

perform and is amenable to large scale screening of patient specimens. Thus, the antigen capture ELISA shows considerable promise clinically, although it remains primarily a research tool at present. ELISAs utilizing p70 and p80 fusion proteins electroeluted from SDS polyacrylamide gels have also been described recently [16]. Of the 22 sera that were positive in either assay, 13 (59%) were also positive by double immunodiffusion. One serum that was positive for anti-p80 antibodies by AMAN-C2.3/4

western blot was negative in the anti-p80 ELISA, possibly due to differences in the conformation of p80 in the two assays. There is no information regarding the correlation of these assays with immunoprecipitation.

4. Prevalence of autoantibodies to Ku

4. 1 Frequency in specific diseases Autoantibodies to Ku have been detected in a variety of autoimmune conditions (Table 2). The initial report of the anti-Ku specificity was in Japanese patients with polymyositis-scleroderma overlap syndrome [1]. Precipitating antibodies to Ku were found in sera of 6 of 11 patients (55%) is this subset, compared with 3 of 319 Japanese patients (1%) with other connective tissue diseases (one of whom had SLE-scleroderma-polymyositis overlap). The high frequency of autoantibodies in the Japanese polymyositis-scleroderma overlap subset has been confirmed in subsequent studies [7, 16, 17]. However, by immunoprecipitation and ELISA, nonprecipitating autoantibodies to Ku are relatively common in Japanese patients with SLE or scleroderma [7]. In American and European patients, anti-Ku antibodies are most strongly associated with SLE, scleroderma, and MCTD, but have also been detected in polymyositis, Sjögren’s syndrome, rheumatoid arthritis, Grave’s disease and primary pulmonary hypertension (Table 2) [2, 3, 5, 6]. The prevalence of anti-Ku antibodies in American SLE patients has been estimated to be 10–40% by ELISA and western blot assays [2–4, 7]. In addition. a high frequency (9/1 6 patients) of anti-Ku antibodies in Grave’s disease was reported in one study [5], although it was not confirmed in a subsequent study [7]. Differences in the frequency of autoantibodies to Ku in various diseases may reflect differences in the methods utilized to detect the antibodies, the ethnic composition of the subjects studied, or both.

4.2 Influence of ethnic background and HLA Although initially, anti-Ku antibodies appeared to be associated primarily with polymyositis-scleroderma overlap syndrome in Japanese [ 1, 17], and with SLE, MCTD and scleroderma in Americans [2, 3], more recent studies suggest that autoantibodies to Ku are at least as common in Japanese as in American patients with lupus [7]. In fact, when the frequency of autoantibodies to Ku was compared in the two groups, a somewhat higher prevalence was found in the Japanese population. Data regarding the frequency of these autoantibodies in Americans of different ethnic origins are not available at present. AMAN-C2.3/5

Table 2. Prevalence of autoantibodies to Ku Reeves¹

Yaneva²

Chan³

Isern4

Satoh5

SLE MCTD PM/DM PM-Scl Overlap Scleroderma Sjögren’s RA Grave’s disease PPH

20151 6/11 n.d. n.d. 6/15 n.d. n.d. n.d. n.d.

13/69 n.d. 2/23 n.d. 9/57 2/10 n.d. n.d. n.d.

n.d. n.d. n.d. n.d. n.d. n.d. n.d. 9116 n.d.

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 7/31

7156 n.d. 0/2 1/3 012 0/2 1/18 0/22 n.d.

Disease Subset*

Mimori6

Hirakata7

Satoh5

Suwa(p70)8

Suwa(p80)8

11150 0125 0/30 7/14 1145 n.d. 0154

1/126 n.d. 0/52 10/39 1/1/3 n.d. n.d.

21178 1122 1/26 11/18 4135 1/13 1/30

4157 2/17 0/10 8/11 4117 n.d. n.d.

Disease Subset* A. American Patients

B. Japanese Patients SLE MCTD PM/DM PM-Scl Overlap ScIeroderma Sjögren’s RA

2/57 1/17 1/10 5/1 1 2/17 n.d. n.d.

* Abbreviations: SLE, systemic lupus erythematosus; MCTD. mixed connective tissue disease; PM/DM. polymyositis or dermatomyositis: PM-Scl overlap. polymyositisscleroderma overlap syndrome: RA, rheumatoid arthritis; PPH, primary pulmonary hypertension; n.d., not done 1 Reeves [2] [ELISA] 2 Yaneva and Arnett [3] [western blot] 3 Chan et al. [5] [immunoprecipitation] 4 Isern et al. [6] [western blot] 5 Satoh et al. [7] [ELISA] 6 Mimori et al. [1] [double immunodiffusion] 7 Hirakata et al. [17] [immunoprecipitation] 8 Suwa et al. [16] [p70, p70 fusion protein ELISA: p80, p80 fusion protein ELISA]

Information regarding HLA associations is also limited. Only one study has examined HLA alleles associated with autoantibodies to Ku [3]. In that study, there were no associations with HLA-A, B, or C alleles, but the class II allele DQwl was present in 17 of 19 (89%) anti-Ku positive patients, compared with 58% and 61% in white and black controls, respectively (relative risk 5.8). However, the number of patients was small, and HLADQwl is a common allele in healthy individuals, and is found at increased frequency in white SLE patients in general. Additional studies involving AMAN-C2.3/6

larger numbers of patients will be necessary to further define the influence of ethnic background and HLA alleles on the production of autoantibodies to Ku.

4.3 Diagnostic sensitivity and specficity of the tests In both the Japanese and American populations, anti-Ku titers may be extremely high, ranging up to 10–7 or more by ELISA in some cases [2, 8]. When present at these titers, detection of autoantibodies to Ku is not difficult by double immunodiffusion, ELISA, or immunoprecipitation or, in many cases, by western blot. There is limited comparative information regarding the sensitivity and specificity of these assays compared with the standard immunoprecipitation assay. In one study, of ten immunoprecipitation positive sera having an ELISA titer of anti-Ku antibodies ≥ 1:12,500, only three (30%) were reactive with p70 or p80 by western blot. Thus, the sensitivity of the western blot assay is relatively low, probably due, in large part, to the preferential reactivity of anti-Ku antibodies with the native form of the antigen. In another study, 30 of 38 immunoprecipitation positive sera were also positive by ELISA (sensitivity = 79%) whereas 217 of 230 immunoprecipitation negative sera were negative by ELISA (specificity 94%) [7]. There is little information regarding the sensitivity and specificity of double immunodiffusion, but our experience suggests that the specificity is high, whereas sensitivity is low.

4.4 Associations of anti-Ku with other autoantibodies Autoantibodies to Ku are found at a significantly higher frequency in human autoimmune sera containing anti-Su autoantibodies than in anti-Su negative sera [ 18]. An association with anti-Sm antibodies has been reported [3], and anti-Ku antibodies may also be associated with anti-RNA polymerase II antibodies in SLE and SLE-overlap syndromes [19], although the numbers of sera evaluated were relatively small. The explanation for the association of anti-Ku, anti-Su, and anti-RNA polymerase II antibodies is uncertain, but may not reflect a physical association between the antigens [18]. Recent studies show that Ku and RNA polymerase II carry a crossreactive epitope recognized by a monoclonal antibody (M Satoh et al., manuscript in preparation), raising the possibility that immunological crossreactivity between Ku, Su, and RNA polymerase II might explain the frequent associations between these specificities. Finally, autoantibodies to the p350 subunit of the DNA-dependent protein kinase (DNA-PK) are associated with anti-Ku autoantibodies (M Satoh et al., submitted). The p350 protein carries the catalytic activity of DNA-PK, and is physically associated with Ku, the DNA binding subunit of the enzyme [20]. AMAN-C2.3/7

5. Clinical significance of autoantibodies to particular epitopes or isoforms

Information about the clinical significance of autoantibodies to either p70 or p80, or to particular epitopes of Ku is limited to a single brief report [14]. In that study of Japanese patients with polymyositis-scleroderma overlap syndrome, all five patients whose sera contained autoantibodies to p70 but not p80 by western blot had Raynaud’s phenomenon, sclerodermatous skin changes and myositis. In contrast, anemia and leukopenia were more common in patients with both anti-p70 and p80. All three patients with autoantibodies to p80 (amino acids 608–697), but not p80 (amino acids 707–732), had pulmonary fibrosis and esophageal hypomotility, whereas 4 out of 5 of those having autoantibodies to both p80 epitopes had anemia, leukopenia, and proteinuria. Based on these data, the first group may have “typical” polymyositis-scleroderma overlap syndrome, the second may belong to a subset with dominant features of scleroderma, and the third to a subset more closely related to SLE. However, although this study suggests that autoantibodies to different epitopes of Ku might be associated with different disease manifestations, the numbers were very small, and additional studies are needed to confirm these associations.

6. Future trends and prospects

6.1 Clinical associations The availability of a simple ELISA should make large scale screening for autoantibodies to Ku possible. This may ultimately lead to more accurate estimates of the frequency of these autoantibodies in sera of patients from different ethnic backgrounds having different clinical syndromes. It is too early to know if autoantibodies to Ku will be useful diagnostically, but the brief report mentioned in Section 5 above raises the possibility that autoantibodies to different epitopes of Ku may prove valuable in this respect. The current ELISA does not distinguish between these specificities, however, and additional large scale screening assays based either on recombinant Ku antigens or competitive binding with murine monoclonal antibodies specific for a variety of different epitopes [2, 8] will be necessary to investigate this important issue.

6.2 Disease activity There is also interest in the relationship of autoantibodies to Ku with autoantibodies specific for other chromatin components. In some cases, anti-Ku and anti-DNA antibodies increase and decrease in parallel, but it is not known how well autoantibodies to Ku correlate with disease activity AMAN-C2.3/8

compared with the standard antibodies to double stranded DNA. The ELISA should be especially useful in investigating this question.

6.3 Disease therapy There are no data at present to indicate whether the presence of anti-Ku antibodies predict a response, lack of response, or adverse reactions to drugs.

Acknowledgements

This work was supported by grants R01-AR40391, P60-AR30701, P50AR42573 and RR00046 from the United States Public Health Service.

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References 1. Mimori T, Akizuki M. Yamagata H, Inada S, Yoshida S & Homma M (1981) J Clin Invest 68: 611–620 2. Reeves WH (1985) J Exp Med 161: 18–39 3. Yaneva M & Arnett FC (1989) Clin Exp Immunol 76: 366–372 4. Francoeur AM, Peebles CL, Gompper PT & Tan EM (1986) J Immunol 136: 1648–1653 5. Chan JYC, Lerman MI, Prabhakar BS, Isozaki O, Santisteban P, Kuppers RC, Oates EL, Notkins AL & Kohn LD (1989) J Biol Chem 264: 3651–3654 6. Isern RA, Yaneva M, Weiner E, Parke A. Rothfield N. Dantzker D, Rich S & Arnett FC (1992) Am J Med 93: 307–312 7. Satoh M, Langdon J & Reeves WH (1993) Clin Immunol Newslett 13: 23–31 8. Reeves WH, Sthoeger ZM & Lahita RG (1989) J Clin Invest 84: 562–567 9. Wang J. Chou CH, Blankson J, Satoh M, Knuth MW. Eisenberg RA. Pisetsky DS & Reeves WH (1993) Mol Biol Rep 18: 15–28 10. Celis JE, Madsen P, Nielsen S, Ratz GP. Lauridsen JB & Celis A (1987) Exp Cell Res 168: 389–401 1 I. Stuiver MH, Celis JE & Van der Vliet PC (1991) FEBS 282: 189–192 12. Porges A, Ng T & Reeves WH (1990) J Immunol 145: 4222–4228 13. Reeves WH, Pierani A, Chou CH, Ng T, Nicastri C, Roeder RG & Sthoeger ZM (1991) J Immunol 146: 2678–2686 14. Suwa A (1990) Keio Igaku 67: 865–879 15. Mimori T, Hardin JA & Steitz JA (1986) J Biol Chem 261: 2274–2278 16. Suwa A, Mimori T, Hama N, Fuji T, Hirakata M, Ohosone Y, Akizuki M & Homma M (1992) Jpn J Clin Immunol 15: 337–345 17. Hirakata M, Mimori T, Akizuki M, Craft J, Hardin JA & Homma M (1992) Arthritis Rheum 35: 449–456 18. Satoh M, Langdon JJ, Chou CH, McCauliffe DP, Treadwell EL. Ogasawara T, Hirakata M, Suwa A, Cohen PL, Eisenberg RA & Reeves WH (1994) Clin Immunol Immunopathol 73: 132–141 19: Satoh M, Ajmani AK, Ogasawara T, Langdon JJ, Hirakata M, Wang J & Reeves WH (1994) J Clin Invest 94: 1981–1989 20. Gottlieb TM & Jackson SP (1993) Cell 72: 131–142

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Autoantibody Manual C2.4. 1–1 3. 1996 © 1996 Kluwer Academic Publishers Printed in The Netherlands

Clinical significance of autoantibodies to PCNA YOSHINARI TAKASAKI¹ and E.M.TAN²

1 Division of Rheumatology, Department of Medicine, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113, Japan; 2 W. M. Keck Autoimmune Disease Center, Department of' Molecular and Experimental Medicine, The Scripps Research Institute. 10666 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.

1. Introduction

An autoantibody to proliferating cell nuclear antigen (PCNA) was reported as an antibody which was specifically detected in sera from patients with systemic lupus erythematosus (SLE) by Miyachi et al. in 1978 [1]. In their report, anti-PCNA antibodies were detected in only three out of 70 lupus patients so that the clinical characteristics of patients with anti-PCNA was not well defined. Since that time, there have been three reports concerning the clinical significance of anti-PCNA antibodies in lupus patients [1~4]. In 1983, Fritzler et al. reported the clinical and serological features of 7 patients with anti-PCNA antibodies [2]. Five out of Patients had SLE, one had idiopathic diffuse proliferative glomerulo-nephritis (DPGN) and the other had seronegative arthritis. In a study of various connective tissue diseases, antiPCNA antibodies were detected in 2% of SLE sera, but not found in the sera of patients with other rheumatic diseases. Those patients with anti-PCNA antibodies showed high prevalence of renal involvement and central nervous system (CNS) lupus, and 3 out of 4 lupus patients with DPGN were negative for anti-DNA antibodies. In addition, the longitudinal study was performed to clarify the relationship between anti-PCNA antibodies and disease activity, and showed the possible relation of anti-PCNA antibodies to the flare of renal involvement, and the high sensitivity of anti-PCNA antibodies to corticosteroid therapy. In 1987, Ohata in our group also studied the clinical characteristics of anti-PCNA positive patients, and found the significant relation of antiPCNA antibodies to thrombocytopenia and seizure [3]. They studied 584 patients with various connective tissue diseases and anti-PCNA antibodies were detected in 2.1% of patients with SLE. All those patients had renal involvement and one out of 7 lupus patients developed DPGN without anti-DNA the same as the report obtained by Fritzler et al. [2]. In addition, a higher incidence of DPGN and hematological disorders in anti-PCNA positive SLE patients was reported by Asero et al. [4]. The longitudinal study showed that the elevation of anti-PCNA titer was strongly related to the activity of renal involvement. AMAN-C2.4/1

In spite of the limited number of patients, the clinical characteristics of the patients with anti-PCNA in these reports was quite similar. In this section, the authors refer to mainly those three reports and the clinical characteristics of patients with anti-PCNA antibodies will be discussed.

2. Methods for detection of anti-PCNA

Methods to detect antibodies to PCNA are listed in Table 1 and described in detail in the A section of this Manual. Double immunodiffusion (DID) is usually used to test the anti-PCNA in patients with various rheumatic diseases and has the high disease specificity. Counter immunoelectrophoresis (CIE) is also available to detect antibodies to PCNA and shows higher Table 1. Methods to detect anti-PCNA antibodies Methods

Relative advantage

limitation of methods

Immunofluorescence (IF)

IF has high sensitivity for anti-PCNA.

Specificity for anti-PCNA is low. IF can be used only when autoimmune sera have only anti-PCNA antibody or higher titer of anti-PCNA than other co-existing autoantibodies.

Double immunodiffusion (DID)

Anti-PCNA detected by DID shows high disease specificity.

Anti-PCNA standard serum is always needed to identify antiPCNA.

Counterimmunoelectrophoresis (CIE)

CIE requires smaller amounts of serum and antigen solution than DID and has higher sensitivity than DID.

Anti-PCNA standard serum is always needed the same as DID.

Immunoprecipitation (IP)

Anti-PCNA sera detected by DID usually precipitate 34 kDa PCNA polypeptide

Radioisotope such as 35Smethionine is required. 1P cannot be used to quantify and identify anti-PCNA.

lmmunoblotting (IB)

IB has higher sensitivity than DID.*

About 50% of anti-PCNA positive sera detected by DID do not react with 34 kDa PCNA polypeptide in IB.

Enzyme-linked immunosorbent assay (ELISA)

ELISA is useful to quantify anti-PCNA.

Some anti-PCNA sera detected by DID do not react with purified or recombinant PCNA antigens in ELISA. AntiPCNA detected by ELISA shows lower disease specificity than that by DID.

*

As long as sera can react with 34 kDa PCNA band

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sensitivity than that of DID [5]. In immunofluorescence (IF), anti-PCNA shows the characteristic staining pattern (see this Manual chapters A.2 and B.2.7). But it is possible to detect anti-PCNA antibodies by IF only when the patients sera contain exclusively anti-PCNA or has much higher titer of antiPCNA than other coexisting antinuclear autoantibodies. Although the sera obtained typical anti-PCNA staining pattern in IF, DID is necessary to identify anti-PCNA because certain autoantibodies like anti-Na antibodies can show a similar staining pattern as anti-PCNA antibodies on HEp 2 cell smears [6]. Immunoblotting (IB) can not be routinely used to test anti-PCNA antibodies because about 50% of anti-PCNA positive lupus sera do not react with 34 kDa PCNA polypeptide (see Chapter B.2.7). Enzyme-linked immunosorbent assay (ELISA) can be also used to detect anti-PCNA antibodies. This method has an advantage to quantify the titer of anti-PCNA [7, 8]. However some anti-PCNA sera show weak reactivity to purified PCNA or recombinant PCNA by ELISA [7]. DID is the simple method but the most useful for the detection of antiPCNA antibodies and for the diagnosis showing the high disease specificity for SLE, as long as the standard serum for anti-PCNA antibody is available. Because of low frequency in patients with SLE, the test for antiPCNA is not routinely performed, but the test system for anti-PCNA antibodies is commercially available.

3. Indications for requesting a test for anti-PCNA antibodies

Although the frequency of anti-PCNA antibody is low, this autoantibody is useful as a diagnostic marker for SLE. The incidence of clinical features such as renal involvement, CNS lupus and thrombocytopenia is significantly higher in patients with anti-PCNA [3]. The sequential detection of antiPCNA antibodies may be useful to evaluate disease flare associated with renal involvement and the response for therapy in SLE patients. In addition to anti-PCNA antibodies, the detection of PCNA positiveactivated peripheral blood mononuclear cells obtained from lupus patients is also useful to evaluate the disease activity [9].

3.1 Disease specificity of anti-PCNA antibodies Miyachi et al tested sera from 70 patients with SLE, 30 with rheumatoid arthritis (RA), 33 with Sjögren’s syndrome (SjS), 10 with progressive systemic sclerosis (PSS), 19 with polymyositis (PM) and 26 normal controls in DID, and anti-PCNA antibodies were detected in only 3 patients with SLE [1]. Fritzler et al. tested 270 sera from patients with various connective tissue diseases and anti-PCNA antibodies were detected in 2 out of 100 lupus patients (Table 2A) [2]. Ohata et al. also tested 584 sera from patients with a AMAN-C2.4/3

variety of rheumatic diseases and found that anti-PCNA antibodies were positive in 7 out of 328 lupus patients (Table 2B) [3]. Two out of 7 antiPCNA positive patients who were reported by Fritzler et al. did not meet the 1982 revised criteria for SLE. One had DPGN without anti-DNA antibodies and the other patient had seronegative arthritis. These patients had at least two items of the criteria for lupus and their clinical features did not contradict those of lupus patients. Although the reported prevalence of anti-PCNA antibodies is not high (2–4.3%), it is shown that this autoantibody is predominantly found in patients with lupus in the different ethnic groups. The association of HLA with anti-PCNA antibody is not known.

3.1 Clinical characteristics of patients with anti-PCNA antibodies Because of the limited number of patients with anti-PCNA, Miyachi et al. could not find any particular clinical features associated with anti-PCNA [1]. Ohata studied the clinical characteristics of patients with anti-PCNA Table 2. Frequency of anti-PCNA and other autoantibodies in systemic rheumatic diseases*

A Disease

Patients PCNA dsDNA Sm

SLE 100 Rheumatoid arthritis 100 Mixed connective tissue disease 20 Diffuse scleroderma 50

nRNP

SS-B

Scl-70

Histone

2

43

14

26

17

0

36

0

6

0

0

6

0

38

0 0

0 0

0 0

20 2

0 1

0 11

2 2

B Disease

Patients PCNA Ki

SLE 328 Rheumatoid arthritis 115 Mixed connective tissue disease 30 PSS 58 DM/PM 23 Sj S 15 Behcet 15

2.1%

Sm

nRNP

SS-A

SS-B

Scl-70 Centro Jo-I mere

10.4% 10.4% 39.0% 40.2% 6.1%, 0%

0%)

0% 0

0

0

0

1.7

17.4

4.3

0

0

0 0 0 0 0

0 0 0 0 0

0 0 0 0 0

100 15.5 4.3 13.3 0

16.7 10.3 13.0 46.7 6.7

0 0 0 20.0 0

0 31.0 0 0 0

0 12.1 0 0 0

0 0 8.7 0 0

* PCNA = proliferating cell nuclear antigen; dsDNA = double-stranded DNA: Sm = Smith antigen; nRNP = nuclear ribonucleoprotein antigen: SS-A = Sjögren’s syndrome antigen A: SS-B = Sjögren’s syndrome antigen B; Scl-70 = scleroderma 70 antigen; SLE = systemic lupus erythematosus: SjS = Sjögren’s syndrome; DM = Dermatomyositis; PM = Polymyositis AMAN-C2.4/4

comparing the clinical features of patients with and without anti-PCNA (Table 3) [2]. Patients with anti-PCNA showed higher percentage of typical clinical features of lupus such as malar rash, photosensitivity, renal involvement, seizure and thrombocytopenia. Especially the incidence of seizure and thrombocytopenia was significantly higher than that of patients without anti-PCNA. The clinical features of 7 patients with anti-PCNA antibodies reported by Fritzler et al were shown in Table 4 [2]. The high prevalence of CNS involvement (42.9%) was also observed in those patients. Renal involvement was observed in 5 out of 6 anti-PCNA positive lupus patients and all those patients had DPGN. The noteworthy observation was that 4 anti-PCNA positive patients with DPGN did not have antibodies to DNA. This is quite interesting because one of anti-PCNA positive lupus patients with DPGN reported by Ohata was also negative for anti-DNA during her clinical course [3]. In contrast, thrombocytopenia which was Table 3. Clinical features and laboratory findings of SLE patients with and without antiPCNA Clinical features and laboratory findings Malar rash Discoid rash Photosensitivity Oral ulcer Arthritis Serositis Raynaud’s phenomenon Alopecia Renal disorder proteinuria cellular casts Neurologic disorder seizure psychosis Hematologic disorder hemolytic anemia leukopenia lymphopenia thrombocytopenia ANA Anti-dsDNA Anti-Sm Anti-UI RNP Anti-SS-A Anti-SS-B Anti-asialo GMI RA-test λ STS

Patients With anti-PCNA (n = 7) 100.0%, 14.3 85.7 42.9 85.7 14.3 42.9 71.4

78.5% 14.3 43.7 38.5 92.8 11.0 53.9 65.9

100.0 100.0

89.4 76.5

42.9* 28.7

7.8 23.9

14.3 100.0 42.9 57.1** 100.0 85.7 14.3 14.3 71.4 14.3 50.0ϕ 28.6 28.6

10.6 65.1 46.8 10.6 99.7 76.5 27.6 43.0 44.1 6.8 54.6δ 36.7 18.4

* p < 0.005; ** p 10.05; ϕn = 4: δn = 66: AMAN-C2.4/5

Without anti-PCNA (n = 293)

λ

Serological test for syphilis

significantly higher in patients reported by Ohata was not observed in 7 antiPCNA positive patients reported by Fritzler et al. [2]. Table 4. Clinical features of 7 patients with anti-PCNA antibodies Patients

Age/sex/ race*

Diagnosis§ Oralcuta neous

AH IF EA FC MC PH MM

26/F/W 36/f/W 36/M/B 49/M/W 30/F/W 22/F/W 35/F/O

SNA DPGN SLE SLE SLE SLE SLE

– – + – – – +

Arthritis

Renalπ

CNS

Kidneybiopsy

+ – + + + + +

– + – + + + +

– – + + – + –

ND DPGN ND DPGN DPGN DPGN DPGN

* B = black; O = oriental; W = white § SNA = seronegative arthritis; DPGN = diffuse proliferative glomerulonephritis S Renal parameters include at least one of the followings: microscopic hematuria, red cell casts, or proteinuria ¶ Central nervous system (CNS) include at least one of the followings: dementia, seizures, coma, psychosis, or focal neurologic disease

3.3 Disease activity and anti-PCNA antibody To study the relationship between anti-PCNA antibodies and activity of clinical features which are associated with anti-PCNA, retrospective longitudinal studies were performed by Ohata and Fritzler et al. [2, 3]. The representative results are shown in Fig. 1 [3]. Case Y.O. was diagnosed as having SLE because of malar rush, alopecia, Raynaud’s phenomenon, proteinuria and positive ANA in 1975. She was given corticosteroid at that time and those clinical features improved. In mid-1979, the anti-PCNA antibodies became positive with anti-dsDNA antibodies, and then the titer rose with decreasing of levels of serum complement preceding the appearance of profuse proteinuria. Thrombocytopenia which has been shown to be significantly higher in patients with anti-PCNA was also observed with the elevated titer of anti-PCNA. All symptoms quickly responded to the corticosteroid therapy with decreasing the titer of anti-PCNA. Case SE (Fig. 1) was negative for anti-dsDNA antibodies through out her clinical course, even when she had the high titer of anti-PCNA with active clinical features such as high fever, proteinuria, leukopenia, and thrombocytopenia. Fritzler et al. also reported the relationship between the titer of anti-PCNA and disease flare of renal involvement, and the high sensitivity of anti-PCNA antibodies to corticosteroid therapy [3]. These data suggest that the sequential detection of anti-PCNA antibodies in anti-PCNA positive lupus patients may give us useful information to evaluate disease activity such as renal involvement, and to evaluate the response for the therapy. Therefore the test for anti-PCNA should be AMAN-C2.4/6

Fig. 1. Clinical course of patients Y.O. and S.E. and titer of anti-PCNA antibodies [3].

performed repeatedly when we treat the lupus patients with anti-PCNA. Although these observations suggest the strong relation of anti-PCNA antibodies to renal involvement and anti-PCNA positive patients sometimes have DPGN without anti-DNA antibodies, direct evidence to show the pathogenic role of anti-PCNA antibodies in renal involvement has not been established.

3.4 Disease activity and activated peripheral blood mononuclear cells detected by anti-PCNA antibodies Detection of peripheral blood mononuclear cells (PBMC) expressing PCNA is also useful for the evaluation of disease activity in lupus patients. Murashima et al. reported that PCNA was detected in PBMC from lupus patients with the high disease activity using monoclonal antibody to PCNA (TOB7) [9]. Forty-four of 58 patients with SLE had PBMC expressed PCNA and the percentage of PCNA-positive PBMC in patients with SLE was 0–20% (mean: 2.63%) which was significantly higher (P <0.01) compared with normal controls (mean: 0.18%), patients with rheumatoid arthritis (mean: 0.83%), and patients with mixed connective tissue disease (mean: AMAN-C2.5/7

0.38% (Fig. 2). They studied the relationship between the percentage of PCNA positive-activated PBMC and the disease activity score estimated the method reported by Ginzler et al. [10], and found that the percentage of PCNA-positive cells in SLE patients significantly correlated with the disease activity as shown Fig. 3. The lymphocyte subsets of PCNA-positive PBMC were examined in these patients, and most of those cells belonged to CD4- or CD8-positive T-cell populations (data not shown).

Fig. 2. Percentage of PCNA-positive cells of patients with SLE, RA, and MCTD. The horizontal bars indicate the mean values. The percentage of PCNA-positive PBMC in patients with SLE was significantly higher, compared with that in patients with RA (P < 0.01), MCTD (P < 0.01, and normal controls (P < 0.01). AM AN-C2.4/8

Fig. 3. Correlation between percentage of PCNA-positive cells and disease activity in SLE patients. Disease activity was scored by Ginzler’s method with modification. There was a significant correlation (P < 0.01) between percentage of PCNA-positive cells and disease activity.

These findings indicate that PCNA-positive activated PBMC are present in SLE patients and the percentage of PCNA-positive PBMC may be used as an indicator of disease activity in addition to anti-PCNA antibodies.

4. Epitopes of anti-PCNA antibodies and clinical significance

There have been several reports concerning the analysis of the epitopes which are recognized by autoantibodies to PCNA as described in ‘Antigen’ Section (Chapter B.2.7). It has been suggested that autoantibodies to PCNA do not recognize sequential linear epitopes but recognize certain conformationdependent epitopes [11–13]. Epitope mapping using deletion mutants of PCNA was performed and showed the heterogeneity of human autoantibodies to PCNA. The relationship of PCNA epitopes to clinical manifestations has not been clarified because of the limited number of patients studied.

5. Serum PCNA and titer of anti-PCNA antibody

Recently we have developed a sandwich type enzyme-linked immunosorbent assay (sELISA) to quantify the amount of PCNA in activated cells [8]. This system was found to be sensitive enough to measure levels of serum PCNA in patients with SLE, and the relationship between the serum PCNA and the titer of anti-PCNA antibodies in lupus patients was studied longitudinally [14]. The levels of serum PCNA in 115 lupus patients were measured by AMAN-C2.4/9

sELISA, and the serum PCNA was detected in 75 patients (6 to 350 ng/ml). The relationship between the level of serum PCNA and anti-PCNA antibody titer was studied in three lupus patients with anti-PCNA longitudinally over ten years (a representative result is shown in Fig. 4). In those patients, levels of serum PCNA fluctuated between 6 and 170 ng/ml and the increase of PCNA level always induced the elevation of anti-PCNA antibody titer within two months. After anti-PCNA titer started to increase, the serum level of PCNA decreased in parallel with the serum level of complements, then patients developed proteinuria or thrombocytopenia. These data indicated that detection of the level of serum PCNA can predict the elevation of anti-PCNA titer and the flare of lupus nephritis in patients with anti-PCNA. In addition these results suggest that the antigen presentation is playing an important role in the production of anti-PCNA antibody.

Fig. 4. Correlation between anti-PCNA antibody and serum level of PCNA in lupus patient Y.O. Anti-PCNA antibodies were detected by ELISA using PCNA purified from rabbit thymus extract Serum level of PCNA was measured by the sandwich type ELISA using monoclonal anti-PCNA antibodies

AMAN-C2.4/10

6. Conclusion

Previous reports concerning anti-PCNA antibodies show low frequency of anti-PCNA of 2–4.3% in lupus patients. The frequencies of anti-PCNA were the same in different ethnic groups. In spite of the low frequency, all reported data revealed that the occurrence of anti-PCNA antibodies is predominantly in lupus patients or patients with clinical features associated with SLE. The higher prevalence of renal involvement and CNS lupus was observed in two different reports. The strong relation of the antibody titer to the activity of renal involvement was revealed in both reports. In addition to anti-PCNA antibodies, detection of PCNA expressing PBMC is show to be useful for the evaluation of the disease activity in lupus. The mechanism of anti-PCNA production is not known but recent studies suggest that the serum autoantigen might play an important role.

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. IO. 11. 12. 13. 14.

Miyachi K, Fritzler MJ & Tan EM (1978) J Immunol 212: 2228–2234 Fritzler MJ, McCarty GA, Ryan JP & Kinsella TD (1983) Arthritis Rheum 26: 140–145 Ohata N (1987) Ryumachi 27: 79–87 Asero R, Origgi L, Crespi S, Bertetti E, D’Agostino P & Riboldi P (1987) Clin Exp Rheumatol 5: 241–246 Takasaki Y , Fishwild D & Tan EM (1984) J Exp Med 159: 981–992 Okano T, Mimori T & Akizuki M (1991) Jpn J Clin Immun 14: 258–266 Takasaki Y, Ohata N, Kodama A, Ishido T, Daidouji H, Hashimoto H & Hirose S (1987) Arthritis Rheum 30: S86 Takasaki Y, Ohgaki M, Kodama A, Ogata K, Hashimoto H, Shirai T & Hirose H (1990) J Immunol Meth 132: 227–237 Murashima A, Takasaki Y, Ohgaki M. Hashimoto H, Shirai T & Hirose S (1990) J Clin Immunol 10: 28–37 Lahita RG, Bradlow HL, Ginzler E, Pang S & New M (1987) Arthritis Rheum 30: 241–248 Ogata K, Kurki P, Celis JE, Nakamura RM & Tan EM (1987) Exp Cell Res 168: 475–486 Huff JP, Roos G, Peebles CL, Houghten R. Sullivan KF & Tan EM (1990) J Exp Med 172: 419–429 Tsai W-M, Roos G, Hugli TE & Tan EM (1992) J Immunol 149: 2227–2233 Takasaki Y, Yamanaka K, Hashimoto H & Hirose S (1992) Arthritis Rheum 35: S119

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Autoantibody Manuals C2.5, 1-14, 1996 © 1996 Kluwer Academic Publishers Printed in The Netherlands

Antiphospholipid antibodies: Clinical aspects VANESSA MORRIS and CHARLES MACKWORTH-YOUNG Kennedy Institue of Rheumatology, 6 Bute Gardens, London W6 7DW, U. K.

1. Introduction

It has been known for about twenty years that raised serum levels of antibodies to phospholipids are associated with a number of clinical disorders, most notably thrombosis (both venous and arterial), recurrent foetal loss and a moderate thrombocytopenia. However, interest in this area has increased considerably over the last decade since the development of sensitive immunoassays for detecting such antibodies. These methods are relatively easy to perform, and have enabled better delineation of the associated clinical features. It has emerged that a number of different organ systems may be involved, and the subject has therefore become of interest to a variety of clinical disciplines. This so-called ‘antiphospholipid syndrome’ (APS) occurs chiefly in association with raised levels of antibodies to negatively-charged or anionic phospholipids. Its name is a little misleading, since it might be taken to imply that the antibodies are themselves pathogenic. This has certainly not been proven, although, as described below, there are several lines of in vitro evidence to suggest that this may possibly be the case.

1.1 History There are three principal methods of detecting antiphospholipid antibodies in serum: the VDRL test (serological test for syphilis), the lupus anticoagulant (LA) test, and the solid-phase immunoassay for anticardiolipin antibodies (aCL). The VDRL test is a development of the original Wasserman reaction. It is a flocculation reaction, which uses a mixture of phospholipids as an antigen. It was recognised in the early 1950s that there were individuals who had a ‘false-positive’ VDRL test, ie more specific tests for treponemal infection were negative. Moore and Mohr showed that these patients tended to fall into two groups: those who had a transiently positive VDRL, in whom there was generally an infection; and those who had a chronically positive test, and who commonly had an autoimmune disorder, such as systemic lupus erythematosus (SLE) [1]. At about the same time, the lupus anticoagulant test was first described [2]. This is an in vitro coagulation abnormality, and is detected as a prolongation of the partial thromboplastin time (PTT) [3]. Although APA AMAN-C2.5/1

are associated with thrombosis in vivo, they may paradoxically cause a prolongation of both the PTT and (less strikingly) the prothrombin time. This is probably due to an interaction between the antibody and the phospholipids in the prothrombinase and factor X-converting complexes of the clotting cascade. This ‘anticoagulant’ activity is distinguished from other coagulation abnormalities, such as factor deficiencies, by the finding that the abnormality cannot be corrected by the addition of normal human plasma. In addition to thrombosis, other clinical associations of the lupus anticoagulant were established, notably thrombocytopenia and a tendency to recurrent abortion. In vitro studies demonstrated that LA activity could be mediated by antibodies of IgG or IgM class. It was also noticed that there was an association between the presence of the LA and a positive VDRL test, suggesting that the LA might be due to activity against certain phospholipids [4]. This was supported by the finding that LA activity could be partially abolished by pre-absorption of serum with the negatively-charged phospholipid cardiolipin, which is one of the antigens in the VDRL reagent. Later it was shown that a monoclonal antibody with LA activity was able to precipitate negatively-charged phospholipids [5]. These observations gave the impetus to the development of solid-phase immunoassays for antiphospholipid – in particular anticardiolipin – activity [6]. The use of these assays confirmed that raised levels of negatively-charged phospholipids are associated with not only with thrombosis, foetal loss and thrombocytopenia, but also with a variety of other clinical disorders (see next Section). The antiphospholipid syndrome thus emerged as clinical entity. Criteria for its diagnosis have now been established (see below).

1.2 Clinical features of the antiphospholipid syndrome Raised serum levels of aPL may be found in a wide variety of conditions. These include infections (particularly viral and protozoal), malignancies and certain haematological disorders. However, raised levels have been most commonly described in connective tissue diseases, most notably systemic lupus erythematosus (SLE) and related conditions. It is in these disorders that the antiphospholipid syndrome tends to occur. Although closely related to connective tissue diseases, the syndrome may exist in isolation as a primary disorder [7–9]. The main clinical manifestations of this syndrome are related to vascular events (Table 1) (10–12). Venous occlusions, often recurrent, are the dominant feature. They most frequently affect deep calf veins, but any part of the venous system may be involved. Occlusion of certain vessels may lead to organ dysfunction, as in retinal or renal vein thrombosis, hypoadrenalism or, in the case of hepatic vessels, the Budd-Chiari syndrome. Pulmonary embolisation often occurs. Occlusion of arteries is somewhat less common. In the case of large AMAN-C2.5/2

Table 1. Clinical features of the antiphospholipid syndrome Established Venous thrombosis Arterial thrombosis Foetal loss Thrombocytopenia Probable Neurological disorder (excluding stroke) e.g. epilepsy chorea migraine multi-infarct dementia lupoid sclerosis Heart valve vegetations Livedo reticularis Raynaud’s phenomenon (Positive direct Coombs’test)

arteries this may result in stroke, transient ischaemic attacks, ischaemia or gangrene of the limbs, myocardial infarction, and ischaemia to kidneys or other areas. Occlusion of smaller vessels may cause different patterns of damage: examples include multi-infarct dementia or ischaemic encephalopathy in the brain, a type of vascular cardiomyopathy in the heart, and a renal microangiopathy. Foetal loss may be striking. It is not uncommon to see patients who have lost as many as six or eight pregnancies before the diagnosis has been made. The thrombocytopenia is usually mild or moderate, and only rarely results in a significant bleeding disorder. A number of other features may occur, most of them related to the hypercoagulable state. Thrombotic vegetations may develop on heart valves, resulting in valvular incompetence or stenosis. Thrombus may form in the cardiac chambers themselves. In both of these situations there is a risk of embolisation leading to strokes or transient ischaemic attacks. A number of other neurological problems may occur, including migraine, epilepsy, chorea, transverse myelitis, Guillain-Barre syndrome and optic atrophy. A wide variety of skin manifestations may be seen, the best described being the blotchy rash of livedo reticularis. In addition, there is probably an association with the presence of a positive direct Coombs’ test, although frank haemolytic anaemia is uncommon. A small minority of patients with high levels of aPL develop widespread vascular occlusions, often without any other apparent predisposing factors. This serious, often fatal condition has been termed the ‘catastrophic’ antiphospholipid syndrome [ 13].

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1.3 Pathogenesis The majority of these clinical features tend to occur more commonly with increasing levels of aPL in the serum, particularly of the IgG isotype [ 14, 15]. However, it is still not clear whether the antibodies themselves play a part in pathogenesis. There is considerable evidence that aCL may interfere with clotting mechanisms, although much of it derives from in vitro work, and is controversial (reviewed in [16]). For instance, there have been studies to suggest that aCL may modulate the release of prostacyclin from endothelial cells, reduce protein C, protein S, thrombomodulin or anti-thrombin III activity, interfere with thrombolysis by reducing the activity of tissue plasminogen activator, or bind to activated platelets. It has also been suggested that aCL may affect clotting through their ability to bind to B2 glycoprotein I, which has anticoagulant properties [ 17]. The finding that normal mice immunized with aCL develop a condition similar to the antiphospholipid syndrome provides some in vivo support for the possibility that aCL are indeed pathogenic [ 18, 19].

2. Indications for requesting a test

Patients whose serum should be tested for the presence of antiphospholipid antibodies include those who have suffered from any from of thrombosis for which there is no obvious cause, such as young patients with a calf vein thrombosis or myocardial infarction. Other individuals in whom the antiphospholipid syndrome is a possibility should also be tested: for instance women who have suffered recurrent abortion, individuals with idiopathic thrombocytopenia, and patients with SLE and other connective tissue diseases. The most practical test to use for such screening is the ELISA for anticardiolipin antibodies. If a serum proves positive, it is reasonable to test the patient for the presence of the LA, preferably by more than one method. Some authors suggest that the LA test should be used in the initial screen, since there are a number of individuals who are LA-positive, but negative by ELISA. However, this is expensive, and not practical for most laboratories. The demonstration of raised levels of APA may suggest or confirm, in the right clinical context, the presence of the antiphospholipid syndrome. If there is no evidence for the syndrome itself, they may point to the presence of a related autoimmune disorder, such as SLE. Although studies in groups of patients have shown an increased risk of thrombotic events with increasing aCL level, the evidence that this is the case for an individual patient is anecdotal. Therefore, in patients in whom the diagnosis of antiphospholipid syndrome is established, repeated measurements of aCL level or LA activity probably need only be performed occasionally (e.g. every few months), in order confirm continuing risk.

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3. Assays for detecting antiphospholipid antibodies

3.1 The VDRL test This serological test for syphilis is an adaptation of the original Wassermann reaction. It is a flocculation reaction, the antigenic material used containing the anionic phospholipid cardiolipin and the neutral phospholipid phosphatidyl choline, together with cholesterol as a ligand. The VDRL test therefore detects a population of antibodies which bind to cardiolipin, in addition to antibodies with other specificities.

3.2. Lupus anticoagulant Although aCL are associated with thrombosis in vivo, they may paradoxically cause a prolongation of the partial thromboplastin time and, less strikingly, the prothrombin time. This is probably due to an interaction between the antibody and phospholipids in the prothrombinase and factor X-converting complexes of the clotting cascade. This ‘lupus anticoagulant’ activity is distinguished from other coagulation abnormalities, such as factor deficiencies, by the finding that the abnormality cannot be corrected by the addition of normal human plasma. Most lupus anticoagulant tests detect a dose-dependent prolongation of the partial thromboplastin time. The tests which are most commonly used are the activated partial thromboplastin time (APTT), the kaolin clotting time (KCT) and the Russell Viper Venom Test (RVVT) (reviewed in [20–22]). In order to distinguish if such a prolongation is due to the lupus anticoagulant or to another disorder (such as a clotting factor defect), a mixing test is usually performed, in which normal human plasma is added to the assay: this should correct a clotting factor deficiency but not the lupus anticoagulant effect. Most laboratories carry out an initial screen using two of the partial thromboplastin time methods, and follow this with a mixing test if appropriate.

3.3 Solid-phase assays for antiphospholipid antibodies The original solid-phase assays for antiphospholipid activity were radioimmunoassays. Nowadays ELISAs are almost universally used. Phospholipid is air-dried onto the wells of plastic plates, after which test serum is added. Antibodies bound to the phospholipid are detected using an enzymelinked isotype-specific antihuman antibody. A substrate is then added, in which the enzyme induces a colour change. Assays of this kind can give a sensitive, quantitative measurement of antiphospholipid activity. Most laboratories now use an assay which is standardised according to international AMAN-C2.5/5

criteria, using known control sera [23]. The results are usually expressed in units: GPL, MPL and occasionally APL for anticardiolipin antibodies of IgG, IgM and IgA isotypes, respectively. Although cardiolipin is the phospholipid most commonly used, activity against other phospholipids may be detected by this method (Table 2). There has been great discussion over the last four years about the precise nature of the ligand(s) bound by aCL in ELISA and other systems. It is now clear that many aCL require the presence of a serum cofactor, β2 glycoprotein I, for binding to cardiolipin [24, 25]. It has not been established whether the antibodies bind to the cofactor itself, an altered configuration on the cardiolipin molecule, or a neoantigen created by the association of cofactor and phospholipid [26]. However, in clinical laboratories tests for aCL are performed in the presence of human serum or plasma, and the exact identity of the ligand recognised is not of central importance.

3.4 Comparison of the methods Although all three of these methods measure antiphospholipid antibodies, there are important differences in the antibodies which they detect. Most plasmas which are LA-positive are also positive for aCL by ELISA. The reverse, however, is not the case, probably because the two methods detect different, albeit overlapping, antibody populations. This is partly due to the fact that the ELISA is generally (but not always) a more sensitive assay than the LA test. Conversely, most workers find that the LA test is more specific than the ELISA for clinical features of the antiphospholipid syndrome (see below). This difference depends largely on the cut-off points used to determine positivity in the ELISA: increasingly high levels of aCL, particularly of the IgG isotype, are associated with higher prediction rates for features of the syndrome. Although some antibodies positive in the LA or ELISA tests are also Table 2. Phospholipids commonly used in assays for detecting antiphospholipid antibodies Negatively-charged (anionic) Cardiolipin Phosphatidic acid Phosphatidyl inositol Phosphatidyl glycerol Phosphatidyl serine Neutral (zwitterionic) Phosphatidyl choline Phosphatidyl ethanolamine Sphingomyelin Platelet activating factor

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positive in the VDRL test, the latter detects a largely different set of antibodies [27]. This may be because the cardiolipin in the VDRL reagent is somehow altered by its association with phosphatidyl choline and cholesterol. The test is a relatively poor predictor of features of the antiphospholipid syndrome, and is not generally used for screening or monitoring patients. The ELISA, being sensitive and easy to perform, is probably the most useful test in this respect; the LA test is somewhat more laborious and requires fresh plasma, and therefore, despite its sensitivity, is less frequently used for screening (see above).

3.5 Diagnosis of the antiphospholipid syndrome Criteria have been established for the diagnosis of the antiphospholipid syndrome, which involve both clinical and laboratory findings. These are now generally accepted, and are summarised in Table 3. Table 3. Criteria for the diagnosis of the antiphospholipid syndrome Clinical

Laboratory

Venous thrombosis Arterial thrombosis Foetal loss Thrombocytopenia

IgG aCL > 20 GPL units IgM aCL > 40 MPL units Lupus anticoagulant test

The minimum for diagnosis is the presence of one clinical plus one laboratory feature. The more clinical and laboratory features present, the likelier the diagnosis. aCL and lupus anticoagulant tests should be positive on more than one occasion greater than eight weeks apart. Although patients may have a false-positive VDRL test, this does not occur frequently enough to be useful in diagnosing the disorder. (Adapted from [26].)

4. Antiphospholipid antibodies in health and disease

4.1 Healthy individuals As with most autoantibodies, there is a low level of antiphospholipid activity in the blood of most healthy people. Elevated levels are defined by reference to groups of normal controls; thus the prevalence of raised levels of aCL in the general population is arbitrarily determined by the cut-off points chosen. The prevalence for the LA has been reported as 2% [28, 29]; that for aCL measured by ELISA as between 0 and 7.5% [30, 31] (Table 4). Following international standardisation of the ELISA, figures for this method are likely to become more consistent [23].

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Table 4. Prevalence for lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) in healthy individuals and patients with rheumatic diseases Population/disease

Antibody

% positive

Reference

Normal

LA aCL LA/aCL LA aCL aCL LA aCL aCL aCL LA aCL aCL aCL

2 0–7.5 100* 34+ 44+ 4–49 0 25 42 32 0 75 67 53

28, 29 30, 31 7–9 12 12 12 12 12 12 38 12 12 39 39

Primary APS SLE Rheumatoid arthritis Systemic sclerosis Juvenile chronic arthritis Lyme disease Syphilis Leprosy Tuberculosis

APS: antiphospholipid syndrome. SLE: systemic lupus erythematosus. LA: lupus anticoagulant. aCL: anticardiolipin antibodies (detected by ELISA). * by definition + these figures represent mean prevalences derived from a large number of separate studies: the ranges for LA and aCL are 6–73% and 21–63%, respectively. The wide disparity between these studies probably reflects differences in patient selection and assay methods. There are similar differences among the other studies quoted in this table Only among patients with primary antiphospholipid syndrome or SLE has a consistently strong association between raised antibody levels and thrombosis or foetal loss been found

4.2 Systemic lupus erythematosus In a review of 29 series comprising over 1,000 patients with SLE, the average prevalence for the LA was reported as 34%, and for raised aCL levels as 44% [12] (Table 4). There was no difference in the levels of aCL in patients taking corticosteroids compared to those who were not. The frequency of LA positivity was generally lower in the studies in which most of the patients were receiving immunosuppressive therapy. There is a strong correlation between aCL and LA levels: in the same review, 45% of the aCL-positive patients were LA-positive, while 59% of the LA-positive subjects were aCL-positive. 48% of the 319 patients with a falsepositive VDRL test had raised aCL levels detected by ELISA. These results, which are fairly typical for the literature, reflect the observation that these different methods detect overlapping but distinctive antibody populations [11, 27, 32, 33]. Although levels of aCL fluctuate over time, they tend to do so less than levels of anti-DNA antibodies, and often remain relatively stable over periods of months or even years.

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4.3 Primary antiphospholipid syndrome Antiphospholipid antibody levels in patients with primary antiphospholipid syndrome tend to be similar to those in patients with antiphospholipid syndrome in the context of SLE [34]. This applies to LA activity, and to IgG and IgM aCL levels as measured by ELISA. The clinical features of the syndrome are also broadly similar in the two groups of patients [7].

4.4 Infections and other conditions aCL levels are frequently raised in certain infections. Syphilis is the best described example. In patients with this disease, the VDRL and ELISA tests are almost always positive, while the LA test is usually negative. The antibodies appear to be substantially different from those seen in the antiphospholipid syndrome, since they tend to bind to both anionic and neutral phospholipids by ELISA [27, 35, 36]. They also lack the requirement of the cofactor B2 glycoprotein I for binding [37]. Lyme disease is another spirochaetal infection in which aCL levels may be raised: one study looked at 28 patients with the condition, seven of whom had raised IgM levels and four raised IgG levels [38]. In most patients studied, these aCL did not bind to Borrelia burgdorferi, the causative organism. Raised aCL levels have also been reported in patients with leprosy (67%) and tuberculosis (53%) [39]; however, this study used a low cut-off point for determining positivity (2 standard deviations above the mean of the normal controls). aCL levels may also be temporarily raised during acute infections, particularly viral. They are generally weakly positive, of the IgM isotype, and mirror the acute-phase response. They have also been described in chronic viral infections, such as HTLV-I and HIV [40]. In none of these infections are features of the antiphospholipid syndrome usually seen. This may be because the aCL are different from those in the antiphospholipid syndrome.

4.5 Family members There is an increased incidence of raised aCL levels among the relatives of patients with SLE. In one study 8%) of first-degree relatives of patients with SLE were found to be positive, all of them strongly so [41]. Most of these were not related to an aCL-positive proband. All of them had clinical and/or other serological abnormalities, compared with only 30% of the aCL-negative relatives; these included Raynaud’s phenomenon, photosensitivity and a positive antinuclear antibody test. However, in none of them could a diagnosis of SLE be made, and none had features of the antiphospholipid syndrome. AMAN-C2.5/9

4.6 HLA associations No consistent data have emerged concerning associations between the antiphospholipid syndrome and particular HLA antigens or haplotypes. There have been studies showing an association between raised aCL levels and the presence of the HLA antigens DR7, DR4, and DQw7 [42–44]. An association with null alleles of C4A or C4B has also been demonstrated [45]. However, C4 null alleles, like the haplotype A1 B8 DR3, are associated with SLE, which is present in many patients with the antiphospholipid syndrome.

4.7 Associations with other autoantibodies Since antiphospholipid antibodies are frequently found in SLE, they are often seen in the context of raised levels of other autoantibodies seen in the condition, such as anti-dsDNA, anti-Sm and anti-RNP antibodies, and antibodies to other intracellular proteins. No particular patterns or negative associations have emerged involving aCL or the LA and these other lupusassociated antibodies. Although many monoclonal antibodies have been described which bind to both cardiolipin and DNA, serum antibodies to these two antigens constitute largely separate populations [ 10].

4.8 Sensitivity and specificity There are considerable differences in both sensitivity and specificity between the LA and aCL (ELISA) tests, and between individual LA and aCL methods. In general, ELISA methods for detecting aCL have been found to be more sensitive, and the LA tests more specific, for the diagnosis of thrombosis and pregnancy loss. Several studies have addressed this issue, with generally similar, though not identical, results [30, 46–48, reviewed in [22]. One of them illustrates the findings well [47]. The authors compared four different LA assays, together with ELISAs for both IgM and IgG ACA, as markers in SLE patients for the various clinical features of the APS. They found that there were differences in sensitivity, specificity and detection rate among the various LA tests, depending on the clinical feature. Thus, for example, a LA method using human brain as a source of phospholipid gave a sensitivity for predicting thrombosis of 65%, a specificity of 87% and a detection rate of 61%, while respective figures for foetal loss were 58%, 89% and 79%. By comparison, the IgM and IgG ELISAs were relatively sensitive (greater than 77% in all cases) but specificity (less than 51%) and detection rate (less than 52%) were relatively low. A panel of three different LA tests was able to identify almost all individuals in this population with thrombosis, foetal loss and

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thrombocytopenia. However, such an approach would be impractical for most centres. One advantage of ELISA methods is their ability to discriminate between different isotypes. Many studies (but not all [49]) have found that raised levels of IgG aCL are more closely associated with thrombosis [14], foetal loss [50] and thrombocytopenia [51] than raised IgM levels. However, most series contain patients with the antiphospholipid syndrome whose aCL are chiefly of the IgM isotype. In general, the higher the aCL level (especially of the IgG isotype), the greater is the risk of clinical features of the syndrome [26]. The use of the internationally standardised ELISA test should help to resolve discrepancies which have been previously reported between different ELISA methods [32].

4.9 Disease expression The observation that increasingly high aCL levels or LA activity are associated with increasing risk of thrombosis or foetal loss is based on a large number of retrospective studies. More recently, prospective studies have emerged which support this finding. For instance, in a study of 15,000 apparently healthy adult males, those with aCL titres above the 95th percentile had a relative risk for developing deep venous thrombosis or pulmonary embolism of 5.3 [52]. Similarly, among a group of 451 low-risk, nulliparous pregnant women, 15.8%) of those who were aCL-positive experienced foetal loss, compared with 6.5% of those who were aCL-negative [53]. These studies confirm that raised antiphospholipid antibody levels may be detected many years prior to the expression of thrombosis or foetal loss.

4.10 Relationship to treatment The question arises as to whether the magnitude or character of the antiphospholipid response (e.g. aCL isotype or LA activity) has a relationship to the effectiveness of therapy. For instance, does a very high IgG aCL level imply indicate that aspirin will be unlikely to prevent arterial thrombosis, and that formal anticoagulation is required? There are as yet no controlled data in this field. Part of the problem is that prophyllaxis itself is an area of considerable controversy, particularly for the prevention of abortion, and good clinical trials are awaited.

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5. Future trends and prospects

It seems likely that important advances in our understanding about the pathogenicity of antiphospholipid antibodies will emerge in the next few years. This may well come about through further in vivo studies, and a clearer picture about the role of cofactors such as β2 glycoprotein I in the binding and pathogenicity of these antibodies. Ultimately, such information may lead to changes in the assay systems used to diagnose and monitor the antiphospholipid syndrome. For the time being, however, the ELISA and lupus anticoagulant are likely to retain their places in the laboratory assessment of this disorder.

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Autoantibody Manual C3.1, 1-10. 1996 © 1996 Kluwer Academic Publishers. Printed in The Netherlands.

Anti-UlsnRNP antibodies and clinical associations FRANK H.J. VAN DEN HOOGEN and LEVINUS B.A. VAN DE PUTTE Department of Rheumatology, University Hospital Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands

1. Introduction

In 1972 Sharp et al. described a syndrome, which they called mixed connective tissue disease (MCTD), that was clinically denoted by features of two or more defined autoimmune connective tissue diseases, namely systemic lupus erythematosus (SLE), systemic sclerosis (scleroderma, SSc) and dermato- or polymyositis (PM), and the presence of a high titre of circulating antibodies to an RNAse sensitive extractable nuclear antigen [1]. This antigen proved to be a ribonucleoprotein complex (RNP) that plays an important role in a process known as pre-messenger ribonucleic acid (RNA)splicing [2] and is composed of a small nuclear (sn) RNA designated U1, associated with a number of common proteins as well as with three distinct U 1 -snRNA binding proteins, namely U1-70K, U1 -A and U 1 -C [3] (reviewed by Manual B 3.1). Anti-UlsnRNP positive sera have been found to react primarily with the 70 kD and A protein, and with lower frequency also with the C protein [4], as well as with the UlsnRNA [5]. Antibodies directed against components of the UlsnRNP complex (anti-UlsnRNP antibodies) are considered to be the hallmark of MCTD. Additional clinical features of MCTD appeared to be infrequent renal disease, good responsiveness to low doses of corticosteroids and a favourable prognosis [1]. Several sets of diagnostic criteria have been proposed in order to distinguish MCTD from other connective tissue diseases [6–9]. Three of these sets of criteria have been compared with each other [10] and are presented in Table 1. However, the concept of MCTD being a truly distinct disease entity has been questioned in many reports. Some hold that MCTD should be viewed as a part of the spectrum of SLE rather than a different disease subset [11], while others have not found a close association between the mixed clinical characteristics of the different autoimmune connective tissue diseases and antibodies to RNP [12–14]. It is evident that anti-UlsnRNP antibodies are not restricted to MCTD, but occur in other connective tissue diseases as well [11, 15-18]. Occasionally anti-U 1 snRNP antibodies have been reported in other autoimmune diseases [19]. Furthermore, the majority of patients with antiU1 snRNP antibodies develop well classified connective tissue disease such as SLE, SSc, rheumatoid arthritis or a combination of these diseases [20–22]. AMAN-C3.1/1

Table 1. Preliminary diagnostic criteria for mixed connective tissue disease Requirements for diagnosis of MCTD Sharp [6]* A. Major Criteria

B. Minor Criteria

1. Myositis, severe 2. Pulmonary involvement (a) CO diffusing capacity <70% normal (b) Pulmonary hypertension (c) Proliferative vascular lesion or lung biopsy 3. Raynaud’s phenomenon or oesophageal hypomotility 4. Swollen hands observed or sclerodactyly 5. Highest observed anti-ENA ≥ 1:10000 and anti-UIRNP positive and anti Sm negative

1. Alopecia 2. Leukopenia 3. Anemia 4. Pleuritis 5. Pericarditis 6. Arthritis 7. Trigeminal neuropathy 8. Malar rash 9. Thrombocytopenia 10. Myositis, mild 11. Swollen hands

a) 4 majors anti-UIRNP ³ 1:4000 exclusions anti Sm + a) 3 majors b) 2 majors from 1.2 and 3 and 2 minors with anti-UIRNP ≥ 1:1000

a) 3 major without anti-UIRNP b) 2 majors, 1 major and 3 minors with anti-UIRNP ≥ 100

Kasukawa et al. [7]* I Common Symptoms

II Anti-UlRNP antibody

1. Raynaud’s phenomenon 2. Swollen fingers or hands III Mixed findings (a) SLE-like findings 1. Polyarthritis 2. Lymphadenopathy 3. Facial erythema 4. Pericarditis of pleuritis 5. Leukopenia of thrombocytopenia (c) PM-like findings 1. Muscle weakness 2. Increased serum levels of myogenic enzymes (CPK) 3. Myogenic pattern in EMG

(b) SSc-like findings 1. Sclerodactyly 2. Pulmonary fibrosis, restrictive change of lung or reduced diffusion capacity 3. Hypomotility or dilatation of oesophagus

1. Positive in either 1 of 2 common symptoms 2. Positive anti-UIRNP antibody 3. Positive in 1 or more findings in 2 or 3 disease categories or (a), (b) and (c)

Alarcón-Segovia and Villarreal [8]* 1. Serological: positive anti-UIRNP at a hemagglutination titre of 1:1600 or higher

1. Serological 2. At least three clinical data

2. Clinical: Oedema of the hands Synovitis Myositis Raynaud’s phenomenon Acrosclerosis * For references: see text AMAN-C3.1/2

3. The association of oedema of the hands, Raynaud’s phenomenon and acrosclerosis requires at least one of the other two criteria

These observations have led to the concept that MCTD is a transient phase of a connective tissue disease, not yet having reached its final expression (Fig. 1). Although it can be concluded from this that anti-UlsnRNP antibodies have predictive value towards the development of a connective tissue disease, it is impossible to make any reliable statement about specificity of antiUlsnRNP antibodies for a certain disease.

2. Indications for requesting a test for anti-UlsnRNP antibodies

Testing for anti-UlsnRNP antibodies is indicated in patients with signs or symptoms of a connective tissue disease and a positive test for anti-nuclear antibodies. Anti-U1 snRNP antibodies cause a speckled fluorescent antinuclear staining pattern (see Manual Parts A and B). Anti-UlsnRNP antibodies may precede disease expression and can already be present years before a diagnosis of a well defined connective tissue disease can be made [20–22]. The clinical implication of this is, that once anti-UlsnRNP antibodies have been demonstrated in a patient suspected of having a connective tissue disease, check-ups are necessary. Although follow-up studies of patients with anti-U1 snRNP antibodies are scarce, there is evidence that anti-U 1 snRNP antibodies persist during periods of active and inactive disease [22, 23]; repeated testing is not necessary. Titers of anti-UlsnRNP antibodies do not appear to have a predictive value towards disease activity; studies examining a possible relation between titers and disease activity are, however, hampered by both difficulties in quantitating anti-Ul snRNP antibodies and properly assessing disease activity. Although in one study the fall or disappearance of antiU 1 snRNP antibodies seemed to occur in association with prolonged remission [24], other studies failed to demonstrate a correlation of antiU 1 snRNP antibody titre and disease activity as determined by predefined criteria [25–26]. In one prospective study major disease exacerbations seemed to be associated with peaks in anti-U1 snRNA antibody level [27]. At first anti-UlsnRNP antibodies were thought to be associated with a favourable prognosis but follow-up studies demonstrated that not only the Transient phase

Definite state Rheumatoid Arthritis

Anti-UlsnRNP antibodies

MCTD

Systemic Lupus Erythematosus SystemicSclerosis

Fig. 1. The majority of patients with anti-UlsnRNP antibodies will develop well classified connective tissue diseases. AMAN-C3.1/3

majority of patients with anti-U1 snRNP antibodies, initially diagnosed MCTD, evolved into well defined connective tissue diseases but that also the disease course was similar to the disease course of patients with these connective tissue diseases but without anti-U 1 snRNP antibodies [20–22]. Antibodies that can be found together with anti-UlsnRNP antibodies are rheumatoid factor in approximately 50% of the cases, and in low frequencies anti-Sm, anti-dsDNA, anticentromere, anti-La and anti-Ro antibodies [22, 23]. When apart from anti-U lsnRNP antibodies antidsDNA- or anti-Sm antibodies are present, the clinical picture will most likely be that of SLE. Whenever anti-dsDNA antibodies de novo appear in a patient with anti-UlsnRNP antibodies, an evolution towards or a flare of SLE must be suspected [22, 28–30]. Anti-UlsnRNP antibodies have been described in all races and approximately 80% of patients with anti-U1 snRNP antibodies are of female gender [31]. The antibodies may occur in all age groups and have been reported in children as young as 4 years [32].

3. Methods for detection (see also Manual A)

3.1 Immunofluorescence The immunofluorescence test can be used as a screening test for the detection of anti-U 1 snRNP antibodies. This test is readily available in general hospitals, gives a coarse speckled pattern in the presence of anti-UlsnRNP antibodies, but does not distinguish between anti-U1snRNP and anti-Sm antibodies. A negative immunofluorescence test makes the presence of antiU1snRNP antibodies unlikely.

3.2 Immunodiffusion or counterimmunoelectrophoresis These tests are at the moment the most widely used techniques to detect antiU1snRNP antibodies. Both tests readily distinguish between anti-UlsnRNPand anti-Sm-antibodies. A reference serum is necessary to define which activity is present. At the current time, these tests are the preferred assays for clinical use.

3.3 Immunoblotting Immunoblotting is a technique that can be used for detection of antiUlsnRNP antibodies as well as a broad range of other autoantibody activities directed against nuclear or cytoplasmic antigens. It is a more laborious test than immunodiffusion or counter immunoelectrophoresis and AMAN-C3.1/4

requires experience concerning the interpretation of the test results. With the immunoblotting technique, it can be shown that the anti-U 1 snRNP antibodies are in most cases directed against the U1-70K- or Ul-A protein, and in 60% of cases against the Ul-C protein as well; the technique is able to distinguish anti-U1 snRNP activity from anti-Sm activity. The major advantage of the immunoblotting technique in clinical practice is that with one test result information can be obtained on the range of autoantibodies associated with connective tissue diseases.

3.4 ELISA ELISA assays offer the possibility to detect and quantitate autoantibodies. Recombinant antigens are available to be used as substrate to make a distinction between antibodies directed against the U1-70K, U 1-A and U 1-C protein. Dot-blot assays have been developed for the detection of anti-U1RNA antibodies. Since there is no evidence that higher levels of antiUlsnRNP antibodies correlate or predict disease activity, there is no need to apply ELISA assays for quantitative purposes.

3.5 RNA and protein immunoprecipitation Both techniques are very sensitive in detecting low titers of anti-UlsnRNP antibodies. They are mostly performed in research laboratories.

4. Antibody activities directed to individual UlsnRNP components

Antibodies to the 70 kD, A en C proteins of the UlsnRNP all contribute to the anti-UlsnRNP response. Antibodies to the 70 kD component of the U1 snRNP particle are detectable in most patients with MCTD, but have also been described in patients with SLE [24, 33, 34]. In patients with MCTD, preselected because of the presence of anti-U1 snRNP antibodies, antibodies to the 70 kD protein occur in 75–95%; anti-70kD antibodies can be detected in 12% of patients with SLE and occasionally in patients with RA, PM/DM or SSc (Table 2) [35]. Interestingly, several studies showed that the absence of antibodies specific for the 70 kD band in patients with antibodies to UlsnRNP particles is strongly associated with SLE [24, 33, 34, 36-38]. Antibodies to the A protein occur in the same frequency as anti-70 kD antibodies in patients with MCTD, and were shown in 23% of patients with SLE [35]. Antibodies to the C protein are detected in approximately the same frequency as anti-70K and anti-A antibodies in patients with MCTD and SLE; anti-A or anti-C antibodies are rare in RA, PM/DM or SSc [35]. AMAN-C3.1/5

Table 2. Occurrence of antibodies directed to individual UlsnRNP components in rheumatic diseases

anti-70 kD anti-A anti-C

MCTD

SLE

RA

SSc

PM/DM

75-95% 75-95% 75-95%

12% 23% 23%

rare rare rare

rare rare rare

rare rare rare

According to Craft and Hardin [35]

Antibodies to the U1 snRNA component of the UlsnRNP particle have been found in 38% of patients with anti-UlsnRNP antibodies [5]. The antiU1snRNA antibodies were always accompanied by anti-U1snRNP antibodies [5]; peaks in anti-UlsnRNA antibody titers seemed to be associated with major disease exacerbations [27].

5. HLA associations and anti-U1snRNP antibodies

Patients with mixed connective tissue disease are reported to have a higher incidence of DR4 compared with normal controls, but this increase appears to be restricted to the subgroup of patients with arthritis [39]. The presence of anti-UlsnRNP antibodies reacting with the 70 kD protein was found to be associated with HLA-DR4/HLA-DRw53 or HLA-DR2 [40, 41]. Several studies have examined immunoglobulin allotypes in patients with anti-U 1 snRNP antibodies. A strong association was found between the Km(1) phenotype and a susceptibility to SLE [42]. In one study the association with the Gm (1,3;5,21) allotype was reported [43], which could not be confirmed in another study [39].

6. Future trends

The cDNA cloning of the autoantigens has facilitated the synthesis of large amounts of human autoantigens to be used in qualitative and quantitative ELISA tests. A sensitive and reliable ELISA test will help to establish an earlier diagnosis with subsequent earlier treatment. Prospective clinical trials will be necessary to disclose any relation between antibody titers and disease activity.

Acknowledgements

We thank W.J. van Venrooij for his critical reading of the manuscript.

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References 1. Sharp GC, Irvin WS, Tan EM, Could RG & Holman HR (1972) Am J Med 52: 148–159 2. Steitz JA, Black DL, Gerke V, Parker KA, Kramer A, Frendewey D & Keller W (1988) In: Birnstiel ML (Ed) Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles, pp. 115–154. Springer Verlag, Heidelberg 3. Lührmann R (1988) In: Birnstiel ML (Ed) Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles, pp. 71–99. Springer Verlag, Heidelberg 4. Van Venrooij WJ & Sillekens PTG (1989) Clin Exp Rheumatol 7: 635–645 5. Van Venrooij WJ, Hoet R. Castrop J, Hageman B, Mattaj IW & Van de Putte LB (1990) J Clin Invest 86: 2154–2160 6. Sharp GC (1987) In: Kasukawa R & Sharp GC (Eds) Mixed Connective Tissue Disease and Anti-Nuclear Antibodies, pp. 23–32. Elsevier, Amsterdam 7. Kasukawa R, Tojo T & Miyawaki S (1987) In: Kasukawa R & Sharp GC (Eds) Mixed Connective Tissue Disease and Anti-Nuclear Antibodies, pp. 41–47. Elsevier, Amsterdam 8. Alarcón-Segovia D & Vilarreal M (1987) In: Kasukawa R & Sharp GC (Eds) Mixed Connective Tissue Disease and Anti-Nuclear Antibodies, pp. 41–47. Elsevier. Amsterdam 9. Porter JF, Kingsland LC, Lindberg DAB, Shah I, Benge JM, Hazelwood SE, Kay DR. Homma M. Akizuki M, Takano M & Sharp GC (1988) Arthritis Rheum 31: 219–226 10. Alarcón-Segovia D & Cardiel MH (1989) J Rheumatol 16: 328–334 11. Reichlin M (1976) N Eng J Med 21: 1194–1195 12. Bresnihan B, Grigor R & Hughes GRV (1977) Ann Rheum Dis 36: 557–559 13. Gaudreau A, Amor B, Kahn MF, Ryckewaert A, Sany J & Peltier AP (1978) Ann Rheum Dis 37: 321–327 14. Notman DD, Kurata N & Tan EM (1975) Ann Intern Med 83: 464–469 15. Lemmer JP, Curry NH, Mallory JH & Waller MV (1982) J Rheumatol 9: 536–542 16. Maddison PJ, Mogavero H & Reichlin M (1978) J Rheumatol 5: 407–411 17. Ginsburg WW, Conn DL, Bunch TW & McDuffie FC (1983) J Rheumatol 10: 235–241 18. Ter Borg EJ, Groen H, Horst G, Limburg PC, Wouda AA & Kallenberg CGM (1990) Sem Arthritis Rheum 20: 164–173 19. Konikoff F, Shoenfeld Y, Isenberg DA. Barrison I, Sobe T, Theodor E & Slor H (1987) Clin Exp Rheumatol 5: 359–361 20. Nimelstein SH, Brody S, McShane D & Holman HR (1980) Medicine 59: 239–248 21. De Clerck LS, Meijers KAE & Cats A (1989) Clin Rheumatol 8: 29–36 22. Van den Hoogen FHJ, Spronk PE, Boerbooms AMT, Bootsma H, de Rooij DJRAM. Kallenberg CGM & Van de Putte LBA (1994) Br J Rheum 33: 1117–1120 23. Lundburg I, Nyman U, Petterson I & Hedfors E (1992) Br J Rheum 31: 811–817 24. Pettersson I, Wang G, Smith EI, Wigzell H. Hedfors E, Horn J & Sharp GC (1986) Arthritis Rheum 29: 986–996 25. De Rooij DJRAM. Habets WJ, Van de Putte LBA. Hoet MH, Verbeek AL & Van Venrooij WJ (1990) Ann Rheum Dis 49: 391–395 26. Ter Borg EJ. Horst G, Limburg PC, Van Venrooij WJ & Kallenberg CGM (1991) J Rheumatol 18: 363–367 27. Hoet RM, Koornneef I, De Rooij DJ, Van de Putte LB & Van Venrooij WJ (1992) Arthritis Rheum 35: 1202–1210 28. Grant KD. Adams LE & Hess LE (1981) J Rheumatol 8: 587–598 29. Sullivan WD, Hurst DJ, Harmon CE, Esther JH, Agia GA, Maltby JD, Lillard SB. Held CN, Wolfe JF, Sunderrajan EV. Maricq HR & Sharp GC (1984) Medicine 63: 92–107 30. Frank MM et al. (1983) Ann Intern Med 98: 206–218 31. Sharp GC et al. (1976) N Engl J Med 295: 1149–1154 32. Singsen BH, Bernstein BH, Kornreich HK, King KK, Hanson V & Tan EM (1977) J Pediatr 90: 893–900 33. Habets WJ, De Rooij DJ, Salden MH, Verhagen AP, Van Eekelen CAG, Van de Putte LB & Van Venrooij WJ (1983) Clin Exp Immunol 54: 265–276 AMAN-C3.1/7

34. Netter HJ, Guldner HH, Szostecki C, Takomek HJ & Will H (1988) Arthritis Rheum 31: 616–622 35. Craft J & Hardin J (1993) In: Wallace DJ & Hahn BH (Eds) Dubois’ Lupus Erythematosus, pp. 216–224. Lea & Febiger, Philadelphia 36. Guldner HH. Netter HJ, Szpostecki C. Takomek HJ & Will H (1988) J Immunol 141: 469–475 37. De Rooij DJ, Van de Putte LB, Habets WJ, Verbeek AL & Van Venrooij WJ (1988) Scand J Rheumatol 17: 353–469 38. Reichlin M & Van Venrooij WJ (1990) Clin Exp Immunol 83: 286–290 39. Black CM, Maddison PJ, Welsh KI, Bernstein R, Woodrow JC & Pereira RS (1988) 31: 131–134 40. Hoffman RW, Rettenmaier LJ, Takeda Y, Hewett JE, Pettersson I, Nyman U, Luger AM & Sharp GC (1990) Arthritis Rheum 33: 666–673 41. Kaneoka H, Hsu K. Takeda Y, Sharp GC & Hoffman RW (1992) Arthritis Rheum 35: 83–94 42. Hoffman RW. Sharp GC, Irvin WS, Anderson SK, Hewett JE & Pandey JP (1991) Arthritis Rheum 34: 453–458 43. Genth E, Zrnowski H, Mierau R, Wohltmann D & Hartl PW (1987) Ann Rheum Dis 46: 189–196

AMAN-C3.1/8

Autoantibody Manual C4.1. 1–14, 1996 © 1996 Kluwer Academic Publishers Printed in The Netherlands

Clinical significance of autoantibodies to Ro/SSA and La/SSB A.G. TZIOUFAS and H.M. MOUTSOPOULOS Department of Pathophysiology, School of’ Medicine, National University of Athens, Greece

1. Introduction

Sjögren’s syndrome (SS) is a common, chronic autoimmune disorder with strong female preponderance. It affects primarily the lacrimal and salivary glands, but systemic manifestations as well as lymphoma development can occur in these patients. The disease can be present alone (primary SS) or in association with the majority of the autoimmune disorders (secondary SS). The major autoimmune phenomena of the syndrome are a local lymphocytic infiltration of the exocrine glands and B-lymphocyte hyperreactivity manifested by hypergammaglobulinemia, autoantibodies directed against organ-specific antigens, such as salivary, thyroid glands and gastric mucosa, as well as non-organ-specific antigens like immunoglobulins (Rheumatoid factors) and cellular antigens Ro/SSA and La/SSB. In 1958, Jones detected a serum factor in patients with SS which precipitated salivary and lacrimal gland extracts. In 1961, Anderson and colleagues identified two immunologically distinct antibody systems in the sera of SS patients, termed SjT and SjD [1]. Later on, two more precipitin systems were discovered and characterized by Reichlin and colleagues [2, 3] using serum samples from systemic lupus erythematosus (SLE) patients. They were designated Ro and La, respectively. Although no direct comparison has ever been performed, their similar physical and serologic characteristics suggest that SjD corresponds to the Ro and SjT to the La antigen. Two new antibody activities in sera from SS patients, designated SS-A and SS-B, were reported in 1975 by Alspaugh and Tan [4]. In 1977 Akizuki detected a precipitin, termed Ha, in sera from patients with pSS and SLE with SS [5]. However, it was soon shown that Ha was identical to the La antigen, whereas in 1979 interlaboratory studies revealed that SSA corresponds to the Ro and SS-B to the La antigen [6]. In recent years, the application of modern techniques and interlaboratory consensus studies have improved the sensitivity and specificity of the assays used in the every day clinical practice, while the molecular biology methods offered new insights in the understanding of these antigen-antibody systems.

AMAN-C4.1/1

2. Methods

A strong association between anti-Ro/SSA and anti-La/SSB autoantibodies has been observed. Anti-La/SSB is always accompanied by anti-Ro/SSA antibodies, while the latter can be found alone in many sera. For the detection of these antibodies several methods have been used. Classically, these autoantibodies are detected by immunodiffusion and counterimmunoelectrophoresis, methods which are qualitative. ELISA and related assays are quantitative, more sensitive and can provide information regarding the isotype and complement fixing ability of the antibody [7]. These methods, however, require purity of the antigens. Western blot offers the advantage of direct visualization of the Ro/SSA and La/SSB antigenic polypeptides. Purified antigens, as well as crude extracts of antigen can be used. RNA precipitation assay is based on the sensitive detection of radiolabelled small Y RNA containing particles precipitated by anti-Ro/SSA antibodies. Anti-La/SSB antibodies, precipitate also a number of other small RNAs including pre-tRNA, 5S RNA, 4.5S RNA, VA-RNA, all products of RNA polymerase III [8]. The precipitation of these RNAs is a confirmation for the presence of anti-Ro/SSA or anti-La/SSB autoantibodies [9].

2.1 Anti-Ro/SSA In recent years, a comparison of all currently available methods for the detection of anti-Ro/SSA antibodies, has been reported from different laboratories. Manoussakis et al. [10] compared five methods including Table 1. Anti-Ro/SSA positive results obtained by four different methods in selected sera from patients with autoimmune rheumatic diseases (modified from [IO]) Percent of positive samples Method

Counterimmunoelectrophoresis

Primary SS SLE

RA

(n = 28)

(n = 26)

(n = 39)

Normal Controls (n = 25)

62

39

0

57

ELISA

50

46

36

0

Immunoblot (Hela extract) anti-60 kD and/or 52 kD anti-60 kD anti-52 kD

46 14 46

31 15 19

13 5 8

0 0 0

RNA precipitation

61

65

49

0

AMAN-C4.1/2

counterimmunoelectrophoresis, ELISA, two immunoblot methods against Hela and erythrocyte extracts and RNA precipitation assay in 93 sera selected from various autoimmune diseases (Table 1). The RNA precipitation assay, showed the highest sensitivity and was selected as the reference method. Counterimmunoelectrophoresis exhibited a specificity of 100% and a sensitivity of 89%. ELISA, showed a comparable specificity (95%) but lower sensitivity (72%). Finally antibodies to 52 or 60KD by immunoblot in Hela extract demonstrated a high specificity (95% and 97%, respectively) but very low sensitivity (36%) and 17%, respectively). In another study, 50 sera, all positive for antiRo/SSA antibodies by counterimmunoelectrophoresis were evaluated by RNA precipitation assay, ELISA and immunoblot for the presence of anti-Ro/SSA and La/SSB antibodies [11]. In this study, also, RNA precipitation assay exhibited the highest sensitivity and specificity. Anti-Ro/SSA ELISA and anti-Ro/SSA immunoblot showed lower sensitivities (96% and 80%, respectively). Sensitivity was even lower for anti-Ro60 antibodies as detected by immunoblot (66%). The consensus workshop for the detection of autoantibodies was initiated in 1988 with major aims to define the interlaboratory consensus in detecting autoantibodies and extract conclusions which could lead to improvements of both sensitivity and specificity of the assays used. The detection rate of anti-Ro/SSA antibodies has improved from 77% in 1989 to 92% in 1992 [12]. The most sensitive technique for anti-Ro/SSA antibodies detection was counterimmunoelectrophoresis (97% vs. 42% for immunodiffusion and 50%) for immunoblot). The above data led to the conclusion that RNA precipitation assay has the highest sensitivity and specificity for the detection of both anti-Ro/SSA and anti-La/SSB autoantibodies. This method, however, cannot be used in the everyday clinical practice but only as a reference and confirmatory method. On the other hand, counterimmunoelectrophoresis appears to be the best and most reliable method for the detection of autoantibodies to Ro/SSA, since it is simple, sensitive and specific, suitable for the every day clinical practice.

2.2 Anti-La/SSB antibodies Parallel data for anti-La/SSB antibodies revealed that ELISA exhibits high sensitivity (98%) but very low specificity (14%)). The immunoblot showed 86% sensitivity and the counterimmunoelectrophoresis 67% [11]. Results from the consensus workshop for the detection of autoantibodies showed that anti-La/SSB specificity was correctly detected by almost all laboratories participating in the study (97%) in 1989 and by all laboratories in 1991. In this study, both counterimmunoelectrophoresis and immunoblot exhibited 100% sensitivity [12]. Counterimmunoelectrophoresis, however, AMAN-C4.1/3

should be the method of choice for routine evaluation, because of its simplicity as compared with immunoblot. Table 2. Prevalence of anti-Ro/SSA and anti-La/SSB in Greek patients with autoimmune rheumatic diseases Diseases

Anti-Ro/SSA (% positive)

Primary Sjögren’s syndrome (n = 101)

63

40

52

10

10

1

SLE

Anti-La/SSB

(n = 112) RA (n = 350) Normal Controls (n = 200)

0.5

0

Table 3. Indications for anti-Ro/SSA and anti-La/SSB testing 1. Patients with suspected diagnosis of primary SS (according to criteria suggested by the European Union Study group) 2. Patients with skin involvement compatible with SCLE 3. Patients with RA, prior to D-penicillamine administration 4. Women with SS. SLE or RA before and during pregnancy

3. Clinical associations

Both anti-Ro/SSA and anti-La/SSB antibodies were initially related with SS or SLE. It soon became evident that anti-Ro/SSA antibodies are detected in sera from patients with several autoimmune diseases (Table 2). In fact anti-Ro/SSA is one of the most prevalent autoantibodies among many autoimmune disorders. In contrast, the presence of anti-La/SSB antibodies is more closely associated with SS. Indications for antibody testing are given in Table 3.

3.1 Anti-Ro/SSA 3.1.1 Primary Sjögren ’s syndrome Antibody to Ro/SSA is the most common autoantibody directed against cellular antigens in sera of patients with primary SS, and found in 60% of patients [13, 14]. Autoantibodies to the 52 kD component are frequently AMAN-C4.1/4

found in sera of SS patients, whereas antibodies to 60 kD component are observed more often in sera of SLE patients [15, 16]. Anti-Ro/SSA is more frequently detected in sera from patients with early disease onset, long disease duration, parotid or major salivary gland enlargement, and intensive lymphocytic infiltration of the minor salivary glands [13]. In fact, anti-Ro/SSA antibodies increased proportionally with grade of minor salivary gland biopsy, starting from 20%) in SS patients with grade 1+ (according to Tarpley’s criteria [17]) and reaching up to 100% in patients with grade 3+ [13]. Table 4. Correlation of anti-Ro/SSA and anti-La/SSB with clinical features in primary SS patients (modified from [13]) Anti-Ro/SSA negative

positive

Anti-La/SSB negative

positive

(n = 23)

(n = 31)

(n = 14)

(n = 40)

Disease onset (years)

39 ± 12

50 ± 9**

38 ± 14

48 ± 15

Disease duration (years)

8 ± 6

4 ± 2 **

9±6

5±4

Positive Shirmer’s test (%)

96

68 *

NS

NS

Parotid gland enlargement (%)78

42 *

93

45**

Splenomegaly/ lymphadenopathy (%)

44

10 *

8 ***

71

Vasculitis (%)

26

0 **

36

0 ***

NS: Non significant p < 0.05 * ** p < 0.01 *** p < 0.001

Furthermore, their presence correlates well with the presence of extraglandular manifestations such as lymphadenopathy, splenomegaly and vasculitis (Table 4). In addition to the close association of antiRo/SSA with vasculitis, Molina et al. have described that the histologic pattern of vasculitis varies in association with anti-Ro/SSA antibodies. In fact, anti-Ro/SSA antibodies are most commonly related with the neutrophilic inflammatory vascular disease, rather than the mononuclear type vasculitis [18]. In summary, antibodies to Ro/SSA are reflecting the extention of the disease in SS and are associated in particular with the extraglandular manifestations and serologic findings of the syndrome. The frequency by which anti-Ro/SSA and anti-La/SSB antibodies are AMAN-C4.1/5

detected in sera from SS patients prompted the European study group to investigate their specificity and sensitivity and to incorporate them in the recently proposed preliminary diagnostic criteria of the disease [19]. In fact, among the serologic tests, the presence of anti-Ro/SSA and anti-La/SSB antibodies showed high specificity but low sensitivity. In contrast, testing for rheumatoid factor or antinuclear antibodies was not specific enough to differentiate between SS and other connective tissue diseases. 3.1.2 Systemic lupus Erythematosus Antibodies to Ro/SSA are detected in 10-50% of patients with SLE depending upon the method used [20]. Antibodies to 60 kD Ro protein, without anti-Ro 52 kD may be found in 10% of SLE patients sera [15, 16]. Furthermore, anti-Ro/SSA antibodies could be found in 21% of SLE sera in the absence of anti-La/SSB antibodies [16]. SLE patients with positive anti-Ro/SSA antibodies present with certain clinical manifestations, such as subacute cutaneous lupus erythematosus (SCLE) and SLE with secondary SS. SCLE is an annular form of lupus dermatitis exacerbated by the sunlight. Cutaneous lesions are non fixed and non scarring and occur usually in sun-exposed areas. The lesions are erythematous papules or small plaques, evolving further into a plaque and scale, the papulosquamous variant, forming polycyclic or erythema annular centrifugum, mimicking psoriasis or lichen planus. SCLE patients present also musculoskeletal problems, most commonly arthralgias and arthritis. The most common serologic abnormality in these patients are antibodies to Ro/SSA, found in 70% of patients. Anti-Ro/SSA antibodies have also been demonstrated in the annular lesions of SCLE patients [21]. Secondary SS may be found in approximately 10% of SLE patients. Eighty percent of SLE and SS patients have anti-Ro/SSA antibodies [22]. 3.1.3 Rheumatoid arthritis Anti-Ro/SSA antibodies are usually found in 2-3% of RA patients. In Greece, a higher incidence of anti-Ro/SSA antibodies in RA patients has been described. Greek RA patients with anti-Ro/SSA antibodies, represent a subgroup comprised primarily of females with erosive symmetric polyarthritis, high incidence of histopathologic evidence of SS and intolerance to D-penicillamine treatment [23]. In fact, from 62 consecutive patients with RA who received D-penicillamine, 32 developed side effects. The clinical picture in both subgroups was similar, but the group with D-penicillamine toxicity was characterized by a high incidence of anti-Ro/SSA antibodies (p < 0.01). The most common side effect in these patients are skin rashes (p < 0.001) [24]). 3.1.4 Neonatal lupus syndrome Neonatal lupus is characterized by the presence of dermatitis resembling the lesion of SCLE, as well as a variety of systemic and hematologic AMAN-C4.1/6

abnormalities, including isolated congenital heart block, hepatitis, hemolytic anemia and thrombocytopenia [25, 26]. The neonatal lupus syndrome is considered to result from the transplacental passage of maternal anti-Ro/SSA or anti-La/SSB autoantibodies into the fetal circulation, producing damage to the otherwise normally developing tissues [27]. Among the clinical features, congenital heart block is the most serious one, since it is irreversible. The non-cardiac manifestations are transient, resolving at about six months of life, coinciding with the disappearance of maternal autoantibodies from the neonatal circulation. In a longitudinal cohort study, carried out to determine the initial clinical picture and the long term outcome of mothers and their children with anti-Ro/SSA and/or anti-La/SSB associated congenital heart block, it was found that 23/60 woman studied were asymptomatic, 15/60 had SLE, 8 had SS and 11 had an undifferentiated connective tissue disease [28]. Seventeen out of 55 affected children (33%) died (12 within the first month after birth). Pacemakers were implanted in 37 (67%) of the 55 children, 27 within 3 months after birth. Eleven (48%) of the 23 initially asymptomatic mothers developed symptoms of a rheumatic disease, while congenital heart block was observed in 4 out of 25 subsequent pregnancies in the 22 women studied (16%). This study, clearly demonstrates that the clinical manifestation of an autoimmune rheumatic disease in asymptomatic mothers, who are identified after the birth of a child with congenital heart block, is common. One third of children with congenital heart block die during the early neonatal period, and of those who survive most require pacemakers. The risk for congenital heart block in subsequent pregnancies, however, is low. The presence of anti-Ro/SSA antibodies in pregnant women may precede neonatal lupus expression in the newborn. Therefore anti-Ro/SSA antibodies should be evaluated in pregnant women with autoimmune rheumatic diseases and particularly SS and SLE.

3.2 Anti-La/SSB 3.2.1 Sjögren’s syndrome Antibodies to La/SSB are detected in approximately 40% of SS patients sera. The presence of anti-La/SSB antibodies is almost always associated with antibodies to Ro/SSA and particularly the 52 kD component [13, 15]. These autoantibodies are more frequently detected in patients with vasculitis as well as in patients with heavy lymphocytic infiltration of the minor salivary glands (60% in patients with grade 3+ according to Tarpley's classification vs. 20% in patients with grade 1+) [13, 18]. Recently it was demonstrated that the total number of immunoglobulin containing plasma cells in labial salivary glands of SS patients with serum anti-La/SSB

AMAN-C4.1/7

antibodies is significantly greater than in SS patients without these autoantibodies [29]. 3.2.2 Systemic lupus erythematosus Antibodies to La/SSB are detected in 10–20% of SLE patients sera. AntiLa/SSB positive SLE patients present very often clinical features of secondary Sjögren’s syndrome. In fact, more than 50% of SLE patients with secondary Sjögren’s syndrome have in their sera antibodies to La/SSB [22, 30]. 3.2.3 Neonatal lupus syndrome Antibodies to La/SSB are also implicated in the development of congenital heart block in neonatal lupus syndrome. It is not known, however, if the tissue damage results from the presence of these autoantibodies or the concomitant presence of anti-Ro/SSA antibodies. In the longitudinal study mentioned above, it was found that all children (100%) who died from congenital heart block had anti-La/SSB antibodies, as compared with 78% of children who survived [28]. Although this difference is not statistically significant, it points to the possible pathogenetic role of anti-La/SSB antibodies.

4. Serologic associations

In primary SS, the association of autoantibodies with each other and hypergammaglobulinemia is very close. Indeed, both anti-Ro/SSA and antiLa/SSB antibodies, are detected more frequently in patients with rheumatoid factor, antinuclear antibodies, cryoglobulinemia and hypergammaglobulinemia [31]. A stepwise logistic regression analysis revealed that neither rheumatoid factor or anti-La/SSB antibodies made an independent contribution to hypergammaglobulinemia. Anti-Ro/SSA, however, showed a higher logistic regression coefficient, leading to the conclusion that antiRo/SSA is the primary autoantibody related to hypergammaglobulinemia in primary Sögren’s syndrome [32, 33].

5. Genetic associations of anti-Ro/SSA immune responses

The association of different products of the MHC loci with certain autoimmune diseases, among them SS, has been studied extensively. Primary SS is associated with the HLA-B8, DR3, and DRw52 alloantigens, and SS in RA patients with HLA-DR4 and DRw52 [34]. Analysing the HLA association in Greek patients it was shown that primary SS in Greece is associated with HLA-DR5 and not with HLA-B8 or HLA-DR3, while patients with SS and RA lack the association with the DR4 antigen [35]. AMAN-C4.1/8

Several studies have shown an increased prevalence of HLA-DR8, DR3 in SS, as well as in SLE patients with anti-Ro/SSA and anti-La/SSB antibodies [32, 36]. An association between DQ1 and DQ2 antigens and high levels of anti-Ro/SSA and anti-La/SSB antibodies has also been reported in patients with pSS [37]. Anti-Ro/SSA antibodies not accompanied by anti-La/SSB are more strongly associated with HLA DR2. Patients with this profile have more often SLE, rather than SS, a young age of disease onset and lower levels of anti-Ro/SSA antibodies [38]. Mothers bearing infants with congenital heart block are invariably HLA-DR3 positive [39]. HLA DR3 is in linkage disequilibrium with DQ2 and DR2 with DQ1. Further studies with restriction fragment length polymorphism (RFLP) techniques have shown that anti-Ro/SSA precipitins are found simultaneously with a particular pair of RFLPs. A DQa RFLP, associated with HLA DQ1 and a DQb RFLP, associated with HLA DQ2 were more closely related with antiRo/SSA antibodies, hence suggesting a gene complementation mechanism operating for the autoantibody response [40]. Molecular analysis of DQB1 and DQA1 alleles found in American Caucasian and American Blacks SS patients revealed high frequencies of DQBl*0201 and DQA1*0501. Recently, a DNA-sequence specific oligonucleotide probe typing and a sequence analysis of Israeli Jewish and Greek non-Jewish patients with SS has been carried out [41]. The majority of patients in both groups presented either DRB1*1101 or DRB1*1104 alleles that are in a linkage disequilibrium with DRB1*0301 and DQA1*0501. Therefore, the majority of SS patients, independent of racial and ethnic differences carry a common allele, specifically the DQA1*0501 allele. Furthermore, it has been shown that a glutamine residue at position 34 of the outermost domain of the DQA1 chain and/or a leucine residue at position 26 of the outermost domain of the DQB1 chain have a 'gene dosage' role in antiRo/SSA and anti-La/SSB antibody response [42]. The DQA1*0501 gene is one of the genes which possesses glutamine at position 34 and is found in the majority of anti-Ro/SSA and anti-La/SSB patients. Taken together, it appears that the DQA1*0501 molecule plays an important role in the autoimmune responses observed in SS.

6. Genetic associations of anti-La/SSB immune responses

Antibodies to La/SSB are almost invariably associated with antibodies to Ro/SSA. Anti-La/SSB has been closely associated with DR3 in several studies, [43–45] while anti-Ro/SSA is associated with HLA DR2. Both autoantibodies are more strongly associated with HLA DR3 and HLA DR2 than their parent disease, SS or SLE [45]. The dissociation between class II antigens observed in anti-Ro/SSA and anti-La/SSB response. suggest that they are probably under independent genetic control, despite AMAN-C4.1/9

the fact that anti-La/SSB responses are not observed usually in the absence of anti-Ro/SSA production. On the molecular level, anti-La/SSB responses seem to be promoted together with anti-Ro/SSA responses in the presence of specific DQα (DQA1) and DQβ (DQβ1) alleles possessing a glutamine residue at position 34 and a leucine residue at position 26, respectively, as mentioned previously [42]. These amino acid residues are located in the second hypervariable region of the HLA DQ molecule and map to the floor of the peptide binding cleft. Heterozygosity of these DQA1 and DQB1 alleles were more strongly associated with anti-La/SSB and anti-Ro/SSA responses in both whites and blacks with SLE and SS when compared with anti-Ro/SSA negative patients with these diseases and to normal, race-matched controls.

7. Pathogenetic role of anti-Ro/SSA and anti-La/SSB autoantibodies

Antibodies to Ro/SSA and La/SSB have stimulated a great interest in the immunology of certain autoimmune diseases such as SS, SLE and neonatal lupus syndrome since their response demonstrate a remarkable strength and persistence, and they are related to the genetic background. The mechanisms underlying the autoimmune response to these antigens remain unknown. It seems, however, that this is an antigen-driven reaction, since: a) a coordinated expression of different anti-La/SSB population exists, and b) human autoantibodies react preferentially with human antigens [46]. Furthermore, it is not yet clear how the antigens trigger the immune system. The onset of the immune response can be accomplished in two different ways. One mechanism is that of molecular mimicry. There is evidence to suggest that regions of both Ro/SSA and La/SSB proteins have sequences identical with viral proteins (see Chapters B.4.1 and 4.2). Second, the La antigen is able to translocate to the epithelial cell membrane after viral infection [47]. Increased concentration of La/SSB antigen in the nucleus and cytoplasm has been also observed in acinar and conjunctival epithelial cells of patients with primary and secondary SS [48, 49]. This aberrant immunostaining pattern was not observed in healthy controls and RA patients. This finding in conjuction with the inappropriate expression of HLA-DR molecules in epithelial cells of SS salivary glands may explain the autoimmune response observed in this disease [50]. The relationship of these autoantibodies with certain clinical manifestations of SS strongly suggests that they are involved in tissue damage. Direct evidence, supporting this notion is weak. Only anti-Ro/SSA antibody have been eluted from the parotid gland of a patient with primary billiary cirrhosis and SS [51]. A similar study has examined the kidneys of two SLE patients with anti-Ro/SSA antibodies and lupus nephritis [52]. Furthermore, Ro/SSA 60 kD inhibits the autologous mixed lymphocyte reaction in a doseAMAN-C4.1/10

dependent manner without similar effects in allogenic mixed lymphocyte reactions or non-specific T-cell proliferation, induced by phytohaemaglutinin [53]. The strongest support for a pathogenetic role of anti-Ro/SSA and antiLa/SSB antibodies comes from the studies of neonatal lupus. Congenital heart block, observed in neonatal lupus is a model of passively acquired autoimmunity. Several lines of evidence support the concept that fetal injury is mediated by anti-Ro/SSA and/or anti-La/SSB autoantibodies. In fact, congenital heart block usually develops between 16 and 24 weeks of gestation, coinciding with the increased transfer of maternal IgG into the fetal circulation [54]. IgG anti-Ro/SSA and anti-La/SSB antibodies and antigens, have been isolated from fetal heart-conducting system [SS]. Finally, infusion of antibodies to Ro/SSA in rabbits may alter the plateau phase of repolarization of the action potential of neonatal, but not in adult rabbit ventricular papillary muscles [56].

8. Future trends and prospects

A great effort is underway for the identification of disease specific B- and T-cell epitopes on Ro/SSA and La/SSB autoantigens. This will allow the better understanding of the immunology of these molecules and will improve the methods used for the detection of autoantibodies directed against them.

Acknowledgment

The authors express their appreciation to Dr. T.A. Kordossis for critical review of the manuscript.

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References 1. Harley JB (1987) In: Talal N, Moutsopoulos HM & Kassan SS (Eds) Sjögren’s Syndrome, pp. 2 18–234. Springer-Verlag. Berlin 2. Clark G, Reichlin M & Tomasi T (1969) J Immunol 102: 117–122 3. Mattioli M & Reichlin M (1974) Arthritis Rheumatism 17: 421–429 4. Alspaugh MA & Tan EM (1975) J Clin Invest 55: 1067–1073 5. Akizuki M, Powers R & Holman HR (1977) J Clin Invest 59: 796–798 6. Alspaugh M & Maddison PJ (1979) Arthritis Rheum 22: 796–798 7. Maddison P, Skinner RP, Vlachoyiannopoulos P, Breunaud BM & Hough D (1985) Clin Exp lmmunol 62: 337–345 8. Hendrick JP, Wolin SL, Rinke J, Lerner MR & Steitz JA (1981) Mol Cell Biol 1: 1138–1149 9. Forman MS, Nakamura M, Mimori T, Gelpi C & Hardin JA (1985) Arthritis Rheum 28: 1350–1363 10. Manoussakis MN, Kistis KG, Liu X, Aidinis V, Guialis A & Moutsopoulos HM (1993) Br J Rheumatol 32: 449–455 1 I. Meilof JF, Bantjes I, De Jong J, Van Dam AP & Smeenk RJT (1990) J Immunol Meth 133: 215–226 12. Charles PJ. Van Venrooij WJ & Maini RN (1992) Clin Exp Rheumatol 10: 507–511 13. Manoussakis MN, Tzioufas AG, Pange PJE & Moutsopoulos HM (1986) Scand J Rheumatol (Suppl) 61: 89–92 14. Harley JB, Alexander EL, Bias NB et al. (1986) Arthritis Rheum 29: 196–206 15. Ben-Chetrit E, Fox RI & Tan EM (1990) Arthritis Rheum 33: 349–355 16. Slobbe RL, Pruijn GJM, Damen WGM, Van Der Kemp JWCM & Van Venrooij WJ (1991) Clin Exp Immunol 86: 99–105 17. Tarpley TM, Anderson LG & White CL (1974) Oral Surg 37: 64–74 18. Molina R, Provost TT & Alexander EL (1985) Arthritis Rheum 28: 1251–58 19. Vitali C, Bombardieri S, Moutsopoulos HM et al. (1993) Arthritis Rheum 36: 340–348 20. Hochberg MC, Boyd RE, Ahearn TM et al. (1985) Medicine (Baltimore) 64: 285–295 21. Sontheimer RD, Maddison PJ, Reichein M et al. (1982) Ann Int Med 97: 664–671 22. Andonopoulos AP, Skopouli FN, Dimou GS, Drosos AA & Moutsopoulos HM (1990) J Rheumatol 17: 201–204 23. Moutsopoulos HM, Giotaki HJ, Maddison PS, Mavridis AC, Drosos AA & Skopouli FN (1984) Ann Rheum Dis 43: 285–287 24. Vlachoyiannopoulos PG, Zerva LV, Skopouli FN, Drosos AA & Moutsopoulos HM (1991) J Rheumatol 18: 44–49 25. Laxer RM, Roberts EA, Gross KR et al. (1990) J Pediatr 116: 238–242 26. Watson R, Kang JE. May M, Huda KM, Kickler T & Provost TT (1988) Arch Dermatol 124: 560–563 27. Scott JS, Maddison PJ. Taylor RV, Esscher E. Scott 0 & Skinner RP (1983) N Engl J Med 309: 209–212 28. Waltuck J & Buyon JP (1994) Ann Int Med 120: 544–551 29. Bodeutsch C. De Wilde PCM. Kater L et al. (1992) Arthritis Rheum 35: 1075–1087 30. Moutsopoulos HM, Klippel JH, Pavlidis N et al. (1980) Arthritis Rheum 23: 36–40 31. Alexander EL, Arnett FC, Provost TT & Stevens MB (1983) Ann Int Med 98: 155–160 32. Harley JB, Alexander EL, Bias WB et al. (1986) Arthritis Rheum 29: 196–206 33. Harley JB (1987) In: Talal N, Moutsopoulos HM & Kassan S (Eds) Sjögren’s Syndrome. Clinical and Immunological Aspects. pp. 218–234. Springer-Verlag. Berlin 34. Moutsopoulos HM, Mann DL, Johnson AH & Chused TM (1979): N Engl J Med 301: 761–763 35. Papasteriades CA, Skopouli FN, Drosos AA, Andonopoulos AP & Moutsopoulos HM (1988) J Autoimmunity 1: 85–90 36. Wilson RN, Provost TT, Bias WB et al. (1984) Arthritis Rheum 27: 1245–1253 AMAN-C4.1/13

37. Harley JB, Reichlin M, Arnett FC, Alexander EL, Bias WB & Provost TT (1986) Science 232: 1145–1147 38. Hamilton RG, Harley JB. Bias WB et al. (1988) Arthritis Rheum 31: 496–508 39. Arnett FC (1991) In: Talal N (Ed) Molecular Autoimmunity. pp, 33–46. Academic press. New York 40. Fujisaku A, Frank MB, Neas B, Reichlin M & Harley JB (1990) J Clin Invest 86: 606–611 41. Roitberg-Tambur A, Friedmann A, Safirman C et al. (1993) Human Immunol 36: 235–242 42. Reveille JD, Mateod MJ, Whirrington K & Arnett FC (1991) J lmmunol 146: 3871–3875 43. Whittingham S, Mackay IR & Tait BD (1983) Aust NZ J Med 23: 565–570 44. Harley JB, Sestak AL, Willis LG et al. (1989) Arthritis Rheum 32: 826–836 45. Bell DA & Maddison PJ (1980) Arthritis Rheum 23: 1268–1273 46. Slobbe RL, Pruijn GJM & Van Venrooij WJ (1991) Ann Med Interne 142: 592–600 47. Baboonian C, Venables PJW, Booth J, Williams DG, Roffe LM & Maini RN (1989) Clin Exp Immunol 78: 454–459 48. Bodeutsch C, De Wilde P, Van den Hoogen F & Van Venrooij W (1992) Clin Rheumatol 11: 122 49. Yannopoulos DI, Roncin S, Lamour A, Pennec YL, Moutsopoulos HM & Youinou P (1992) J Clin Immunol 12: 259–265 50. Moutsopoulos HM (1993) LUPUS 2: 209–211 51. Penner E and Reichlin M (1982) Arthritis Rheum 22: 1250–1253 52. Maddison PJ and Reichlin M (1979) Arthritis Rheum 22: 858–863 53. Karsh J, Harley JB, Coldstein R & Lazarovits AI (1993) Clin Exp Immunol 91: 103–109 54. Herreman G & Galezewski N (1985) N Engl J Med 312: 1329 55. Horsfall AC, Venables PJW, Taylor PW & Maini RN (1991) J Autoimmunity 4: 165–176 56. Alexander EL, Buyon JP, Provost TT & Guarnieri T (1992) Arthritis Rheum 35: 176–189

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Autoantibody Manual C5.1. 1–7, 1996 © 1996 Kluwer Academic Publishers Printed in The Netherlands

Autoantibodies to scleroderma-associated antigens NAOMI F. ROTHFIELD The School of Medicine of the University of Connecticut Health Center, Department of Medicine MC-1310, 263 Farmington Avenue, Fammington, Connecticut 06032, U.S.A.

1. Anti-centromere Antibodies (ACA)

1. Introduction Speckled and nucleolar patterns of nuclear fluorescence using mouse liver as substrate were first reported in 60 percent of scleroderma patients by Rothfield and Rodnan in 1968 [1]. Since then the characteristics of the speckles have been defined and many of the autoantigens have been identified. The anti-centromere antibody (ACA) was identified when tissue culture cells in division instead of resting rodent liver or kidney were used as substrate in the indirect immunofluorescence assay [2]. ACA since then have been separated into specific antibodies directed against specific centromeric proteins (CENPs), i.e. anti-CENP-A, anti-CENP-B, and anti-CENP-C [3]. ACA along with anti-topoisomerase I are very specific for scleroderma/ CREST and are the major autoantibodies found in this group of diseases. The autoantibodies are easily detected by assays available in nearly all clinical laboratories. (see also Chapter A and Chapter B.5.2)

1.2 Assays ACA is detected in most laboratories by the indirect immunofluorescence assay using dividing cells (such as HEp-2 cells) as substrate. The test is very simple to perform but time must be spent by the reader of the slides so that dividing cells are found. The test is very simple, very sensitive and moderately specific for the diagnosis of SSc/CREST. A positive test is also found in about 10% of patients with pure Raynaud’s diseases and in a small percentage of patients with other connective tissue diseases who have Raynaud’s syndrome, i.e. SLE, Sjögren’s syndrome with Raynaud’s syndrome. polymyositis with Raynaud’s syndrome. The test is not a required criterion for diagnosis of SSc/CREST. ACA are found primarily in CREST/SSc/Raynaud’s patients and may be found in patients with primary biliary cirrhosis. ACA is not found in normal individuals even in low titers. ACA is more common in patients with CREST who do not have proximal scleroderma than in patients with CREST and AMAN-C5.1/1

proximal scleroderma. Thus far, SSc/CREST patients with ACA have been found to have a longer disease duration and to be older than the SSc/CREST patients without ACA [4, 5]. ACA(+) patients have less internal organ and less extensive skin involvement, and are associated with skin thickening limited to the fingers [6], the development of calcinosis or ischemic digital loss [7, 8]. Using the severity index devised by Hughes et al. [9], we found that there was significantly lower mean disease severity score in patients with ACA than in those without ACA. In patients with proximal scleroderma, those with ACA are less likely to have facial skin thickening or arthritis and more likely to have telangiectasias and be female than those who are ACA(–) [10]. ACA(+) is more likely to occur in white females [11, 12]. ACA has been reported to occur more often in older patients [4, 5, 13] although analysis of our own patients did not support this finding [10]. In Raynaud’s disease, we found the prevalence of ACA in 194 patients is 13.4%. In a prospective study, ACA predicted the development of telangiectasias and the development of a connective tissue disease [14].

Prevalence rates of ACA

No+/No tested

%+

CREST (without tight skin) SSc Raynaud’s disease * Overlap diseases + Raynaud’s syndrome

42189 621222 261253 32197

47.0 28.0 10.0 33.0

*

Includes SLE, SLE/SSc, Primary Sjögren’s disease, rheumatoid arthritis, polymyositis

ACA are more common in Caucasians than in Blacks [15]. No other ethnic, geographic or racial differences incidence has been reported. ACA are rarely found in the same patient as anti-topoisomerase. ACA are found in association with other autoantibodies in SLE patients and in Sjögren’s patients. ACA is basically associated with the presence of Raynaud’s syndrome and is infrequently found in patients who do not have Raynaud’s syndrome. In Sjögren’s syndrome, patients who also have Raynaud’s syndrome, may have anti-Ro, anti-La and ACA. In Raynaud’s disease, the presence of ACA predicts the development of telangiectasias [14] and, therefore, it is worthwhile checking on the presence of ACA in patients with isolated RD. ACA does not reflect disease activity but is present without fluctuating very much in titer for many years [16]. As described above, the presence of ACA does predict telangiectasias in RD patients and mild disease in those with CREST/SSc. Patients without ACA do not become positive over the course of the disease unless Raynaud’s syndrome suddenly appears in the course of a connective tissue disease. In my opinion, there is no reason to repeat the assay once a positive ACA has been found. AMAN-C5.1/2

There is no data at present to conclude that ACA predicts a response or lack of response or adverse reactions to drugs.

Future trends Recombinant CENPS are being used in ELISA for screening of large populations of patients. It may be that the dissection of autoantibody reactivity may lead to our ability to predict response to drugs on the basis of the specific CENP to which the patient’s ACA reacts.

2. Anti-topoisomerase I (Anti-topo I)

Anti-topo I was previously known as anti-Scl-70 [17]. Douvas and Tan first describe Scl-70 as 70 kDa antigen extracted from calf thymus which gave a precipitin line in immunodiffusion with some SSc sera. We identified Scl-70 as topoisomerase I and thus further studies of this autoantibody was made possible [18].

2.1 Assays Anti-topo I is most commonly measured using the classical double diffusion in agar and an antigen extracted from calf thymus. A more sensitive technique is the use of immunoblotting on nuclei where topo I is identified as a 100 kDa band. However, there are more than one nuclear antigen which are 100 kDa protein so that the more definitive assays should be carried out. The enzyme inhibition assay is very sensitive and specific. In this assay, the ability of sera to inhibit the action of topoisomerase I in uncoiling supercoiled DNA is determined [18]. A simple assay is the ELISA using purified topo I as substrate. This is more sensitive than double immunodiffusions. For example, in our studies, if inhibition of enzyme activity is taken as the gold standard, then the sensitivity of the double diffusion is 79%, of the ELISA is 100% and of the immunoblot 100%). The sensitivity of the double diffusion assay is 100%. ELISA 98% and immunoblot 97%. The presence of anti-topo I is not a required criterion for diagnosis. Anti-topo I is associated with tight skin and is more common in SSc with diffuse than with limited skin involvement [5]. It is not seen in other diseases or in normals, except for an occasional patient with isolated Raynaud’s disease where it predicts the future development of tight skin [14]. It is associated with SSc patients with facial skin involvement and heart involvement [5]. Steen et al. also found an association with kidney involvement, pulmonary fibrosis and ischemic ulcers of the fingertips. The AMAN-C5.1/3

association with pulmonary fibrosis has also been noted by others [6, 19, 20]. We have reported the association of anti-top0 I and the development of malignancies [11, 43 VR], although we found no anti-topo I in 150 cancer patients who did not have SSc. Prevalence rates of anti-topo I

No+/No tested

%+

CREST (without tight skin) SSC Raynaud’s disease *Overlap diseases with Raynaud’s syndrome

3/89 501222 21253 97**

3.3 23.0 0.8 4.1

* includes diseases as above for ACA ** The four patients had SSc plus either SLE or Sjögren’s syndrome

Anti-topoisomerase I is found equally among males and females [21]. It is more common in African Americans than in American Caucasians [22]. It is not detected in healthy relatives of SSc patients [11, 23, 24]. There appears to be an association with HLA DQBl allel encoding for tyrosine at position 30 of the first domain [25–27]. Anti-topoisomerase I does not occur in the same patients as ACA. However, anti-topo I may occur in association with other antigens such as anti-Ro if the patient has associated Sjögren’s syndrome or anti-DNA if the patient has both SLE and SSc. Anti-topo I may preceed the development of SSc in some patients with Raynaud’s disease. It also is more common in SSc patients who will develop a malignancy. Thus, all patients with RD should have anti-topo I assay performed. The antibody does not reflect disease activity and persists for years. If the antibody is not present when originally tested it will not appear in later years. It is not clear whether the antibody predicts a response or lack of response or adverse reactions to drugs. Future trends include the widespread use of ELISA assays using either purified topoisomerase I or recombinant topoisomerase I. This method is more sensitive than the usual double immunodiffusion test which is used by most laboratories.

3. Anti-RNA polymerase

3.1 Introduction Anti-polymerase I was first described by Reimer et al. in 1987 as occurring in 4% of 208 SSc sera [28]. Later, anti-RNA polymerase III was reported to AMAN-C5.1/4

occur in 23% of 252 SSc patients from North America [29]. Most of the sera containing anti-RNA polymerase II also contain anti-RNA polymerase I and/or anti-RNA polymerase III. Anti-RNA polymerase I, II, III produce a variable punctate nucleolar pattern on immunofluorescence but immunoblotting on nuclei does not yield bands. Thus, purified antigens must be used for immunoblotting and this antibody is rarely detected since very few laboratories have purified antigen. In limited studies, anti-RNA polymerase was reported to be absent from patients with other connective tissue diseases, normals or Raynaud’s disease [29]. Anti-RNA polymerase is limited to patients with SSc/CREST in which it is found in 28%. It is also found in scleroderma overlap diseases. It is associated with diffuse SSc and with cardiac involvement and extensive skin involvement. Patients with anti-RNA polymerase have less telangiectasias, myositis and pulmonary fibrosis [29]. In studies of Japanese patients, anti-RNA polymerase was associated with older age of onset, cardiac and renal involvement and a worse prognosis [30, 31]. Anti-RNA polymerase is more common in North-American Caucasians than in Japanese or in African Americans [29]. Anti-RNA polymerase is not found in sera containing ACA or antitopo I [29, 30]. There is no easy way at present to detect anti-RNA polymerase and it is not necessary to determine the presence of this autoantibody in order to diagnose SSc. It is not known whether this autoantibody precedes disease expression or reflects disease activity. Since it is not present in RD, it does not seem to predict outcome in that group of patients. The assay should not be used repeatedly if at all. Nothing is known about the association of the autoantibody with response or adverse reactions to drugs.

4. Anti-nucleolar and other autoantibodies

Small numbers of patients with anti U1 RNP, anti-Ku, anti-U3RNP (fibrillarin), anti-Th, anti-PMiSCL, anti-Nor90, anti-U4/U6RNP and antiU2RNP have been described. These autoantibodies have been detected in from 35% (anti-U1RNP) to 14% (anti-U2RNP) to less than 10% of SSciCREST patients (anti-Ku, anti-U3RNP, anti-Th, anti-PM/SCL, antiNOR90). Most have little known clinical significance and most are not specific for SSc. The clinical significance of anti-U1 RNP, anti-U2RNP and anti-U3RNP (anti-fibrillarin) is described below. Anti-U3RNP or anti-fibrillarin has been reported in 6% of American SSc patients and in 4%) of Japanese SSc patients [32, 33]. In these limited studies, it was more frequent in African American females and in patients with diffuse rather than limited SSc. The clinical subgroup identified was those with pulmonary hypertension, myositis, and small bowel disease [33]. AMAN-C5.1/5

Anti-U1RNP is present in patients with many rheumatic diseases. It has been reported in 35% of Japanese SSc patients [33] and in 28% of SSc patients in the United States [29]. It is also present in SSc overlap diseases and in patients with Raynaud’s disease. It is more frequently found in Japanese than in North American Caucasians or African Americans. Anti-U2RNP was first described by Mimori in 1984 in a serum from a patient with SSc and polymyositis [34]. It is found in 14% of SSc patients [35] but is also found in a similar percentage of patients with SLE, MCTD and SSc/myositis. These autoantibodies are only detected in specialized laboratories and are of little use in the usual clinical setting.

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Rothfield NF & Rodnan GP (1968) Arthritis Rheum 11: 607–617 Morio Y, Peebles C, Fritzler MJ et al. (1980) Proc Natl Acad Sci USA 77: 1627–1631 Earnshaw WC & Rothfield N (1985) Chromosoma (Berl.) 91: 313-321 McCarty GA, Rice JR, Bembe ML et al. (1983) Arthritis Rheum 26: 1–7 Steen VD, Ziegler GL, Rodnan GP et al. (1984) Arthritis Rheum 27: 125–131 Giordano M, Valentini G, Migliaresi S et al. (1986) J Rheumatol 13: 911–916 Nishikai M, Itoh K & Sato A (1992) Br J Rheum 31(1): 9-12 Wigley FM, Wise RA, Miller R et al. (1992) Arthritis Rheum 35: 688–693 Hughes P, Holt S, Rowell NR et al. (1976) Brit. J Dermatol 95, 469–473 Weiner ES, Earnshaw WC, Senecal JL, Bordwell B, Johnson P & Rothfield NF (1988) Arthritis Rheum 31, 378–385 1 1. McHugh NJ, Whyte J, Artlett C et al. (1994) Clin Exp Immunol 96: 267–274 12. Vazquez-Abad D & Rothfield NF. Unpublished 13. Vlachoyiannopoulos PG, Drosos AA, Wiik A & Moutsopoulos HM (1993) Brit J Rheum 34(4), 297–301 14. Weiner ES, Hildebrandt S, Senecal J-L et al. (1991) Arthritis Rheum 34: 68–77 15. Reveille JD, Durban E, Goldstien R, Moreda R & Arnett FC (1992) Arthritis Rheum 35(2), 216–218 16. Vazquez-Abad D, Russell CA, Cusick SM, Earnshaw WE & Rothfield N (1995) Clin Immunol Immunopath 74: 257–270 17. Douvas AS. Achten M & Tan EM (1979) J Biol Chem 254: 10514–10522 18. Shero JH. Bordwell BJ, Rothfield NF & Earnshaw WC (1986) Science 231. 737–740 19. Catoggio LJ, Skinner RP & Maddison PJ (1983) Rheum Int 3, 19–21 20. Kuwana M, Kaburaki J, Mimori T et al. (1994) Arthritis Rheum 37: 75–83 21. Rothfield N, Kurtzman S, Vazquez-Abad D, Charron C. Daniels L & Greenberg B (1992) Arthritis Rheum 35, 724 22. Reveille JD & Arnett FC (1993) Arthritis Rheum 36: 1332–1334 23. Barnett AJ & McNeilage LJ (1993) Ann Rheum Dis 52: 365–368 24. Maddison PJ, Stephens C, Briggs D, Welsh KI, Harvey G, Whyte J, Mchugh N, Black CM, Bacon P, Bernstein R, Jayson MIV & Silman A (1993) Medicine 72, 103–112 25. Whyte J, Arlett C, Harvey G, Stephens CO, Welsh K, Black C, Maddison PJ, McHugh NJ et al. (1994) J Autoimmunity 7: 509–520 26. Reveille JD, Durban E, MacLeod-St. Clair R, Goldstein R. Moreda R, Altman D & Arnett FC (1992) J Clin Invest 90: 973–980 27. Kuwana M, Kaburaki J, Okano Y, Inoko H & Tsuji K (1993) J Clin Invest 92: 1296–1301 28. Reimer G, Rose KM, Scheer U & Tan EM (1987) J Clin Invest 79: 65–72 29. Okano Y, Steen VD, Medsger TA (1993) Ann Int Med 119: 1005–1013 30. Kuwana M, Okano Y, Kaburaki J et al. (1993) J Clin Invest 92: 1296–1301 31. Kuwana M, Okano Y, Kaburaki J et al. (1994) Arthritis Rheum 37: 75–83 32. Okano Y, Steen VD & Medsger TA (1992) Arthritis Rheum 35: 95–100 33. Kuwana M, Okano Y, Karuraki J, Tojo T & Medsger TA (1994) Arthritis Rheum 37: 902–906 34. Mimori T, Hinterberger M & Pettersson I (1984) J Biol Chem 259: 560–565 35. Craft J, Mimori T, Olsen TL (1988) J Clin Invest 8: 1716–1724

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Autoantibody Manual C6.1. 1–12, 1996 © 1996 Kluwer Academic Publishers. Printed in The Netherlands

The autoantibody system: Anti-aminoacyl-tRNA synthetase antibodies IRA N. TARGOFF and PAUL H. PLOTZ The University of Oklahoma, Oklahoma City, OK 73104, U.S. A.

1. Introduction

In 1980, Nishikai and Reichlin defined the anti-Jo-1 precipitin, the most common precipitin in polymyositis (PM) or dermatomyositis (DM) sera, as a unique system with a strong specificity for PM/DM [1]. Although later observations refined the clinical picture of patients with these autoantibodies [2], the major clinical associations – myositis with interstitial lung disease and arthritis – were recognized in the early series of cases [3–6]. The first clue to the identity of the antigen was the recognition that antiJo- 1 sera immunoprecipitated tRNA from 32P-labelled HeLa cells [7], that this was a tRNA for histidine [8], and that the antibodies reacted with a protein rather than directly with the tRNA [1,8]. Mathews and Bernstein shortly thereafter showed that a protein of molecular weight 50 kD coprecipitated with the tRNAhis and that IgG from anti-Jo-1 positive antisera blocked charging of tRNA with histidine but not with other amino acids, thus establishing that the Jo-1 antigen was histidyl-tRNA synthetase (HRS) [9]. They recognized the extraordinary interest of this observation because it suggested a possible connection between a target autoantigen and viruses that had been suspected of causing PM and DM. The nucleic acid of several viruses, including the two picornaviruses encephalomyocarditis and Mengo, could serve as substrate for HRS or other aminoacyl-tRNA synthetases, and both of those viruses, as well as closely related picornaviruses such as Coxsackie and Echo viruses, were candidate viruses for human PM and DM. A decade later, direct evidence of a role for picornaviruses, or indeed any other viruses, remains elusive despite extensive and sensitive searching [IO]. The question of the reason for the appearance of autoantibodies to HRS grew more intriguing, however, with the discovery that some patients with PM or DM could have autoantibodies directed against other aminoacyl-tRNA synthetases, including: threonyl-tRNA synthetase (PL-7) [11]; alanyl-tRNA synthetase (PL-12) and tRNAala [12]; glycyl-tRNA synthetase (EJ) [ 13]; and isoleucyl-tRNA synthetase (OJ) [ 13]. Anti-OJ sera may also contain autoantibodies to certain other synthetases that are part of the multi-enzyme complex of which isoleucyl-tRNA synthetase is a component [14]. AMAN-C6.1/1

2. Assays

2.1 Preferred assays for clinical use 2.1.1 Anti-jo-1 Most commercial laboratories test for anti-Jo-1 by double immunodiffusion, confirming the identification of the precipitin line with standard sera. Concentrated tissue extracts are commonly used as antigen source, often from bovine liver or thymus, sometimes partially purified. This is a sensitive and specific method for this purpose. Direct-binding methods such as enzyme immunoassays using affinity-purified antigens are also available. These have high sensitivity and provide quantitation, which may be helpful in certain circumstances. Recombinant antigens should soon become more widely used for this purpose. Western blot has also been used for detection of anti-Jo-1 [15]. 2.1.2 Non-jo-1 anti-synthetases The best method for detection of these antibodies is protein A-assisted immunoprecipitation for nucleic acids and proteins, a method generally used in research laboratories. It is both highly sensitive and specific, and can simultaneously screen for all anti-synthetases [13, 16]. Almost all samples of all five anti-synthetases specifically inhibit the function of the enzyme at which they are directed, and assays based on this have also been used to detect the presence of these antibodies [15, 17, 18], but each must be tested for separately. Anti-PL-7 can be identified by immunodiffusion in most sera with the antibody, but other non-Jo-1 anti-synthetases are not reliably detected by this method. Anti-PL-7 and many sera with anti-PL-12 can be detected by counter-immunoelectrophoresis; as with immunodiffusion, confirmation with standard serum is necessary. Immunoblotting is not reliable for detection of non-Jo-1 anti-synthetases, since many sera appear to react exclusively with conformational epitopes [ 14, 19]. Anti-synthetases commonly give a cytoplasmic pattern by indirect immunofluorescence on human cultured cells (antinuclear antibody testing), and this may be more evident with non-lo-1 anti-synthetases [1 1, 18]. It may be accompanied by nuclear or nucleolar fluorescence, presumably due to other autoantibodies in the sera [20, 21]. While not specific for antisynthetases, the report of such a cytoplasmic pattern in the absence of antiribosomal or anti-mitochondrial antibodies should increase suspicion that these antibodies may be present. Absence of cytoplasmic staining does not exclude the presence of anti-synthetases, but makes it much less likely.

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2.2 Use of testing 2.2.1. Availability Testing for anti-Jo-1 by routine methods is widely available from rheumatology reference laboratories. Testing for non-Jo-1 anti-synthetases is less accessible, but can be obtained through research laboratories, and is worthwhile clinically when results would affect treatment decisions. The need for specialized techniques such as immunoprecipitation or synthetase enzyme inhibition assays limits availability. Even testing by routine methods, such as immunodiffusion for anti-PL-7, is not usually offered due to the low frequency of the antibodies. The availability of recombinant antigens should make anti-synthetase testing more accessible. Recombinant HRS has been made in a baculovirus vector, and the crude extract of the cytoplasm of the insect cell line in which it is produced can serve as a highly sensitive and specific antigen in an ELISA assay ([22], patent pending), and that assay is expected to be more widely available in the near future. Clones for the human forms of the synthetases for isoleucine [23], glycine [24], and threonine [25] have been reported, and recombinant human glycyl-tRNA synthetase reacted similarly to the natural protein by blot [24]. It is likely that recombinant forms of all of the antigenic human synthetases will be available for ELISA assays before long. 2.2.2 Sensitivity and specificity of the assays for detection of antibody Immunodiffusion is sensitive for detection of anti-Jo-l , in that it detects almost every patient with the antibody. ELISA or Western blot can detect quantitatively smaller amounts of antibody, but few additional patients are identified by these methods that would be missed by immunodiffusion [ 15]. Immunodiffusion is highly specific for the antibody when confirmed against a standard serum. The specificity of direct-binding methods for the antibody depends on the purity of the antigen and the methodology employed. Immunodiffusion is less sensitive than immunoprecipitation for anti-PL7, but still detects most sera with the antibody. Immunodiffusion should not be used for anti-PL-12, anti-EJ, or anti-OJ. CIE has been used for antiPL-12 with some success [16].

2.3 Antibody as a criterion for diagnosis The most commonly used criteria for establishing a diagnosis of PM or DM are those of Bohan and Peter [26]. These are based on clinical findings, muscle enzymes, electromyography, and muscle biopsy, and do not include myositis-specific autoantibodies or other serological tests. In view of the high specificity of anti-synthetases for PM/DM, however, the AMAN-C6.1/3

finding of an anti-synthetase in a patient with a compatible clinical picture provides strong support for the diagnosis, and in some cases may obviate the need for a muscle biopsy. Since the anti-synthetases, even when taken together, have low sensitivity, the absence of anti-synthetases should not be considered evidence against the diagnosis of PM or DM. The methods discussed above should be used for establishing a positive result. For anti-Jo-1, immunodiffusion against a standard serum can be definitive. Use of a combination of tests, such as immunodiffusion and enzyme immunoassay, can provide additional confirmation when results are pivotal.

3. Autoantibody

3.1/2 Disease association and clinical subset The clinical associations of anti-Jo- 1 have been studied most thoroughly. Few patients with other anti-synthetases have been described in detail, but patients with autoantibodies to PL-7 [11, 17], PL-12 [18], EJ [27], or OJ [14] appear to have a very similar clinical picture, as do patients with antiKJ, which reacts with a still undefined translation component [28]. The clinical picture is so homogeneous that it is reasonable to think that there is a unique clinical syndrome [2, 4, 11, 12, 29, 30]. Some have used the term ‘anti-synthetase syndrome’ to refer to the set of clinical features that have been associated with these antibodies [27, 31]. Almost all of the patients have myositis, and most also have interstitial lung disease/fibrosing alveolitis [3, 4], and one or more of the following: arthralgias/arthritis [3, 30]; Raynaud phenomenon; fevers; and a roughening of the sides of the index or other fingers sometimes referred to as mechanic’s hands [2, 32]. Even the few patients without clinical myositis usually have subtler indications of muscle disease, such as an elevated creatine kinase, and invariably have one of the other features (usually interstitial lung disease) [18, 33]. The arthritis is usually non-deforming, but a more severe, subluxing arthropathy may occur [18, 30].

3.3 Prevalence rates Anti-Jo-1 has been found in approximately 15-30% of unselected adult PM and DM patients from rheumatic disease centers in most studies [1, 2, 4, 16, 20, 29, 30, 34]. It accounts for well over half of anti-synthetases. The frequency of each of the other anti-synthetases is low (<5%) [2, 11, 12, 14, 16, 18, 27, 29]. Even when anti-Jo-1 is screened for routinely in sera referred for autoantibody testing, the vast majority of patients with anti-Jo-1 have myositis. AMAN-C6.1/4

Occasionally, anti-Jo-1 or another anti-synthetase is identified in patients with only interstitial lung disease [29, 33], or even less often arthritis, and some of these patients have increased creatine kinase suggesting subclinical myositis. Thus far, a higher proportion of patients with anti-PL-12 and antiOJ than with anti-Jo-1 have had interstitial lung disease without overt myositis [18, 33], and among such patients with anti-synthetases, only 2 of 10 had anti-Jo-1 [33], but the number of patients with these antibodies that have been described remains limited. Rare patients with anti-synthetases have had prolonged arthritis, without other manifestations of the syndrome until years later [14, 17, 18]. The prevalence of anti-synthetases among unselected patients with isolated idiopathic interstitial lung disease has not been adequately studied. One report found a frequency of anti-Jo-1 of about 3% in cryptogenic fibrosing alveolitis [4], but given the under-representation of anti-Jo-1 among such patients noted above, the prevalence of any anti-synthetase is undoubtedly higher, and would be higher still among those with features such as arthritis, Raynaud’s, or elevated creatine kinase. Other features may also vary in patients with different anti-synthetases, such as the rash of DM [27], but it is possible that even these apparent differences are the result of the small number of cases so far described. For example, despite early suggestions to the contrary, DM and PM were about equally common among those with anti-Jo-1 autoantibodies in a recent larger series [2]. Probably about a fifth of the patients with antisynthetases have another connective tissue disease in addition to myositis; lupus, scleroderma, Sjögren’s syndrome, and rheumatoid arthritis are all well described [17]. None have inclusion body myositis, but a few have had an associated cancer [29]. Anti-synthetases occur in a far smaller proportion of children with myositis than of adults, but a small number of children with anti-synthetases have now been observed [29, 35, 36], with a syndrome similar to that in adults with anti-synthetases.

4. Environmental and genetic associations

4.1 Geographic, environmental differences Anti-Jo-1 has been reported from geographically and ethnically diverse populations at similar frequency in PM/DM, including the U.S., the U.K., Japan, and Australia [2, 16, 34, 37]. Although the frequency of other antisynthetases is low in all populations, studies have suggested differences in the relative frequency of anti-PL-7 vs. anti-PL-12 in different parts of the U.S., with anti-PL-7 more common in the Northeast [2], while anti-PL-12 was more common by >5:1 among patients identified in Houston, Texas [18. 38]. An intriguing observation is that one study has shown a strong trend for weakness to manifest itself in the first half of the calendar year in AMAN-C6.1/5

patients with anti-Jo-1 autoantibodies, in contrast to those with the myositis-specific autoantibody anti-SRP (directed at the signal recognition particle), who had onset in the last few months of the year, or cases without myositis-specific autoantibodies, where no seasonal preference was seen [39]. Preliminary observations in a larger number of patients supported the finding [38]. These observations suggest the possibility of an environmental agent triggering the illness.

4.2/3 HLA/genetic associations Patients with anti-Jo-1 are highly likely to have HLA-B8 and DR3, or another member of the DR52 group [2, 5,40,41]. The association of anti-Jo1 with DR3 is strong in Caucasians (75%), but is low or not seen in AfricanAmericans [41]. However, the association with DR52 persists in both white and black populations [2, 41], present in close to 100%, Non-Jo-1 antisynthetases also appear to be associated with DR52, but not with DR3, possibly due to a lower frequency of white patients in this group [18, 42]. Anti-synthetases have been associated with HLA-DQA1*0501 or *0401 in whites and blacks. but with DQA1*0101, *0102, or *0103 in Japanese patients [43].

5.1 Other autoantibodies A striking observation with regard to the occurrence of anti-synthetases is that individual patients almost always have only one of the five antisynthetase autoantibodies. As noted, sera with anti-OJ may have antibodies to other synthetase components of the multi-enzyme complex, but generally not to the four autoantigenic non-complexed synthetases. Considering the similarity in clinical associations of the antibodies, and in the cellular function of the antigens, this is surprising, and speaks against their arising secondary to muscle injury. It is also very rare to see other myositis autoantibodies, such as anti-Mi-2, anti-PM-Scl, and anti-SRP, in sera with anti-synthetases. However, patients with anti-synthetases may have other autoantibodies. Anti-Ro/SSA occurs significantly more often in sera with anti-synthetases than in those without myositis autoantibodies (25% vs. 7% [2]), sometimes with anti-LaiSSB. These patients do not necessarily have overt overlap syndromes with Sjögren’s syndrome or lupus, the usual conditions associated with these antibodies [2], although they may. Anti-UlRNP may also occur in sera with anti-synthetases as well as in those without, but appears to be more common in association with non-Jo- 1 anti-synthetases, often in overlap patients. Anti-Sm, usually with anti-UlRNP, may also occur, almost always in connective tissue overlap syndromes [2]. AMAN-C6.1/6

5.2 Recommendation regarding the use of anti-synthetases in diagnosis of myositis PM and DM are often not easily distinguishable from other myopathies, and the presence of immunologic abnormalities such as autoantibodies, particularly those which are myositis-specific, can point sharply towards an inflammatory myopathy. The anti-synthetases can thus be a valuable diagnostic aid in evaluation of patients with weakness, particularly when electromyography or muscle biopsy are normal or equivocal. It is important to remember, however, that a negative test is of no value in diagnosis. Some patients with anti-synthetases have presented with myositis, pulmonary fibrosis or arthritis and have later developed other manifestations of the anti-synthetase syndrome. It is therefore of some clinical value to look for anti-synthetases even if the diagnosis of PM or DM is firmly established. It is also worthwhile to look for these antibodies in patients with pulmonary fibrosis who have features of connective tissue disease (such as Raynaud’s phenomenon, suggestive rash, or arthritis) or elevated creatine kinase.

6. Anti-synthetases in the course of disease

6.1 At onset At the time the patient presents to the physician, the anti-Jo-1 test is positive. The initial appearance of anti-Jo-1 after myositis has been present has not been documented. For the few patients for whom stored serum was available, the antibody was shown to antedate the disease [27, 44]. However, the antibodies are seldom useful for predicting the onset of myositis in normal individuals because they are so rare. One patient was demonstrated to have the new occurrence of anti-Jo-1 while being treated for another illness, and this was followed several months later by the development of myositis [44]. These observations strongly support the impression that the antibody is not secondary to the effects of disease, but is related to fundamental etiologic and/or pathogenetic factors.

6.2 Antibody and disease activity The antibody usually remains detectable throughout the course of disease, and indefinitely thereafter despite control of disease activity. Occasionally, the antibody becomes undetectable, and in those cases in which this was observed, it was associated with remission of disease [45]. However, a fluctuation of anti-Jo-1 titer in association with disease activity was demonstrated in one study [45]. The clinical utility of this fluctuation requires further investigation, but unless the antibody disappears completely, serial AMAN-C6.1/7

titers would be needed. No significant data are available for the other specificities, although a case report indicated similar changes for anti-PL-7 [46]. 6.3 Prognostic significance As noted, conditions associated with anti-synthetases, such as myositis, interstitial lung disease, or arthritis, may develop at different points in time. Thus, identification of an anti-synthetase in a patient with myositis should alert the clinician to the possible development of other anti-synthetaseassociated manifestations in the future. Patients with anti-synthetases have a relatively rapid onset of disease, and it tends to be severe. Anti-synthetase patients are significantly more likely than patients without myositis-specific antibodies to require cytotoxic agents (62% vs. 30%), to flare during taper of therapy (60% vs. 20'%), and to be unable to discontinue therapy completely, spending only 3% of their time without therapy [2]. They also have a higher mortality than patients without myositis antibodies (21% vs. 7%), although not as bad as that of patients with anti-SRP [2]. Interstitial lung disease was found to worsen the prognosis in myositis patients independent of the antibody [47], consistent with this finding.

6.4 Repeated testing Although, as noted, it would be extremely unusual for an anti-synthetase antibody to develop in a patient who was previously negative for the antibody, repeat testing of such a patient might be considered in order to confirm the accuracy of the previous result in a suggestive clinical situation, such as myositis with interstitial lung disease. If the patient's clinical situation changes significantly, such as with the new appearance of interstitial lung disease, repeat testing might also be considered. In a patient found to be positive, besides confirmatory testing if questions exist, repeat testing might be considered as treatment is being discontinued, since the risk of reexacerbation is high if the antibody persists (the more common situation). Theoretically, the fluctuation with disease activity noted above raises the possibility of using antibody titers in assessment of disease activity or even predicting exacerbations, but the clinical value of this has not yet been demonstrated. Serial samples would be needed, and properly stored samples tested simultaneously with quantitative methods would be preferable.

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7. Use in therapeutic management

Patients with anti-synthetases usually show substantial responses to corticosteroid therapy, but usually not complete responses [48]. As noted, they are more likely to require cytotoxic immunosuppressive agents, and are more likely to have flares of disease activity with reduction of treatment. Early realization of this could be valuable in planning therapy. In addition, when comparing responses to methotrexate with those to azathioprine, patients with anti-synthetases were more likely to respond to methotrexate; while most responded partially (but not completely) to methotrexate, almost half showed no response to azathioprine [48]. However, caution is necessary in the use of methotrexate due to the high frequency of interstitial lung disease in anti-synthetase patients. With potential differences in responsiveness, and the previously noted differences in prognosis among antibody defined subgroups, if it ever becomes possible to conduct a clinical trial large enough to have a statistically meaningful number of patients with this condition, it ought to be separately stratified by autoantibody.

8. Conclusion

While knowledge about the presence of anti-Jo-1 autoantibodies may not be essential to the diagnosis or management of PM and DM, appreciation of the distinctive subgroups marked by these and other myositis autoantibodies will be important in understanding the etiology and discovering the optimal therapy. The mystery of the relationship between autoantibodies and autoimmune disease has been a stimulus to research in this field. The case of the anti-synthetase syndrome, in which something closely links myositis with autoantibodies to a family of proteins which are structurally distinct but functionally homologous, epitomizes the mystery and is likely to continue to stimulate basic research until it is explained.

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References 1. Nishikai M & Reichlin M (1980) Arthritis Rheum 23: 881–888 2. Love LA, Leff RL, Fraser DD, Targoff IN. Dalakas MC, Plotz PH & Miller FW (1991) Medicine 70: 360–374 3. Yoshida S, Akizuki M, Mimori T, Yamagata H, Inada S & Homma M (1983) Arthritis Rheum 26: 604–611 4. Bernstein RM, Morgan SH, Chapman J, Bunn CC, Mathews MB, Turner-Warwick M & Hughes GRV (1984) Br Med J 289: 151–152 5. Arnett FC, Hirsch TJ, Bias WB, Nishikai M & Reichlin M (1981) J Rheumatol 8: 925–930 6. Wasicek CA, Reichlin M, Montes M & Raghu G (1984) Am J Med 76: 538–544 7. Hardin JA, Rahn DR, Shen C, Lerner MR, Wolin WL, Rosa MD & Steitz JA (1982) J Clin Invest 70: 141–147 8. Rosa MD, Hendrick JP Jr, Lerner MR, Steitz JA & Reichlin M (1983) Nucl Acids Res 11: 853–870 9. Mathews MB & Bernstein RM (1983) Nature 304: 177–179 10. Leff RL, Love LA, Miller FW, Greenberg SJ, Klein EA, Dalakas MC & Plotz PH (1992) Lancet 339: 1192–1195 11. Mathews MB, Reichlin M, Hughes GRV & Bernstein RM (1984) J Exp Med 160: 420–434 12. Bunn CC, Bernstein RM & Mathews MB (1986) J Exp Med 163: 1281–1291 13. Targoff IN (1990) J Immunol 144: 1737–1743 14. Targoff IN, Trieu EP & Miller FW (1993) J Clin Invest 91: 2556–2564 15. Targoff IN & Reichlin M (1987) J Immunol 138: 2874–2882 16. Bernstein RM, Bunn CC, Hughes GRV, Francoeur AM & Mathews MB (1984) Mol Biol Med 2: 105–120 17. Targoff IN, Arnett FC & Reichlin M (1988) Arthritis Rheum 31: 515–524 18. Targoff IN & Arnett FC (1990) Am J Med 88: 241–251 19. Dang CV, Tan EM & Traugh JA (1988) FASEB J 2: 2376–2379 20. Reichlin M & Arnett FC (1984) Arthritis Rheum 27: 1150–1156 21. Shi MH, Tsui FWL & Rubin LA (1991) J Rheumatol 18: 252–258 22. Raben N, Nichols RC, Dohlman J, McPhie P, Sridhar V, Hyde C. Leff RL & Plotz PH (1994) J Biol Chem 269: 24277–24283 23. Nichols RC, Raben N, Boerkoel CF & Plotz PH (1995) Gene 155: 299–304 24. Ge Q, Trieu EP & Targoff IN (1994) J Biol Chem 269: 28790–28797 25. Cruzen ME & Arfin SM (1991) J Biol Chem 266: 9919–9923 26. Bohan A & Peter JB (1975) N Engl J Med 292: 344–347, 403–407 27. Targoff IN, Trieu EP, Plotz PH & Miller FW (1992) Arthritis Rheum 35: 821–830 28. Targoff IN, Arnett FC, Berman L, O'Brien CA & Reichlin M (1989) J Clin Invest 84: 162–172 29. Marguerie C, Bunn CC, Beynon HLC, Bernstein RM, Hughes JMB, So AK & Walport MJ (1990) Q J Med 77: 1019–1038 30. Oddis CV, Medsger TA Jr & Cooperstein LA (1990) Arthritis Rheum 33: 1640–1645 31. Targoff IN (1992) Rheum Dis Clin North Am 18: 455–482 32. Stahl NI, Klippel JH & Decker JL (1979) Ann Intern Med 91: 577–579 33. Friedman AW, Targoff IN & Arnett FC (1994) Arthritis Rheum 37: S242 (Abstract) 34. Walker EJ, Tymms KE, Webb J & Jeffrey PD (1987) J Immunol Meth 96: 149–156 35. Rider LG, Miller FW, Targoff IN, Sherry DD, Samayoa E, Lindahl M, Wener MH, Pachman LM & Plotz PH (1994) Arthritis Rheum 37: 1534–1538 36. Chmiel JF, Wessel HU, Targoff IN & Pachman LM (1995) J Rheumatol 22: 762–765 37. Hirakata M, Mimori T, Akizuki M, Craft J, Hardin JA & Homma M (1992) Arthritis Rheum 35: 449–456

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38. Love LA, Burgess SH, Hill PC, Oddis CV, Medsger TA Jr, Leff RL, Plotz PH, Reveille JD, Arnett FC, Targoff IN & Miller FW (1992) Arthritis Rheum 35: S40 (Abstract) 39. Leff RL, Burgess SH, Miller FW, Love LA, Targoff IN, Dalakas MC, Joffe MM & Plotz PH (1991) Arthritis Rheum 34: 1391–1396 40. Hirsch TJ, Enlow RW, Bias WB & Arnett FC (1981) Hum Immunol 3: 181–186 41. Goldstein R, Duvic M, Targoff IN, Reichlin M, McMenemy AM, Reveille JD, Warner NB, Pollack MS & Arnett FC (1990) Arthritis Rheum 33: 1240–1248 42. Garlepp MJ (1993) Baillieres Clin Neurol 2: 579–597 43. Reveille JD, Targoff IN, Mimori T, Nguyen HC, Goldstein R & Arnett FC (1992) Arthritis Rheum 35: S84 (Abstract) 44. Miller FW, Waite KA, Biswas T & Plotz PH (1990) Proc Natl Acad Sci USA 87: 9933–9937 45. Miller FW, Twitty SA, Biswas T & Plotz PH (1990) J Clin Invest 85: 468–475 46. Walker EJ, Jeffrey PD, Webb J & Tymms KE (1989) Clin Exp Rheumatol 7: 537–540 47. Arsura EL & Greenberg AS (1988) Semin Arthritis Rheum 18: 29–37 48. Joffe MM, Love LA, Leff RL, Fraser DD, Targoff IN, Hicks JE, Plotz PH & Miller FW (1993) Am J Med 94: 379–387

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Autoantibody Manual C7.1. 1-12. 1996 © 1996 Kluwer Academic Publishers. Printed in The Netherlands

ANCA: their clinical relevance CEES G.M. KALLENBERG Department of Clinical Immunology, University Hospital, Oostersingel 59, 9713 EZ Groningen, The Netherlands

1. Introduction

Anti-neutrophil cytoplasmic antibodies (ANCA) as detected by indirect immunofluorescence (IIF) on ethanol-fixed neutrophils [1] were originally described as sensitive and specific markers for active Wegener’s Granulomatosis (WG) [2]. The antibodies produce a characteristic cytoplasmic staining pattern with accentuation of the fluorescence intensity in the area within the nuclear lobes (c-ANCA) [1]. The antigen recognized by c-ANCA in WG has been characterized as proteinase 3 (Pr3; see Chapter B.7.1). Although c-ANCA are sensitive and specific for WG, their presence is not (yet) included as a criterium for the diagnosis of WG since classification criteria for WG (Table 1) were defined by the American College of Rheumatologists (ACR) just before ANCA-testing was introduced into the U.S.A. [3]. After the description of c-ANCA in 1985, autoantibodies were detected that produced a perinuclear staining pattern on ethanol-fixed neutrophils (pANCA) [4]. The major autoantigen recognized by p-ANCA proved Table 1. ACR criteria for the classification of Wegener’s granulomatosis* Criterion

Definition

1. Nasal or oral inflammation

Development of painful or painless oral ulcers or purulent or bloody nasal discharge.

2. Abnormal chest radiograph

Chest radiograph showing the presence of nodules, fixed infiltrates, or cavities.

3. Urinary sediment

Microhematuria (>5 red blood cells per high power field) or red cell casts in urine sediment.

4. Granulomatous inflammation on biopsy

Histologic changes showing granulomatous inflammation within the wall of an artery or in the perivascular or extravascular area (artery or arteriole).

* For purposes of classification, a patient shall be said to have Wegener’s granulomatosis if at least 2 of these 4 criteria are present. The presence of any 2 or more criteria yields a sensitivity of 88.2% and a specificity of 92.0% AMAN-C7.1/1

myeloperoxidase (MPO) [4], see Chapter B.7.2) Anti-MPO antibodies were detected in the sera of patients with pauci-immune necrotizing and crescentic glomerulonephritis (NCGN) with or without associated vasculitis [4, 5], but also in patients with a variety of idiopathic vasculitic disorders, in particular Churg-Strauss Syndrome and incidentally in polyarteritis nodosa [6]. Their presence is not included in the ACR-criteria for the classification of ChurgStrauss Syndrome (Table 2) or polyarteritis nodosa (Table 3). A proposal for (re-)definition of the idiopathic vasculitides has been formulated [7]. Besides MPO two other antigens may be targets for ANCA in patients with systemic vasculitis. Antibodies to human leukocyte elastase incidentally occur in patients with diverse forms of vasculitis [8]. Recently, antibodies to bactericidal/permeability-increasing protein (BPI) have been described in patients with vasculitis who were either c-ANCA or p-ANCA-positive by IIF but negative for antibodies to MPO or Pr3 [9]. The diagnostic significance of anti-BPI antibodies still has to be established. Further studies have demonstrated that p-ANCA as well as other ANCA that produce an aspecific cytoplasmic staining on ethanol-fixed neutrophils, are present in the sera of patients with a variety of idiopathic inflammatory disorders and, incidentally, in infectious conditions [ 10–12]. The antigens recognized by those ANCA are only in part characterized. Amongst these, lactoferrin is most prominent [ 13–15]. Table 2. ACR-criteria for the classification of Churg-Strauss syndrome* Criterion

Definition

Asthma

History of wheezing or diffuse high-pitched rales on expiration.

Eosinophilia

Eosinophilia >10% on white blood cell differential count.

Mononeuropathy or polyneuropathy

Development of mononeuropathy. multiple mononeuropathies, or polyneuropathy (i.e. glove/stocking distribution) attributable to a systemic vasculitis.

Pulmonary infiltrates, non-fixed

Migratory or transitory pulmonary infiltrates on radiographs (not including fixed infiltrates). attributable to a systemic vasculitis.

Paranasal sinus abnormality

History of acute or chronic paranasal sinus pain or tenderness or radiographic opacification of the paranasal sinuses.

Extravascular eosinophils

Biopsy including artery, arteriole, or venule, showing accumulations of eosinophils in extravascular areas.

* For classification purposes, a patient shall be said to have Churg-Strauss syndrome (CSS) if at least 4 of these 6 criteria are positive. The presence of any 4 or more of the 6 criteria yields a sensitivity of 85% and a specificity of 99.7%. AMAN-C7.1/2

As a major message resulting from these data it can be concluded that ANCA-testing, at present, should include not only IIF on ethanol-fixed neutrophils but also antigen-specific testing, preferably by ELISA.

Table 3. ACR-criteria for the classification of polyarteritis nodosa* Criterion 1. Weight loss

Definition ≥

4 kg

Loss of 4 kg or more of body weight since illness began, not due to dieting or other factors.

2. Livedo reticularis

Mottled reticular pattern over the skin of portions of the extremities or torso.

3. Testicular pain or tenderness

Pain or tenderness of the testicles. not due to infection, trauma. or other causes.

4. Myalgias. weakness, or leg tenderness

Diffuse myalgias (excluding shoulder and hip girdle) or weakness of muscles or tenderness of leg muscles.

5. Mononeuropathy or polyneuropathy

Development of mononeuropathy. multiple mononeuropathies, or polyneuropathy.

6. Diastolic BP >90 mm Hg

Development of hypertension with the diastolic BP higher than 90 mm Hg.

7. Elevated BUN or creatinine

Elevation of BUN >40 mg/dl or creatinine >1.5 mgidl, not due to dehydration or obstruction.

8. Hepatitis B virus

Presence of hepatitis B surface antigen or antibody in serum.

9. Arteriographic abnormality

Arteriogram showing aneurysms or occlusions of the visceral arteries, not due to arteriosclerosis, fibromuscular dysplasia, or other noninflammatory causes.

I0.Biopsy of small or medium-sized artery containing PMN

Histologic changes showing the presence of granulocytes or granulocytes and mononuclear leukocytes in the artery wall.

* For classification purposes, a patient shall be said to have polyarteritis nodosa if at least 3 of these 10 criteria are present. The presence of any 3 or more criteria yields a sensitivity of 82.2% and a specificity of 86.6%. BP = blood pressure; BUN = blood urea nitrogen; PMN = polymorphonuclear neutrophils

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2. Indications for requesting a test

2.1 Diagnostic work-up A test for ANCA should be ordered in every patient suspected of having idiopathic systemic vasculitis. Suspicion of systemic vasculitis will arise in the following conditions: 1. in the presence of visible vascular lesions such as purpura, necrotic fingertips, etc., 2. in the presence of more or less typical symptoms such as necrotizing inflammation in the upper airways (WG), hypereosinophilia with nonallergic asthma (Churg-Strauss Syndrome), etc., 3. in cases of rapidly progressive glomerulonephritis, 4. in the presence of symptoms and signs of inflammation such as fever, raised ESR and C-reactive protein level, etc., without identifiable cause or localization. As stated before, a positive test by IIF should be followed by antigenspecific assays for antibodies to Pr3 and MPO. Generally, anti-Pr3 and anti-MPO do not occur concomitantly although exceptions have been described [16]. The further work-up of a patient suspected of vasculitis and having positive testresults for anti-Pr3 or anti-MPO is given in Figs. 1 and 2 [17]. nose, sinus, and/or lung involvement

Clinical findings

active urine sediment

Possible clinical diagnosis

Biopsy sites

localized Wegeners’s granulomatosis I 1. nose biopsy

generalized Wegeners’s granulomatosis I nose biopsy

active urine sediment

microscopic polyangiitis/ idiopathic NCGN I renal biopsy

2. (open) lung renal biopsy biopsy (in case of abnormalities on chest X-ray)

NCGN = necrotizing crescentic glomerulonephritis. Fig. 1. Work-up of a patient with anti-Pr3 and suspected of vasculitis AMAN-C7.1/4

localized Wegeners’s granulomatosis I 1. ENTinvestigation and biopsy of lesions 2. biopsies from visible lesions or lesions suspected from other studies.

Fig. 2. Work-up of a patient with anti-MPO and suspected of vasculitis

At present, ANCA specificities different from Pr3 and MPO are still under investigation with respect to their identification and clinical correlates [10, 12]. They will not be discussed in this chapter.

2.2 Follow-up Already in the first study on ANCA in WG [2] it was recognized that titers of ANCA were higher during active disease than at remission. Follow-up studies in patients with WG who are positive for c-ANCAianti-Pr3 have shown that rises in titer of ANCA preceed or concur with increasing disease activity [18, 19] although these data have not been fully confirmed by others [20, 21]. Recently, it has been shown that changes in the IgG3-subclass of anti-Pr3 have a higher predictive value for the occurrence of a relapse than changes in the c-ANCA titer alone [22]. Treatment based on increases of ANCA-titer in WG proved to prevent the occurrence of relapses in a rather small group of patients [23]. It can be safely concluded now that increasing titers of c-ANCA/anti-Pr3 in patients with WG should alert the clinician to the possibility of an ensuing relapse. Comparable studies have not been performed in patients with anti-MPO antibodies.

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2.3 Prognostic value and predictive character No solid data are available with respect to the predictive value of anti-Pr3 or anti-MPO. Several observations, however, suggest that patients with antiPr3 and some overlapping signs of necrotizing vasculitides develop extended WG during the course of their disease.

3. Methods for ANCA detection

ANCA are routinely detected by indirect immunofluorescence on ethanolfixed neutrophils [1]. Cytospins of the buffy-coat are preferentially used as a substrate since the simultaneous presence of lymphocytes and neutrophils in the preparation allows the distinction between ANCA, which are myeloid specific, and ANA. The presence of ANCA can, however, not be excluded in ANA-positive sera. Three different patterns of staining are recognized: cytoplasmic- or c-ANCA, perinuclear- or p-ANCA, and aspecific cytoplasmic staining. Anti-Pr3 positive sera usually produce a c-ANCA pattern, and anti-MPO positive sera a p-ANCA pattern (although exceptions do occur [24]). The assay gives reproducible results also between different laboratories [25]. Titration may be useful for follow-up of patients [ 18-23].A positive result should be followed by antigen-specific assays (see before). Although immunoblotting and immunoprecipitation are performed in research laboratories [26], ELISA systems are generally used for detection of anti-Pr3 and anti-MPO. Differences between these assays are primarily based on the antigenic substrate used. The following assays are currently in use:

3.1 ELISA for measuring anti-MPO [27, 28] Most laboratories use MPO obtained from Calbiochem (La Jolla, CA). The purity of this preparation has been questioned [29]; in particular, it seems to contain lactoferrin. MPO can also be produced by standard methods [30]. As mentioned by Wieslander and Wiik (Chapters B.7.1 and B.7.2), DNA can bind to negatively charged MPO [31]. As such, sera from SLE patients with DNA-anti DNA complexes can theoretically give rise to false positive results in the ELISA.

3.2 Capture ELISA for measuring anti-MPO [26, 28] The assay uses a monoclonal antibody to MPO for catching MPO from a crude preparation of α-granules from neutrophils. The assay has been described in detail elsewhere [28]. When performing this assay, wells coated AMAN-C7.1/6

with an irrelevant monoclonal antibody have to be included in order to control for aspecific interactions.

3.3 ELISA for measuring anti-Pr3 Pr3 can be purified from neutrophil granules by dye-ligand affinity and ionexchange chromatography [32], by immunoaffinity chromatography [33], or by high pressure liquid chromatography [34]. Purified protein can be used in ELISA [25]. Assays using different Pr3 preparations are currently compared in a collaborative study sponsored by the EEC [25] in order to standardize the assay.

3.4 Capture ELISA for measuring anti-Pr3 [26, 28] This assay is identical to that mentioned in Section 3.2 for anti-MPO with the exception that a monoclonal antibody specific for Pr3 is used. It is expected that a standardized procedure for anti-MPO and anti-Pr3 by ELISA will be available during 1996 based on the results of the EECstudy mentioned before.

4. Clinical associations

4.1 Anti-Pr3 Three major studies comprising more than 200 patients have found a sensitivity of 90% for so-called extended WG characterized by the triad of granulomatous inflammation of the respiratory tract, systemic vasculitis, and necrotizing crescentic glomerulonephritis [ 18, 35, 36]. The sensitivity of anti-Pr3 for limited WG, i.e. disease manifestions without obvious renal involvement, amounted to 75% [10, 12]. These data concern patients with active disease only. The specificity of anti-Pr3 for active WG has been found as high as 98% [18, 35, 37] when sera were studied from patients with a wide variety of renal, autoimmune, vasculitic, infectious, or lymphoproliferative disorders. Anti-Pr3 are, however, detected in patients with overlapping symptoms of WG and other forms of vasculitis, and in some patients with idiopathic necrotizing glomerulonephritis without systemic involvement [5, 6, 35, 38]. Some of these patients will evolve into definite (extended) WG (see before). Anti-Pr3 have recently been detected in patients with invasive amoebiasis [39] and in patients with thyroid disease treated with propylthiouracil who developed a vasculitic-like disorder [16]. The frequency of HLA-DQw7 was reported to be significantly increased in Caucasian patients with vasculitis AMAN-C7.1/7

(relative risk 2.9). With respect to ANCA, it proved that patients with the DQw7/DR4 haplotype more frequently had transiently positive tests for ANCA during follow-up, whereas patients with DR2 bearing haplotypes more frequently had persistently positive ANCA-tests [40].

4.2 Anti-MPO In contrast to anti-Pr3, anti-MPO is associated with different forms of idiopathic necrotizing vasculitides. Anti-MPO are detected in nearly all patients with pauci-immune crescentic glomerulonephritis who are negative for anti-Pr3 [4, 5, 41, 42]. In one third of the anti-MPO positive patients, crescentic glomerulonephritis occurs without extrarenal manifestations, socalled idiopathic crescentic glomerulonephritis. Most of the remaining patients have crescentic glomerulonephritis in combination with pulmonary infiltrates and/or alveolar hemorrhage, a condition sometimes designated as microscopic polyangiitis, or in combination with a systemic illness with constitutional symptoms, arthralgias, purpura, and/or otorhinolaryngologic symptoms [5, 6, 41]. A number of these patients have a history of asthma and hypereosinophilia, and fulfill the criteria for the Churg-Strauss syndrome [6, 38]. Anti-MPO are also present in patients with polyangiitis or ChurgStrauss syndrome without renal involvement [38]. These data are summarized in Table 4. The specificity of anti-MPO for systemic vasculitis and/or idiopathic crescentic glomerulonephritis is as high as 95% [5, 6, 43] when groups of patients with renal disorders and diseases related to vasculitis are studied. Anti-MPO have, however, also been detected in patients with hydralazine-induced lupus [44], patients with anti-glomerular basement membrane disease [45], and in a minority of patients with SLE [42, 46]. The possibly false positive results for anti-MPO in SLE have already been mentioned. Table 4. Disease associations of anti-proteinase 3 and anti-myeloperoxidase antibodies* Disease entity

Sensitivity of anti-proteinase 3

Idiopathic crescentic glomerulonephritis Microscopic polyangiitis Wegener’s Granulomatosis Churg Strauss Syndrome Classic polyarteritis nodosa Polyangiitis overlap syndrome

30%

70%

50% 80%, 10% 10% 40%

50% 20% 70% 20% 20%

* These data are summarized from the references cited in the text AMAN-C7.1/8

anti-myeloperoxidase

5. Future trends/prospects

Present and future research will be directed at the exploration of the pathophysiological role of ANCA, and on the characterization and clinical significance of ANCA specificities different from Pr3 and MPO.

5.1 Pathophysiological vole of ANCA Several lines of evidence suggest that ANCA are involved in the pathophysiology of the necrotizing vasculitides: 1. Anti-Pr3 can inhibit the irreversible inactivation of the enzyme by α1antitrypsin, its natural inhibitor [47]. Although most anti-Pr3 positive sera not only inhibit inactivation of Pr3 but also its enzymatic activity [47], the reversible antigen-antibody binding may be dissolved at the site of inflammation allowing the enzyme to display its lytic activity and contribute to the inflammatory process. 2. ANCA can interact with MPO or Pr3 expressed on or bound to endothelial cells which may result in endothelial damage. Pr3 can be expressed on the surface of cytokine stimulated endothelial cells [48], and MPO, being a highly cationic protein, can bind to the negatively charged surface of endothelial cells [49]. The in vivo relevance of this phenomenon is doubtful as significant deposition of IgG along capillary walls has not been observed in the aforementioned conditions. 3. ANCA can activate primed neutrophils to the production of reactive oxygen species and the release of lysosomal enzymes [SO]. Primed neutrophils, i.e. neutrophils pretreated with low dosis of TNFα, express the target antigens of ANCA on their surface. In the presence of ANCA those primed neutrophils are further activated. This process is Fc-dependent as it can be inhibited by blocking of the FcII receptor on neutrophils [51]. 4. In vivo experimental data have shown that rat immunized with MPO develop pauci-immune necrotizing crescentic glomerulonephritis after perfusion of the kidney with the products of activated neutrophils [52]. Taken together these data suggest a role for ANCA, i.e. anti-Pr3 and anti-MPO, in the pathophysiology of necrotizing vasculitis. More data are, however, needed to definitely prove the basic role of the autoimmune response to myeloid lysosomal enzymes in these conditions [53].

5.2 Characterization and clinical associations of ANCA specificities different from Pr3 and MPO ANCA have been described that are directed against lactoferrin, cathepsin AMAN-C7.1/9

G, elastase, lysozyme, eosinophil peroxidase, and others [ 10-12]. These specificities have been detected in diverse conditions such as rheumatoid arthritis, Felty’s syndrome, ulcerative colitis, primary sclerosing cholangitis, and others [10–12]. In contrast to anti-Pr3 and anti-MPO which are highly specific for the group of idiopathic necrotizing vasculitides, most of the ANCA directed against the aforementioned antigens are not specific for one particular disorder but appear to occur in different conditions characterized by idiopathic inflammation. E.g. anti-lactoferrin have been detected in patients with rheumatoid arthritis with [ 13] and without [ 14] concurrent vasculitis, but also in patients with ulcerative colitis [15] and primary sclerosing cholangitis [54]. Future studies will be directed at the more precise characterization of those ANCA specificities different from Pr3 and MPO. This will be necessary in order to establish the clinical significance of those ANCA.

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References 1. Wiik A (1989) APMIS 97 (Suppl 6): S12–S13 2. Van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H, Van Es LA, Van der Giessen M, Van der Hem GK & The TH (1985) Lancet ii: 425–429 3. Leavitt RY, Fauci AS, Bloch DA, Michel BA, Hunder GG, Arend WP, Calabrese LH, Fries JF, Lie JT, Lightfoot RW, Masi AT, McShane DJ, Mills JA, Stevens MB, Wallace SL & Zvaitler NJ (1990) Arthritis Rheum 33: 1101–1107 4. Falk RJ & Jennette JC (1988) N Engl J Med 318: 1651–1657 5. Cohen Tervaert JW, Goldschmeding R, Elema JD, Van der Giessen M, Huitema MG. Van der Hem GK, The TH, Von dem Borne AEGKR & Kallenberg CGM (1990) Kidney Int 37: 799–806 6. Cohen Tervaert JW, Goldschmeding R, Elema JD, Limburg PC, Van der Giessen M, Huitema MG. Koolen MI, Hené RJ, The TH, Van der Hem GK, Von dem Borne AEGKR & Kallenberg CGM (1990) Arthritis Rheum 33: 1264–1272 7. Jennette JC, Falk RJ, Andrassy K, Bacon PA, Churg J, Gross WL, Hagen EC. Hoffman GS, Hunder GG, Kallenberg CGM, McCluskey RT, Sinico RA, Rees AJ, Van Es LA, Waldherr R, Wiik A (1994) Arthritis Rheum 37: 187–192 8. Cohen Tervaert JW, Mulder AHL, Stegeman CA, Elema JD, Huitema MG, The TH & Kallenberg CGM (1993) Ann Rheum Dis 52: 115–120 9. Zhao MH, Jones SJ & Lockwood CM (1995) Clin Exp Immunol 99: 49–56 10. Kallenberg CGM, Mulder AHL & Cohen Tervaert JW (1992) Am J Med 93: 675–682 11. Peter HH, Metzger D, Rump A & Röther E (1993) Clin Exp Immunol 91 (Suppl 1): S12S14 12. Kallenberg CGM, Brouwer E, Weening JJ & Cohen Tervaert JW (1994) Kidney lnt 46: 1–15 13. Coremans IEM, Hagen EC, Daha MR, Van der Woude FJ, Van der Voort EAM, Kleijburg-van der Keur C & Breedveld FC (1992) Arthritis Rheum 35: 1466–1475 14. Mulder AHL, Horst G. Van Leeuwen MA, Limburg PC & Kallenberg CGM (1993) Arthritis Rheum 36: 1054–1060 15. Mulder AHL. Broekroelofs J, Horst G, Limburg PC, Nelis GF & Kallenberg CGM (1994) Clin Exp Immunol 95: 490–497 16. Dolman KM, Gans ROB, Vervaart THJ, Zevenbergen G, Maingay D, Nikkels RE, Donker AJM, Von dem Borne AEGKR & Goldschmeding R (1993) Lancet 342: 651–652 17. Cohen Tervaert JW, Kallenberg CGM (1993) In: Elkon K (Ed) Neurologic Aspects of Rheumatic Diseases, Rheumatic Disease Clinics of North America, Vol 19, pp. 913–940. WB Saunders, Philadelphia 18. Cohen Tervaert JW, Van der Woude FJ, Fauci AS, Ambrus JL, Velosa J, Keane WF, Meijer S, Van der Giessen M, The TH, Van der Hem GK, Kallenberg CGM (1989) Arch Intern Med 149: 2461–2465 19. Egner W, Chapel HM (1990) Clin Exp Immunol 82: 244–249 20. Kerr GR, Fleischer THA, Hallahan CW, Leavitt RY, Fauci AS, Hoffman GS (1993) Arthritis Rheum 36: 365–371 21. Cohen Tervaert JW, Stegeman CA, Huitema MG, Kallenberg CGM (1994) Arthritis Rheum 37: 596 (Lett to Ed) 22. Cohen Tervaert JW, Mulder AHL, Kallenberg CGM, Stegeman CA (1994) J Am Soc Nephrol 5: 828 23. Cohen Tervaert JW, Huitema MG, Hené RJ, Sluiter WJ, The TH, Van der Hem GK & Kallenberg CGM (1990) Lancet 336: 709–711 24. Segelmark M. Baslund B, Wieslander J (1993) Clin Exp Immunol 93 (Suppl 1): S32 25. Hagen EC, Andrassy K. Csernok E. Daha MR, Gaskin G, Gross WL, Lesavre PH, Lüdemann J, Pusey CD, Rasmussen N, Savage COS, Sinico A, Wiik A, Van der Woude FJ (1993) J Immunol Meth 159: 1–16 26. Goldschmeding R, Van der Schoot CE, Ten Bokkel Huinink D, Hack CE, Van den Ende AMAN-C7.1/11

ME, Kallenberg CGM & Von dem Borne AEGKR (1989) J Clin Invest 84: 1577–1587 27. Wiik A, Rasmussen N, Wieslander J (1993) In: Van Venrooij WJ & Maini RN (Eds) Manual of Biological Markers of Disease, pp. Ag, 1-14. Kluwer Academic Publishers, Dordrecht 28. Kallenberg CGM, Cohen Tervaert JW & Limburg PC (1991) In: Bullock GR, Van Velzen D, Warhol MJ & Herbrink P (Eds) Techniques in Diagnostic Pathology. Vol 2, pp. 43-60. Academic Press. London 29. Audrain MAP, Baranger TAR, Lockwood CM & Esnault VLM (1993) Clin Exp Immunol 93 (Suppl 1): S18 30. Merrill DP (1980) Prep Biochem 10: 133–137 31. Murao S, Stevens FJ, Ito A & Huberman E (1988) Proc Natl Acad Sci USA 85: 1232-1236 32. Kao RC, Wehner NG, Skubitz KM, Gray BH & Hoidal JR (1988) J Clin Invest 82: 1963–1973 33. Lüdemann J, Utecht B & Gross WL (1988) J Immunol Meth 114: 167–174 34. Gaskin G, Turner AN, Ryan JJ, Rees AJ & Pusey CD (1991) J Am SOC Nephrol 2: 595 35. Nolle B, Specks U, Lüdemann J, Rohrbach MS, Deremee RA & Gross WL (1989) Ann Intern Med 111: 28–40 36. Weber MFA, Andrassy K, Pullig O, Koderisch J & Netzer K (1992) J Am SOC Nephrol 2: 1227–1234 37. Kallenberg CGM & Cohen Tervaert JW (1992) In: Andreucci VE & Fine LG (Eds) International Yearbook of Nephrology 1992, pp. 313–336. Springer Verlag, London 38. Cohen Tervaert JW, Limburg PC, Elema JD, Huitema MG, Horst G, The TH & Kallenberg CGM (1991) Am J Med 91: 59–66 39. Pudifin DJ, Duursma J, Gathiram V & Jackson TFGH (1994) Clin Exp Immunol 97: 48–51 40. Spencer SJW, Burns A, Gaskin G, Pusey CD & Rees AJ (1992) Kidney Int 41: 1059–1063 41. Jennette JC, Wilkman AS & Falk RJ (1989) Am J Pathol 135: 921–930 42. Gueirard P, Delpech A, Gilbert D, Godin M, Le Loet X & Tron F (1991) J Autoimm 4: 517–527 43. Nässberger L, Sjöholm AG & Thysell H (1990) Nephron 56: 152–156 44. Nässberger L, Sjöholm AG, Jonsson H, Sturfelt G & Äkessen A (1990) Clin Exp Immunol 81: 380–383 45. Jayne DRW, Marshall PD, Jones SJ & Lockwood CM (1990) Kidney Int 37: 965–970 46. Gallichio MC, Savige JA (1991) Clin Exp Immunol 84: 232–237 47. Van de Wiel A, Dolman KM, Van der Meer-Gerritsen CH, Hack CE, Von dem Borne AEGKR & Goldschmeding R (1992) Clin Exp Immunol 90: 409–414 48. Mayet WJ, Csernok E, Szymkowiak C, Gross WL, Meyer Zum Buschenfelde KH (1993) Blood 82: 1221–1229 49. Savage COS, Gaskin G & Pusey CD (1993) J Exp Nephrol 1: 190–195 50. Falk RJ, Terrell RS, Charles LA, Jennette JC (1990) Proc Natl Acad Sci USA 87: 4115–4119 51. Mulder AHL, Horst G, Limburg PC, Kallenberg CGM (1994) Clin Exp Immunol 98: 270–278 52. Brouwer E, Huitema MG, Klok PA, De Weerd H, Cohen Tervaert JW, Weening JJ & Kallenberg CGM (1993) J Exp Med 177: 905–914 53. Kallenberg CGM, Cohen Tervaert JW, Van der Woude FJ, Goldschmeding R. Von dem Borne AEGKR & Weening JJ (1991) Immunol Today 12: 61–64 54. Mulder AHL, Horst G, Haagsma EB, Limburg PC, Kleibeuker JH & Kallenberg CGM (1993) Hepatology 17: 411–417

AMAN-C7.1/12

Autoantibody Manual C8.1. 1-18, 1996 © 1996 Kluw er Academic Publishers Printed in The Nertherlands

The autoantibodies of primary biliary cirrhosis: clinico pathological correlations

IAN R. MACKAY¹ and M. ERIC GERSHWIN²

¹ Centre for Molecular Biology and Medicine, Monash University, Wellington Road, Clayton 3168, Australia; ² Division of Rheumatology, Allergy and Clinical Immunology University of California Davis, Davis, CA 95616 U.S.A.

Introduction

1.1 Historical background to primary biliary cirrhosis The first description of primary biliary cirrhosis (PBC) is attributed to Addison and Gull in 1851, but that of Hanot in 1875 as hypertrophic biliary cirrhosis is the more definitive, according to Mann [1]. After 1900, associations between biliary cirrhosis and lipid abnormalities including cutaneous xanthomas led to the designation of xanthomatous biliary cirrhosis, with connotations of xanthomatous obstruction of the biliary system [2]. The name primary biliary cirrhosis became used after 1949 [3] to distinguish the disease from biliary cirrhosis due to extrahepatic obstruction. The classic modern description is that of Ahrens et al. [4] in 1950 which included the occurrence in middle aged or older women of European background, progressive jaundice pruritus and hepatosplenomegaly with, morphologically, a biliary cirrhosis associated with an inflammatory destruction of intrahepatic biliary ductules. This paper was slanted particularly to the lipid abnormalities and no insights into pathogenesis were then available.

1.2 Mitochondrial antigens In 1959 the detection in a case of PBC of high titres of complement-fixing antibodies to tissue homegenates pointed to autoimmunity as the pathogenetic process [5]. In 1965 the application of indirect immunofluorescence with patients' sera on frozen tissue sections revealed a cytoplasmic staining identifiable as antimitochondrial [6], later confirmed by the finding that PBC sera reacted in vitro with isolated mitochondria [7]. The mitochondrial autoantigen proved to be located on the inner mitochondrial membrane [8] and trypsin sensitive [9]. The mitochondrial antibody reaction AMAN-C8.1/1

was soon found in clinical studies to have a high utility for the diagnostic identification of cases of PBC, whether tested for by complement fixation [8, 10] or immunofluorescence [10–12]. During the 1970s, anti-mitochondrial reactions other than that associated with PBC were successively described: either the mitochondrial antibody reaction differed in immunofluorescence characteristics or by chromatographic partition of antigen from that associated with PBC, or there was an accompanying disease that differed from PBC. The first antigen was cardiolipin applicable to serological tests for syphilis which had a mitochondrial location [13], and was subsequently distinguished as M 1 from the PBC-related antigen which was called M2 [9]. Thereafter other putative mitochondrial antigens were reported and numbered M3 to M9; those associated with apparently variant forms of PBC became designated as M4, M8, and M9, and those associated with other diseases as M3, M5, M6 and M7 [14]. Whereas the M1 and M2 antigens are identifiable as cardiolipin and pyruvate dehydrogenase complex respectively, the identity of the other mitochondrial autoantigens is in doubt. Accordingly it has been suggested that the M3-M9 nomenclature be set aside pending clearer information on the character of these antigens and/or their disease associations [15]. The mitochondrion is a complex organelle that contains hundreds of proteins, including numerous enzymes of which some may well be autoantigenic, but consensus should be reached on associations of autoantibodies to such proteins with disease before classification can be recommended. Thus only the M2 autoantigens will be described in this chapter.

1.3 Immunomolecular era During 1985 the new immunomolecular era for PBC began, initially with immunoblotting. This allowed the recognition of discrete polypeptide antigens of defined molecular weight [16–18]. This was followed by the derivation by molecular cloning of a cDNA that encoded the mitochondrial autoantigen [19], and thereby the recognition of enzyme molecules of the 2oxoacid dehydrogenase complexes (2-OADC) as the reactants revealed by immunoblotting [20, 21]. The major mitochondrial autoantigen, reactive in 90-95% of cases of PBC, proved to be the 74 kD E2 subunit, the acyl transferase, of the pyruvate dehydrogenase complex (PDC); the other 2OADC autoantigens are reactive at lower frequency [22]. There is usually a co-association of the autoantibodies that comprise the M2 subset, as shown in Table 1, but the individual autoantibodies are not mutually cross-reactive. The identification of the 2-OADC enzymes as the autoantigenic reactants for PBC sera has allowed for great conceptual advances, but terminological difficulties persist. Thus, unless immunoblotting is performed, or an individual ELISA is set up to detect autoantibodies to each of the three 2-OADC enzyme autoantigens, these will not be separately identified by the AMAN-C8. 1/2

Table 1. Combinations of antibodies to 2-OADC in PBC* PDC. OGDC, BCOADC PDC only PDC, OGDC OGDC and/or BCOADC PDC, BCOADC NIL

47% 27% 15% 5% 3% 3%

Individual reactivities: PDC, 92%; OGDC, 66%: BCOADC, 54% * Based on 129 patients sera studied by immunoblotting [22]

immunofluorescence methods use in routine diagnostic laboratories. In other words, independent specification of the autoantibodies as anti-PDC-E2, anti-OGDC-E2 or anti-BCOADC-E2 is difficult in practice as well as being terminologically awkward. Hence the use is recommended of the older term M2 for a generic specification of the mitochondrial 2-OADC enzyme autoantigens in PBC, and anti M-2 for the autoantibodies to any or all of the three 2-OADC enzyme complexes.

1.4 Clinical diagnostic criteria for PBC ‘Primary biliary cirrhosis’ is clearly a misnomer for a disease that has a long precirrhotic stage, and autoimmune cholangiolitis would be a more apt descriptor; however the former term is too entrenched to alter. Diagnostic criteria for PBC were presented in the ‘Fogarty Manual’ of 1976 [23], and an updated version was published in 1994 by the International Hepatology Informatics Group [24]. Alternatively authors may choose to validate their case selection by reference criteria based on standard clinical descriptions based on published reviews [25–28]. There are no discernible subsets of PBC but there are wide variations in clinical expression attributable to the stage of evolution. There is current emphasis on the high frequency of asymptomatic cases [29], and on cases in which the disease course is more benign and protracted [30].

1.5 Histological criteria The ‘gold-standard’ for the diagnosis of PBC is the liver biopsy which is usually required for full confirmation of the diagnosis. Pathologists agree that the histological evolution of PBC can be described in four stages [31]: (i) Inflammatory destruction of intrahepatic bile ducts; (ii) proliferation of ductules; (iii) fibrosis; (iv) cirrhosis. There are no clear quantitative differences in anti-M2 reactivity according to these four stages of the disease. AMAN-C8.1/3

2. Assays

2.1 Utility of anti-M2 The detection of anti-M2, whether by the older technique of immunofluorescence, or the newer assays (vide infra), has proven to be one of the most clinically useful of all of the autoimmune diagnostic assay systems. There are numerous studies attesting to a positivity rate for anti-M2 of some 95% in histologically proven PBC, so that the sensitivity of the test is gratifyingly high. However, the specificity of anti-M2 for PBC, whilst not systematically assessed, falls well short of 100% Considering data from a UK survey of random serum samples [32], it was found that of 4200 sera referred for diagnostic testing by immunofluorescence, there were 69 positive for anti-M2. Of these 69, there were 9 for which PBC could be established unequivocally as the diagnosis, and 60 (1.3%) for which another cause could be attributed: for 6 the diagnosis was chronic active hepatitis, for 10 there were abnormal liver function tests with no established diagnosis, and for 44 there was no evidence at all of liver disease. A particular problem in establishing specificity is that there can be a long asymptomatic phase in PBC during which even biochemical indices of liver function may remain normal so that, unless biopsy is performed, latent PBC cannot be diagnosed. Hence the significance for disease of a positive serological reaction for anti-M2 is not readily ascertainable, either from random population studies or from serological analysis on patients with other diseases. Thus liver biopsy would be seldom performed merely on the basis of a positive serological test for anti-M2, and there are indeed recorded instances in which the liver was histologically normal despite a positive test for anti-M2 by immunofluorescence [33]. The newer ‘second generation’ tests for anti-M2, ELISA or enzyme inhibition (see below), may improve specificity for the diagnosis of PBC since immunofluorescence probably identifies mitochondrial reactants other than M2.

2.2 Clinical applications of assays The symptoms that would prompt a request for anti-M2 are indolent jaundice, pruritus or even unexplained fatigue in a middle-aged woman. A test is mandatory in cases in which routine biochemical assessment of liver function tests shows abnormalities with an obstructive pattern, and particularly when there is elevation of the serum alkaline phosphatase level disproportionate to other tests. Anti-M2 has particular value in cases of jaundice with ‘obstructive’ liver function tests wherein the test result can discriminate intrahepatic from extrahepatic (surgically remediable) causes. Titration to endpoint of the immunofluorescence reaction is recommended, since there is greater diagnostic certainty attached to higher titres (> 1/320) AMAN-C8.1/4

than to lower titres (1/40-1/80). The prevalence rates for mitochondrial antibody in the index disease PBC, normal populations and certain other diseases are shown in Table 2. In one study, patients with early (stage 1) PBC were found to have a lower incidence of autoantibodies by immunoblotting to each of the M2 antigens [34]. However, tests for anti-M2 have not been widely used for monitoring the course of cases of PBC, since titres by immunofluorescence were found not to alter much with the course over time, or biochemical/clinical features, progression or severity of the disease [31]. Levels by ELISA of anti-M2 of IgG class were stated to correlate significantly with histological stage and certain of the prognostic biochemical variables [35], but much greater experience with the newer assays for anti-M2 is needed before a consensus can be reached (see below). Thus, in routine practice, once a positive test is recorded, repetition is seldom necessary. However, for monitoring of specific therapy including immunoregulatory agents, levels of anti-M2 might well prove useful as a surrogate marker of effectiveness of therapy, and treatment trial designs should consider inclusion of the newer assays, ELISA or enzyme inhibition. Alterations in level of antibody could point to efficacy of the drug under investigation and possibly clarify the relationship of anti-M2 to pathogenesis. Table 2. Prevalences of anti-M2 in PBC and other diseases Disease

Prevalence [ref]

Primary biliary cirrhosis Caucasians Japanese Miscellaneous referred disease sera Scleroderma (CREST) Sjögren’s syndrome (primary) SLE Normal population

>90% [28] 89% [54] 1.3%[32] 1 0% [63] 10% [64] 17% [71] 0.03% [55]

2.3 Methods used for detection of anti-M2 For some 20 years from the mid- 1960s indirect immunofluorescence remained the standard technique for detecting anti-M2. In 1985 a further dimension was added with the introduction of immunoblotting, and in 1987 a cDNA was cloned for the major autoantigen, PDC-E2. This led to the availability of recombinant fusion proteins and their use in automated assays. Reliable ELISAs with high sensitivity have been described for antibody to the 2-OADC enzymes, using either purified intact PDC [35], commercially available PDC (Sigma, St. Louis, MO) [36] or OGDC [37], or a recombinant antigen, PDC-E2 [38]. One disadvantage of ELISA, compared AMAN-C8.1/5

with immunofluorescence or immunoblotting, is that only one of the three 2OADC enzymes can be detected in a single assay system; this could be partly overcome by the use of a hybrid recombinant molecule that expresses reactive sites of two of the 2-OADC-E2 subunits, those of PDC and BCOADC [39]. There are preparations of purified PDC and OCDC available commercially (Sigma St. Louis, MO), but each has minor ‘contamination’ with other of the 2-OADC enzymes and other proteins. ELISA kit formats for testing for antibody to recombinant PDC-E2 are now available commercially. The ELISA marketed by Biotrin International (Dublin, Ireland) has a claimed sensitivity of 83% and specificity of 94% (product literature) for the diagnosis of PBC, based on data from a relatively small panel of sera. The advantages and limitations, and overall applicability of the various assays are, shown in Table 3. Table 3. Comparison of assays for anti-M2 in PBC Assay Immunofluorescence

Comments traditional: sensitivity high; specificity moderate; detects reactivity to all 2-OADC enzymes; non-automated; subjective readout

Immunoblotting

sensitivity high; specificity moderate; detects individual reactivity to all 2-OADC enzymes: non-automated; labour intensive

ELISA

sensitivity high; specificity high but not fully assessed; automated; non-subjective readout; detects reactivity to a single 2-OADC enzyme in each run.

Enzyme inhibition

sensitivity and specificity high but not fully assessed; automated, fewer steps than ELISA: non-subjective readout: detects reactivity to a single 2-OADC enzyme in each run

A promising automated assay is based on the capacity of PBC sera to inhibit specifically the catalytic activity of pyruvate dehydrogenase complex, determined spectrophotometrically [40]. This assay has been ‘miniaturized’ for performance in microtitre plates with assessment of enzyme activity using a plate reader [41, 42], as illustrated in Fig. 1. In a comparative assessment, the enzyme inhibition assay was comparable with ELISA in regard to sensitivity and specificity for the diagnosis of PBC, and has the advantage of fewer procedural steps [42].

AMAN-C8.1/6

Fig. 1. The technique for ‘miniaturized microtitre well assay for the capacity of PBC sera to inhibit the catalytic activity of pyruvate dehydrogenase complex (PDC). The ‘read-out’ depends on inhibition of production of NADH, the end product of the enzyme activity. Technical details, reagent concentrations and assay performance are described by Teoh et al. [41, 42]. (Reproduced with permission from Raven Press.)

2.4 Autoantibodies to isoforms or epitopes of autoantigens Autoantibodies to isoforms or mutants of PDC have been searched for in an attempt to explain the occurrence of autoimmune reactivity to this autoantigen. Among the E2 subunits of pyruvate dehydrogenases derived by cloning from different tissues there were minor polymorphic differences in sequence, but structural isoforms of 2-OADC enzymes are not demonstrable [43, 44]. The epitope on PDC for reactivity with autoantibody, the ‘B cell epitope’, was first postulated on theoretical grounds to be associated with the conserved region within which the lipoyl co-factor is attached to a lysine residue [21]. This was corroborated by the capacity of a 20-mer synthetic peptide to partly absorb out from (dilute) PBC sera the capacity to react with recombinant PDC-E2 [45]. Subsequent studies indicated that the autoantibody epitope was more complex, and conformational rather than linear, as judged by the requirement of some 91 residues for optimal reactivity with PBC serum [46], and by a greater reactivity of PBC sera with the intact PDC enzyme compared with recombinant PDC-E2 (37). Diagnostic assays do not need to utilize specifically the epitope-bearing configuration of PDC-E2, although the enzyme inhibition assay may, more than others, identify serum reactivity with this part of the molecule.

AMAN-C8.1/7

2.5 T lymphocyte responses T lymphocytes derived from blood or from infiltrates in liver biopsy samples give proliferative responses to the 2-OADC enzymes [47, 48], but this response has no diagnostic implications. There are indications that a proliferative response to the PDC-E2 subunit, via-a-vis other subunits, occurs specifically in PBC and that, as with other autoantigenic molecules, there are multiple T cell epitopes on the antigenic E2 molecule.

3. Mitochondrial antibody: disease associations and prevalence

3.1 Numerical values and prevalences of anti-M2 With immunofluorescence, numerical values for levels of autoantibody by immunofluorescence are conventionally expressed by serum titres, noting that cut-off titrations for positivity will vary according to test conditions and substrates used. Moreover since serum levels of many autoantibodies behave as a continuous variable, there is an inevitable overlap between normal and disease. Accordingly a cut-off titre for positivity of anti-M2 by indirect immunofluorescence will be arbitrary: a 1/40 serum dilution is widely used. Sensitivity and specificity will of course depend on the cut-off titres selected. In 1972 the Medical Research Council (U.K.) introduced 'British Research Standard 67/183’ as a reference standard serum as a step towards an international standard against which anti-M2 sera could be calibrated, but the initiative lapsed. More recently there has been the successful example of the pancreatic islet cell antibody for which the potency is now quantitated against a reference standard and expressed as Juvenile Diabetes Foundation (JDF) units [49]. As mentioned above, the generally accepted frequency for a positive reaction for anti-M2 by immunofluorescence in PBC is 95%. With ELISA, numerical values are usually based on a critical optical density (OD) reading at a given serum dilution, with a selection of an OD that appears to give the best discrimination between positive and negative sera [38]. Optimal results will be obtained by titration of sera at the selected OD [42]. Wit the enzyme inhibition assay described above, serial titrations can be made from the initial serum dilution of 1/500. The frequencies of autoantibodies to the 2-OADC enzymes in PBC and other diseases using the newer techniques still require more extensive analysis, but preliminary data are available [38, 42].

AMAN-C8.1/8

4. Mitochondrial antibody: geographic, ethnic, genetic aspects

4.1 Geographic-ethnic differences in an anti-M2 frequency PBC has interesting and at present unexplained differences in prevalence according to different geographic regions and ethnic groups [50]; there are no data on comparative population prevalences of anti-M2 to set alongside data on prevalence data of PBC itself. The highest population frequencies of PBC have been ascertained in Northern England and Sweden, about 128 cases per million, whereas among the Canadian population for which the ‘ethnicity’ would resemble that of the U.K., the figure is very considerably lower, 23 per million [51]; this is similar to that reported for middle Europe. The population frequencies of PBC among Asian populations is substantially lower. PBC is detectable in Japanese at 5.16 per million [52], and is said to be increasing in prevalence, whereas the disease remains virtually non-detectable in India. In actual instances of disease, the frequency of anti-M2 by immunofluorescence seems equally high whatever the population group. However in studies on the comparative frequency by ELISA among Japanese of different components of the 2-OADC enzyme family, the frequency of antibody to PDC-E2 of 65%)–70% is markedly lower than that among Caucasians, whereas the frequency of antibody to OGDC-E2 was relatively higher [53, 54]. The population frequency of antiM2 by immunofluorescence of 1 per 3500 subjects for a rural population in Australia, equivalent to 286 per million [55], is some tenfold above the assumed population prevalence in Australia of PBC itself. In contrast, the frequency of anti-M2 among miscellaneous disease sera referred for routine antibody testing in two surveys in the U.K. was 2%) [11] and 1.3% [32] which is some hundredfold above the regional prevalence of PBC itself.

4.2 Mitochondrial antibody and HLA Many autoimmune diseases are associated with genetic markers, notably HLA alleles. However it can seldom be determined whether the HLArelated predisposition is to the disease itself with autoantibodies as a secondary association, or whether HLA is associated directly with the presence of the disease-related autoantibody. The first major study to show an HLA association in PBC was with DR8 [56]. Other studies confirming this with, in one, an association with a null allele for the fourth component of complement, C4, are reviewed in the report of a study that failed to show any significant MHC class-I1 associations [57]. The high degree of association of anti-M2 with PBC, and the very low frequency of anti-M2 without PBC, precludes identification of associations of anti-M2 itself with particular HLA alleles. There are a sufficient number of case reports of familial association of AMAN-C8.1/9

PBC to suspect a genetic component to the disease [58]. However the basis for this would not appear to be HLA or other known immunogenetic processes. A recent study was performed on the frequency of autoantibodies in 48 first or second degree relatives of 27 index cases of PBC [59]. Overall there were 7 relatives (1 1%) with autoantibodies, but anti-M2 was expressed by only two relatives of whom one had histological PBC and the other sarcoidosis. These results are reminiscent of the earlier family studies in which relatives of patients with PBC compared with controls had a raised frequency of various autoantibodies other than antiM2 [60, 611. Thus the most evident genetic factor would be a ‘soft’ level of natural self tolerance.

5. Co-associations of PBC and anti-M2 with other diseases 5.1 Overlap syndromes There are two ‘overlap’ syndromes with PBC that have attracted attention, the PBC-autoimmune hepatitis overlap [62] and the PBC-scleroderma/ CREST overlap [63]. In addition there are several other diseases with a reported coexistence with PBC, notably Sjögren’s syndrome and autoimmune thyroiditis, and possibly rheumatoid arthritis [25–28]. These overlap syndromes are accompanied by the marker antibodies of both of the overlapping diseases. In the autoimmune hepatitis-PBC overlap, there is in addition to anti-M2 a variant form of mitochondrial autoantibody referred to as anti-M4 [62], but the existence of M4 has not been validated [15]. In the PBC-scleroderma overlap there is anticentromere as well as anti-M2 [63]. The apparent co-association of Sjögren’s syndrome and PBC is interesting. Thus in an analysis of cases of Sjögren’s syndrome [64], anti-M2 was more frequent in the primary sicca syndrome (6 of 60 cases) than in the rheumatoid arthritis-associated sicca syndrome (3 of 71 cases), whereas when cases of PBC were assessed for sicca symptoms, the positivity rate was 72–100%. Notwithstanding, the anti-La-Ro reactivity characteristic of primary Sjögren’s syndrome appears to be infrequent in the PBC-associated sicca syndrome. In other words, the ‘sicca syndrome’ that accompanies PBC cannot be aligned either with primary Sjögren’s syndrome with anti-La-Ro, nor with the type of Sjögren’s disease that accompanies rheumatoid arthritis.

5.2 Antibodies to non-mitochondrial antigens in PBC In addition to anti-M2 there are autoantibodies to non-mitochondrial antigens at high frequency in PBC (Table 4). The most prominent of these non-M2 autoantibodies are those to nuclear antigens, with patterns defined by immunofluorescence as speckled dots, anticentromere, and antilamin. AMAN-C8.1/10

Table 4. Non-mitochondrial autoantibodies in PBC Specificity

Frequency

Comment

1. Nuclei centromere [63] Sp-100 [63]

12–30% ~30%

± CREST syndrome speckled fluorescence; no clinical associations high association with PBC

lamins including gp 210 [68] Ro/La

~30% infrequent

primary biliary cirrhosis with secondary Sjögrens syndrome!

2. Thyroid peroxidase [28] colloid

data variable

autoimmune thyroid disease coexists

3. Stomach parietal cell H+ K+ ATPase

data lacking

pernicious anaemia rarely coexists

4. Smooth muscle [11]

50%

PBC-autoimmune hepatitis overlap

The anticentromere antibody in the PBC-scleroderma overlap has the same pattern of reactivity by immunofluorescence as that present in the CREST syndrome, remains after affinity purification of PBC serum on PDC-E2 [63], and is reactive with the CENP-B recombinant protein [65]. The speckled dot pattern by immunofluorescence specifies a nuclear antigen with a molecular weight of 100 kD by analysis on gels, and hence was called Sp-100 [66]; subsequently a cDNA that encoded the antigenic protein corresponding to Sp-100 was derived, so allowing for a substrate for an ELISA for Sp-100 [67]. Antibodies to proteins of nuclear lamina are present in PBC, and in other autoimmune disorders including autoimmune hepatitis and SLE [63]; the antibodies of greatest interest, because of their apparent total specificity for PBC, are those to the nuclear pore membrane glycoprotein gp210 [68].

5.3 Anti-M2 in a diagnostic context A ‘sensible consensus’ on anti-M2, by whatever technique is used, would be that a positive test in an appropriate clinical or biochemical setting virtually confirms a diagnosis of PBC, although most hepatologists would still wish a liver biopsy for absolute confirmation and disease staging. A clearly positive test discovered incidentally by multiphasic screening, outside of an appropriate setting or in a healthy person, should prompt regular perforAMAN-C8.1/11

mance of biochemical tests of liver function; most hepatologists would be unwilling to perform a liver biopsy on the basis of a positive test for anti-M2 alone, although the degree of positivity of the test and concordance by other assays (immunoblotting, ELISA, enzyme inhibition) might influence this decision. Unfortunately there are no systematic data on liver histology in anti-M2 positive healthy individuals with normal liver function tests. A negative test for anti-M2, by all type of assay, occurs in some 5% of cases of histologically confirmed PBC, so should not dissuade the clinician from the diagnosis in the appropriate clinical setting. The explanation for this 1 in 20 negativity for anti-M2 in confirmed PBC is uncertain. Although antinuclear antibodies (ANA) of one or another specificity occur with some frequency (40–50%) in PBC, and will be available usually in the routine immunological work-up, there is no indication of these having diagnostic utility in PBC (apart from the scleroderma-CREST overlap), since the anti-M2 negative cases are not marked by a specific type of ANA. The well-established M3 to M9 nomenclature introduced by Berg [62] specifies different types of mitochondrial reactivity with either variant expressions of PBC (M4, M8, M9), or disease states other than PBC (M3, M5, M6, M7). These ‘variant’ types of mitochondrial antibody are difficult to identify under routine laboratory conditions. Moreover corroborative identification of candidate antigenic specificities for at least some of the non-M2 antigens has been unsuccessful, leading to a suggestion that the M3-M9 notation be set aside pending further correlative data on the syndromes and their associated antigenic specificities [15].

6. Anti-M2 and course/expression of PBC

6.1 Occurrence of anti-M2 before disease onset The occurrence of autoantibodies before onset of their associated disease has obvious connotations for population screening, identification of at-risk individuals and prophylactic interventions. These considerations are the subject of numerous publications in the diabetes literature pertaining to antibodies to islet cell antigens or glutamic acid decarboxylase [69]. However it is seldom that any given autoantibody can be regarded as a ‘harbinger’ in the sense of existing not only before symptoms but also before subclinical histological evidence of autoimmunity. In the case of PBC, the requirement would be that anti-M2 should be demonstrable despite normal liver histology, but with later evolution to actual PBC; this sequence has not been reported. On the other hand, facilities now for automated testing for anti-M2 will focus attention on screening for presymptomatic PBC among ‘at-risk’ populations i.e. middle-aged women in PBC-endemic regions.

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6.2 Anti-M2 and disease activity The test for anti-M2 (AMA) by immunofluorescence is regarded as generally insensitive and is not usually ‘factored’ into decisions on treatment or prognosis, or into ‘monitoring’ of therapeutic trials. The availability of newer and more quantifiable assays for anti-M2 may alter this perception, noting that results from ELISA and enzyme inhibition assay were predictive of an apparent response of PBC patients to cyclosporine [42].

6.3 Anti-M2 and disease outcome (prognosis) The test for anti-M2 (AMA) by immunofluorescence does not correlate with disease activity, nor with disease outcome in the sense of a strongly positive test signifying a more rapidly evolving or ‘highly active’ type of PBC. Indeed a positive test for anti-M2 usually persists after liver transplantation for PBC but, in this setting at least, is not indicative of recurrence of PBC in the grafted liver. Levels of anti-M2 according to the histologically graded stage of disease did not differ according to immunofluorescence [42], but contrary data have been reported according to Western blotting and ELISA (see Section 2.2).

6.4 Repetitive testing for anti-M2 The recommendation is that, in routine practice, one reliably positive assay for anti-M2 should be sufficient, and that there is little to be gained from repetitive testing. However this has not been thoroughly examined, particularly in the setting of therapeutic trials wherein repetitive testing using at least one of the newer assay formats could be informative. 6.5 Drug reactions and anti-M2 Certain drugs are known to elicit autoimmune serological reactions and, occasionally, diseases with immunopathological features. However, there are no validated examples of drug-initiated PBC accompanied by a typical anti-M2 response. An AMA reaction by immunofluorescence was reported in 1972 in three instances of drug-induced cholangio-hepatitis attributed to halothane, chlorpromazine or sulphonamide [12]. More recently in 1985, AMA reactions by immunofluorescence were included among 157 cases of drug-induced hepatitis with ‘anti-organelle’ antibodies [70]. Hence an antiM2 reaction may occasionally accompany an adverse hepatotoxic drug reaction, and hence should be included in an ‘autoimmune’ work-up of individuals with drug-sensitivity reactions affecting the liver. AMAN-C8.1/13

7. Anti-M2 and therapeutic management of PBC

Anti-M2, according to titre or even simple positivity, has no place per se in therapeutic decisions, once the diagnosis is established on other criteria. In particular, the level of antibody by immunofluorescence appears not predictive of a response ’or lack of response to treatment. Currently most patients with non-advanced PBC will (or should) receive ursodeoxycholic acid, and those with advanced PBC will be on a transplantation assessment programme. The level of anti-M2 would not influence either of these options.

8. Future trends and prospects

Future trends with diagnostic assays for PBC will be towards optimized versions of automated assays based either on recombinant enzyme molecules as in ELISAs or on miniaturised ‘machine-readable’ enzyme inhibition assays. A required technical advance will be the development of assay procedures in which serum reactivity can be ascertained to all three 2-OADC enzymes in combination. There is considerable population survey work required to define better the specificity of positive serological reactions to M2, and to ascertain whether the newer automated assays (ELISA, enzyme inhibition assay) can be calibrated to provide a greater specificity than given by indirect immunofluorescence, without loss of sensitivity. As yet, these assays have not been trialled on a large scale using serum sets of some 1000 samples that would be needed to answer these questions. A welcome prospect would be the regular inclusion of quantitative serum assays for anti-M2 in treatment trials of newer immunomodulatory drugs in PBC, to ascertain whether changes in serological reactivity herald a clinical response.

Note added in proof

Recently attention has been directed to ‘autoimmune cholangitis’, a controversial entity in which biochemical and histological features of PBC coexist with hepatitic features and with autoantibodies to nuclear (ANA) rather than mitochondrial antigens (AMA), and immunosuppressive treatment is beneficial. Since the ANA may resemble ‘PBC types’ more than ‘lupus types’, and anti-M2 (anti-2-OADC enzymes) may be demonstrable by the newer assays, cases of autoimmune cholangitis may align most closely with PBC72.

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22. Bassendine MF, Fussey SPM, Mutimer DJ, James OFW & Yeaman SJ (1989) Identification and characterization of four M2 mitochondrial autoantigens in primary biliary currhosis. Sem Liver Dis 9: 124–131 23. Leevy CM, Popper H & Sherlock S (1976) Diseases of the Liver and Biliary Tract. Standardization of nomenclature. Diagnostic Criteria and Diagnostic Methodology. Fogarty International Centre Proceedings No. 22. DHEW Publication No. (NIH) 76–725. U.S. Government Printing Office, Washington D.C. 24. International Hepatology Informatics Group (1994) Diseases of the Liver and Biliary Tract. Standardisation of Nomenclature, Diagnostic Criteria and Prognosis. Editorial Committee: Leevy CM, Sherlock S, Tygstrop N & Zetterman R, Raven Press, New York. 25. Sherlock S & Scheuer PJ (1973) The presentation and diagnosis of 100 patients with primary biliary cirrhosis. N Engl J Med 289: 674–678 26. Christensen E, Crowe J, Doniach D et al. Clinical pattern and course of disease in primary biliary cirrhosis based on an analysis of 236 patients. Gastroenterology 78: 236–248 27. Kaplan MM (1987) Primary biliary cirrhosis. N Engl J Med 316: 521–528 28. Mackay IR (1985) In: Rose NR & Mackay IR (Eds) The Autoimmune Diseases, pp. 291–337. Academic Press, Orlando 29. James O, Macklon AF & Watson AJ. Primary biliary cirrhosis- a revised clinical spectrum. Lancet i: 1278–-1281 30. Knox TA & Kaplan MM (1991) In: Krawitt EL & Wiesner RH (Eds) Autoimmune Liver Diseases, pp, 167–173. Raven Press, New York 31. Dickson ER, Fleming CR & Ludwig (1979) In: Popper H & Schaffner F (EDS) Progress in Liver Disease VI, pp. 487–502. Grune and Stratton, New York. 32. Triger DR, Charlton CAC & Ward AM (1982) What does the antimitochondrial antibody mean? Gut 23: 814–818 33. Fusconi M, Ghademinejad I, Branchi FB, Baum H, Bottazzo GF & Pisi E (1988) Heterogeneity of antimitochondrial antibodies with the M2-M4 pattern by immunofluorescence as assessed by Western immunoblotting and enzymelinked immunosorbent assay. Gut 29: 440–447 34. Mutimer DJ, Fussey SPM, Yeaman SJ, Kelly PJ, James OFW & Bassendine MF (1989) Frequency of IgG and IgM autoantibodies to four specific M2 mitochondrial autoantigens in primary biliary cirrhosis. Hepatology 10: 403–407 35. Heseltine L, Turner IB, Fussey SPM, Kelley PJ, James OFW, Yeaman SJ & Bassendine F (1990) Primary biliary cirrhosis. Quantitation of autoantibodies to purified mitochondrial enzymes and correlation with disease progression. Gastroenterology 99: 1786–1792 36. Kisand K, Kisand K, Salupere V & Uibo R (1994) Enzyme linked immunosorbent assays for the determination of IgG, IgA, and IgM autoantibodies to pyruvate dehydrogenase in primary biliary cirrhosis. Int J Clin Lab Res 24: 98–101 37. Rowley MJ, McNeiliage LJ, Armstrong J McD & Mackay IR (1991) Inhibitory antibody to a conformational epitope of the pyruvate dehydrogenase complex, the major autoantigen in primary biliary cirrhosis. Clin Immunol Immunopathol 60: 356–370 38. Van de Water J, Cooper A, Surh CD, Coppel R, Danner D, Ansari A, Dickson R & Gershwin ME (1989) Detection of autoantibodies to recombinant mitochondrial proteins in patients with primary biliary cirrhosis. N Engl J Med 320: 1377–1380 39. Leung PSC, Iwayama T, Prindiville T et al. Use of designer recombinant mitochondrial antigens in the diagnosis of primary biliary cirrhosis. Hepatology 15: 367–372 40. Uibo R, Mackay IR, Rowley MJ, Humphries P, Armstrong JMcD & McNeilage J (1990) Inhibition of enzyme function by human autoantibodies to an autoantigen pyruvate dehydrogenase E2: different epitope of spontaneous human and induced rabbit autoantibodies. Clin Exp Immunol 80: 19–24 41. Teoh K-L, Rowley MJ & Mackay IR (1991) An automated microassay for enzyme inhibitory effects of M2 antibodies in primary biliary cirrhosis. Liver 11: 287–291 AMAN-C8.1/16

42. Teoh K-L, Rowley MJ, Zafirakis H, Dickson ER, Wienser RH, Gershwin ME & Mackay 1R (1994) Enzyme inhibitory autoantibodies to pyruvate dehydrogenase complex in primary biliary cirrhosis: applications of a semi-automated assay. Hepatology 20: 1220–1224 43. Moehario LH, Wang L, Devenish RJ. Mackay IR & Marzuki S (1991) The human pyruvate dehydrogenase complex: a polymorphic region of the lipoate acetyl transferase (E2) subunit gene. Biochim Biophys Acta: Mol Basis Dis 1097: 128–132 44. Turchaney JM, Leung PSC, Iwayama T et al. (1993) Comparative metabolism and structure of BCKD-E2 in primary biliary cirrhosis. J Autoimmunity 6: 459–466 45. Van de Water J, Gershwin ME, Leung P, Coppel RL, The autoepitope of the 74-KD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase. J Exp Med 167: 1791–1 799 46. Surh CD, Ahmed-Ansari A & Gershwin ME (1990) Comparative epitope mapping of murine monoclonal and human autoantibodies to PDH-E2, the major mitochondrial autoantigen of primary biliary cirrhosis. J Immunol 144: 2647–2652 47. Van de Water J, Ansari AA, Surh DC, Coppel R, Roche T, Bokovsky H, Kaplan M & Gershwin ME (1991) Evidence for the targeting by 2-oxo-dehydrogenase enzymes in the T cell response in PBC. J Immunol 146: 89–94 48. Lohr H, Fleischer B. Gerken G, Yeaman SJ, Meyer zum Buschenfelde K-H & Manns M (1993) Autoreactive liver-infiltrating T cells in primary biliary cirrhosis recognize mitochondrial epitopes and the pyruvate dehydrogenase complex. J Hepatol 18: 322–327 49. Bonifacio E, Boitard C, Gleichmann G et al. (1990) Assessment of precision. concordance, specificity. and sensitivity of islet cell antibody measurement in 41 assays. Diabetologia 33: 731–736 50. Myszor M & James OFW (1990) The epidemiology of primary biliary cirrhosis in northeast England: an increasingly common disease. Quart J Med (n.s) 75: 377–385 51. Witt-Sullivan H, Heathcote J, Cauch K, Blendis L, Ghent C, Katz A, Miler R, Pappas SC, Rankin J & Wanless IR (1990) The demography of primary biliary cirrhosis in Ontario, Canada. Hepatology 12: 98–105 52. Gershwin ME, Van de Water J, Leung PSC & Coppel R (1993) T cell biology and cell surface expression of PDC-E2 in primary biliary cirrhosis. In: Meyer zum Buschenfelde K-H, Hoofnagle J & Manns M (EDS) Immunology and Liver: Falk Symposium No 70. pp. 373–382. Kluwer Academic Publishers. Lancaster 53. Iwayama T, Leung PSC, Rowley M et al. (1992) Comparative immunoreactive profiles of Japanese and American patients with primary bilary cirrhosis against mitochondrial antigens. Int Arch Allergy Immunol 99: 28–33 54. Omagari K, Rowley MJ, Jois JA, Komatsu K, Maeda T, Yamazaki K & Mackay IR (in press) Immunoreactivity of antimitochondrial antibodies in Japanese patients with primary biliary cirrhosis. J Gastroenterol 55. Hooper B, Whittingham S, Mathews JD, Mackay IR & Curnow DH (1972) Autoimmunity in a rural community. Clin Exp Immunol 12: 79–87 56. Gores GJ, Moore SB, Fisher LD, Powell FC & Dickson ER (1987) Primary biliary cirrhosis: associations with class II major histocompatibility complex antigens Hepatology 7: 889–892 57. Zhang L, Weetman AP, Bassendine M & Oliveira DBG (1994) Major histocompatibility complex class-11 alleles in primary biliary cirrhosis. Scand J Immunol 39: 104–106 58. Mackay IR (1984) Genetic aspects of immunologically mediated liver disease. Sem Liver Dis 4: 13–25 59. Caldwell SH, Leung PSC, Spivey JR et al. Antimitochondrial antibodies in kindreds of patients with PBC: AMA are unique to clinical disease and are absent in asymptomatic family members. Hepatology 16: 899–905 60. Galbraith RM, Smith M. Mackenzie RM, Tee DE, Doniach D & Williams R (1974) High prevalence of seroimmunologic abnormalities in relatives of patients with active chronic hepatitis or primary biliary cirrhosis. N Engl J Med 290: 63–69 AMAN-C8.1/17

61. Salaspuro MP, Laitinen OI & Lehtola J (1976) Immunological parameters. viral antibodies and biochemical and histological findings in relatives of patients with chronic active hepatitis and primary biliary cirrhosis. Scand J Gastroenterol 11: 313–320 62. Berg PA, Wiedmann K-H & Sayers TJ (1980) Serological classification of chronis cholestatic liver disease by the use of two different types of antimitochondrial antibodies. Lancet ii: 1329–1 332 63. Mackay IR, Rowley MJ & Whittingham SF (1993) Nuclear autoantibodies in primary biliary cirrhosis. In: Meyer zum Buschenfelde K-H. Hoofnagle J & Manns M (Eds) Immunology and Liver: Falk Symposium No 70. pp. 397–409. Kluwer Academic Publishers. Lancaster 64. Whaley K, Webb J, McAvoy BA, Hughes GRV, Lee P. MacSween RNM & Buchanan WW ( 1973) Sjögren’s syndrome. 2. Clinical. associations and immunological phenomena. Q J Med 42: 5 13–548 65. Earnshaw WC, Machlin PS. Bordwell BJ, Rothfield NF & Cleveland DN (1987) Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B. Proc Natl Acad Sci USA 84: 4979–4983 66. Szoztecki C, Krippner H, Penner E & Bautz FA (1987) Autoimmune sera recognise a 100 kD nuclear protein antigen (Sp100). Clin Exp lmmunol 68: 108–116 67. Szostecki C, Guldner HH, Netter HJ & Will H (1990) Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies from patients with primary biliary cirrhosis. J Immunol 145: 4338–4347 68. Nickowitz RE & Worman HJ (1993) Autoantibodies from patients with primary biliary cirrhosis recognise a restricted region within the cytoplasmic tail of nuclear pore membrane glycoprotein Gp210. J Exp Med 178: 2237–2242 69. Zimmet PZ, Elliott RB, Mackay IR, Tuomi T, Rowley MJ, Pilcher CC & Knowles WJ (1994) Autoantibodies to glutamic acid decarboxylase and insulin in islet cell antibody positive presymptomatic type 1 diabetes mellilus: frequency and segregation by age and gender. Diabetic Med I: 866–871 70. Homberg JC, Abuaf N, Helmy-Halil S et al. Drug-induced hepatitis associated with anticytoplasmic antoantibodies. Hepatology 5: 722–727 71. Zurgil N. Bakimer K. Moutsopoulos HM et al. (1992) Antimitochondrial (pyruvate dehydrogenase) autoantibodies in autoimmune rheumatic diseases. J Clin lmmunol 12: 201–209 72. Omagari K, lkuno N, Matsuo I, Shirono K, Hara K, Feeney SJ, Whittingham S, Mackay IR (1966) The autoimmune cholangitis syndrome with a bias towards primary biliary cirrhosis (submitted).

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Autoantibody Manual C8.2. 1 -9. 1996 © 1996 Kluw er Academic Publishers Pr inted in The Netherlands

Human autoantibodies to the coiled body and other nuclear bodies LUÍS E.C. ANDRADE Disciplina de Reumatologia, Escola Paulista de Medicina, Rua Botucatu 740, sao Paulo SP 04023, BraziI

The morphological pattern observed in indirect immunofluorescence (IIF) with autoantibody-containing sera can be regarded as an autoantigenic map that provides information on the topographic distribution of the target antigen(s). Some autoantibodies originate in IIF a peculiar type of discrete nuclear speckles that, in contrast to anti-centromere antibodies [1], are not aligned at the metaphase plate in dividing cells. Therefore, the IIF patterns generated by these autoantibodies have been generally named atypical discrete speckled patterns. Other designations include atypical speckles, nuclear dots [2], multiple nuclear dots [3], NSp1 [4], NSp2 [4], and nuclear bodies [5]. According to the criterion of number of speckles per nucleus in human cycling cells, two patterns can be readily recognized, one characterized by 1–6 speckles per nucleus and the other one appearing as 5–20 speckles per nucleus [3]. The first pattern was shown to be associated to the nuclear coiled body, being designated coiled body pattern [5]. The second pattern is designated MND-pattern (for multiple nuclear dot) and the related subcellular functional domain seems to correspond to less wellcharacterized nuclear bodies [6, 7].

1. Autoantibodies to the coiled body

1.1 Introduction Autoantibodies to the coiled body were identified in a group of sera associated to an IIF pattern characterized by 1–6 variable sized round dots throughout interphase nucleus [5] (Fig. 1, left panel). In dividing cells the metaphase plate is not stained and the nuclear dots disappear in early prophase only to reappear in mid-G1 phase [8]. However, remnants of the coiled body may be seen occasionally at the periphery of metaphase and anaphase cells [9]. This group of sera was shown to react uniformly with an 80-kDa nuclear protein, designated p80-coilin [5].

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Fig. 1. Left panel: Coiled body pattern. Indirect immunofluorescence on HeLa cells with human serum containing anti-p80-coilin autoantibodies. Characteristic distribution of coiled bodies in cells in a nonsynchronous culture. Right panel: Multiple nuclear dot pattern. Indirect immunofluorescence with human serum NSpl, kindly provided by M.J. Fritzler and regarded as a paradigm of MND-pattern antibodies [3, 4, 20]. Observe the high number of speckles per nucleus in a nonsynchronous culture of HeLa cells.

1.2 Clinical significance Autoantibodies to the coiled body have been reported in a variety of autoimmune conditions as depicted in Table 1. A few patients, however, presented no signs or symptoms suggesting autoimmune disease. An ELISA screening with the recombinant protein corresponding to the carboxyl terminal end of p80-coilin showed the autoantibody to be rare in systemic autoimmune diseases (Table 2) [5]. It seems to be unusual in healthy individuals, since no coiled body IIF pattern was detected in the sera of 2,500 Table 1. Diagnosis in patients presenting anti-p80-coilin antibodies [5] Primary biliary cirrhosis Sjögren’s syndrome Progressive systemic sclerosis Systemic lupus erythematosus Amyotrophic lateral sclerosis Skin rash/arthralgia Respiratory infection-associated rash Inflammatory brachial plexopathy

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Polymyalgia Dermatomyositis Raynaud’s phenomenon Hashimoto’s disease Cold urticaria Osteoarthritis Soft tissue rheumatism Peripheral nerve palsy

Table 2. Prevalence of anti-p80-coilin antibodies in some systemic autoimmune diseases [5] Disease

Total of patients

Number of positive cases

Scleroderma Systemic Lupus Erythematosus Rheumatoid Arthritis Sjögren’s syndrome Chronic Active Hepatitis Primary Biliary Cirrhosis Multiple Sclerosis

44 44 31 54 32 59 26

0 0 0 2 (3.7%) 0 2 (3.3%) 0

blood donors [12]. Previous reports on autoantibodies with a similar staining pattern but without further characterization of the target antigen refer to patients with systemic sclerosis [2], morphea [2], mixed connective tissue disease [2], PBC [3], HBsAg-negative chronic hepatitis [3], and lupus-like paraneoplastic syndrome [11]. Autoantibodies to p80-coilin have been detected in people from different ethnic background (african, Caucasian, asiatic, hispanic) and from different geographic areas (Japan, U.S.A., Brazil, Europe). Although no study has addressed a possible relationship with MHC alleles or other genetic element, two sisters (one with juvenile rheumatoid arthritis) presented high titer anti-p80-coilin antibodies (Andrade LEC, unpubl.). There is no longitudinal study addressing the relationship between circulating anti-p80-coilin antibodies and prognosis, disease activity or therapeutic response. In conclusion, at the moment autoantibodies to p80-coilin seem not to be specific of any particular clinical condition. Future clinical studies should focus on possible associations of these autoantibodies with a peculiar clinical or pathological trait.

1.3 Immunological characterization Human autoantibodies to p80-coilin so far identified belong to IgG class and may occur in titer as high as 1:40,000 in IIF. The epitope(s) recognized by human autoantibodies seem(s) to be well conserved since they react in IIF and immunoblot with substrate from species as diverse as mice, rat, rabbit and rat kangaroo [5, 8, 13]. Native epitope(s) seem(s) to be recognized, since human autoantibodies immunoprecipitate an 80-kDa protein from 32Plabeled cell extracts [9]. The carboxyl terminal end of p80-coilin contains at least one dominant autoepitope, since most human autoantibodies to p80coilin react in ELISA with a fusion protein derived from the 3’ end of p80coilin cDNA [5]. However, a few sera presenting strong reactivity in IIF and immunoblot react modestly with this fusion protein, suggesting that other epitope(s) outside this region may be involved. In fact, the carboxy-terminal AMAN-C8.2/3

Fig. 2. Competition between human and rabbit antibodies to p80-coilin. Recombinant fusion protein derived from the 3´ end of p80-coilin cDNA [5] was used as substrate in ELISA. Human anti-p80-coilin sera Op. Gr, CI. Pz. Wo and normal human serum (NHS) were tested after pre-incubation of the wells with pre-immune rabbit serum or anti-p80-coilin rabbit serum R288. The reactivity of all human anti-p80-coilin sera was partially inhibited by rabbit anti-p80-coilin antibodies.

end of p80-coilin seems to have an unusual potential for eliciting autoantibodies, since rabbits immunized with the cognate recombinant protein produced high levels of antibodies (R288) reactive with p80-coilin from rabbit as well as from other species [8]. Moreover, rabbit R288 serum and human autoantibodies inhibited each other in ELISA suggesting recognition of the same or close epitope(s) (Fig. 2). Frequently there are coexisting autoantibodies, usually associated with a homogeneous or fine speckled nuclear staining in IIF. However, no association with a particular autoantibody specificity has been detected.

1.4 Methods of detection IIF is the standard screening method for anti-p80-coilin antibodies. However, human autoantibodies to p80-coilin seldom occur as the only autoantibody detected in IIF. As already mentioned, most sera also depict an additional homogeneous or fine speckled nucleoplasmic staining and occasionally a cytoplasmic fine speckled staining. The identification of the coiled body pattern may be hindered by such coexistent antibodies, specially when strong homogeneous or coarse nuclear speckled patterns are present. Nevertheless, the coiled body is usually so brightly stained by anti-p80-coilin AMAN-C8.2/4

sera that a careful observer will identify its additional staining pattern in most cases. It should be noted that the IIF staining is less evident or even absent in tissue sections since the coiled body is usually smaller and less numerous in quiescent cells [8–10, 13]. Although the vast majority of sera staining 1–6 dots per nucleus contain antibodies to p80-coilin, some sera with such IIF pattern fail to recognize p80-coilin in immunoblot with whole cell extracts or ELISA with recombinant p80-coilin. This observation raises the possibility of other autoantigens with coiled body-like cellular distribution. Therefore. the definition of anti-p80-coilin antibodies should rest on the association of the suitable IIF pattern and appropriate results in at least one other test, either immunoblot or ELISA. Substrate for immunoblot should be either human cycling cell whole lysate or nuclear fraction [5]. ELISA should be done with the recombinant protein for the carboxy-terminal end of p80-coilin [5]. 1.5 Future trends and prospects The search for a clinical significance for antibodies to p80-coilin should be fostered through clinical studies of a larger spectrum of diseases, as well as through longitudinal studies addressing correlations with prognosis and disease activity. Another potential practical application is the use of antip80-coilin antibodies as markers of cell proliferation in biopsy specimens. Since the coiled body is larger and more numerous in cycling cells as compared to quiescent cells [8], anti-p80-coilin antibodies may be useful in the immunohistological study of proliferating lesions.

2. Autoantibodies associated to the multiple nuclear dot pattern (MND)

2.1 Introduction This less well-characterized group of autoantibodies has been gathered by means of a collective IIF staining pattern. Immunoblotting data is still not conclusive about the involvement of only one or more autoantigenic systems. The IIF pattern is characterized by discrete variable sized round dots distributed throughout the nucleus of cycling cells in numbers ranging from 5-20. These nuclear domains clearly differ from coiled bodies (Fig. 1, right panel). Mitotic cells present a diffuse staining in the cytoplasm and the metaphase plate contains no speckles. This pattern has been reported by several groups under different designations, such as atypical discrete speckled nuclear pattern [14], multiple nuclear dots [3, 15], NSpl [4], S3 pattern [ 16], nuclear spots [ 17], and discrete nuclear dots [18].

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2.2 Antigen characterization Immunoblot analysis has shown the antigen to be present in the nuclear fraction, with reports of reactivity with two broad bands spanning from 78-92 kDa and 96-100 kDa [7], a 95 kDa-band [18], and a 100 kDa-protein [ 17]. In these reports, antibodies affinity-purified from the relevant bands reproduced the original IIF pattern. The apparent discrepancies in the reported immunoblot reactivity may be due to technical peculiarities. However, the possibility of distinct autoantibody-autoantigen systems should be kept in mind and is stressed by the observation that not all tested sera reacted in immunoblotting [7, 18]. The notion that such nuclear domains may contain several different antigens is supported by a recent IIF data in which the monoclonal antibody 5E10 to a 126 kDa-nuclear matrixassociated protein [6] co-localized precisely with 100 kDa-band reactive serum SUN-3 regarded as representative of MND serum [17]. Besides human substrates, weaker IIF reactivity has been obtained in rat [7] and mouse [4] tissue cells, and PtK2 [17] cycling cells, while some reports claim no staining in tissue sections [4, 15]. The spot-like distribution of the antigen seems to correspond to nuclear bodies [6, 7]. Although the nature of such nuclear bodies is not known, its number increases in diethylbestroltreated rat endometrial cells [7]. No information on nucleic acid interaction is known besides the fact that in IIF the autoantigen is resistant to DNase and RNase [2, 4, 17, 18]. Immunoprecipitation with 35S-methionine-labeled extracts suggested no protein association [18]. No cDNA clone and gene information is available.

2.3 Clinical significance Most reports point out to a close association with primary biliary cirrhosis (PBC), with prevalence ranging from 5 to 44% (Tables 3 and 4). Since this disease is associated with antimitochondria antibodies, it is interesting to observe that MND-pattern autoantibodies do not cross react with mitochondria [ 15], although the two specificities are frequently observed in the same serum. Its prevalence in PBC may be even greater than estimated, since the coexistence of anticentromere antibodies may overshadow the characteristic MND pattern. Indeed, taking advantage of the fact that anticentromere antibodies rarely are IgG2, IIF with secondary antibodies specific for different isotype subclasses showed coexistence of the MND pattern and anticentromere antibodies in 30% of PBC patients and 10% of CREST patients. All the PBC patients in this series had features of CREST syndrome [20]. As observed in Table 3, MND-pattern autoantibodies may occur occasionally in autoimmune hepatitis and other autoimmune conditions. However, many of these cases were shown to have chronic cholestatic liver AMAN-C8.2/6

Table 3. Prevalence of MND-pattern autoantibodies in primary biliary cirrhosis Designation

PBC total/positive (%)

Controls total/positive (%)

“ADSN” [14] Powell et al.

246/60 (24%)

DCTD 26011 (0.4%) Blood donors 2,500/1 (0.0004%)

"MND"[3] Bernstein et al.

110/14 (13%)

CAH(–) 50/0 (0%) CAH(+) 30/0 (0%) Normal 6010 (0%)

"MND" [15] Cassani et al.

83/14 (17%)

CAH(–) 51/0 (0%) CAH(+) 2510 (0%) Cryptogenic Cirrhosis 19010 (0%)

“S3” [16] Mzali et al.

89/11 (12%)

“DND” [18] Evans et al.

50/22 (44%)

DCTD 1240/4 (0.003%) ANA(+) Chronic Liver Disease 49/0 (0%)

"MND"[19] Hansen et al.

5318 (15%)

DCTC 25/1 (4%)* SSj5011(2%1)* CAH 2514 (16%)* Chronic Liver Dis. 300/0 (0%)

“ADSN” = atypical discrete speckled nuclear pattern; "MND" = multiple nuclear dots; “S3” = type 3 speckles; “DND” = discrete nuclear dots; CAH (+) = HBSAg-positive chronic active hepatitis; CAH (–) = HBSAg-negative chronic active hepatitis; SSj = Sjögren’s syndrome: DCTD = diffuse connective tissue disease * reassessment of these patients showed chronic cholestatic liver disease

Table 4. Prevalence of MND-pattern autoantibodies in patients with association of primary biliary cirrhosis and sicca syndrome Author

PBC total/positive (%)

PBC + dry eye total/positive (%)

Cassani et al. [15]

1411 (7%)

8/4 (50%)

Bernstein et al. [3]

7814 (5%)

32/10 (31%)

Mzali et al. [16]

59/8 (14%)

14/1 (7%)

SS alone total/positive (%)

55/1 (2%)

disease, which may represent a boundary in the PBC spectrum [21]. Some authors observed a particularly high prevalence of MND-pattern with serum from patients with PBC and dry eye (Table 4) [3, 15, 19]. AMAN-C8.2/7

Thus, autoantibodies associated to the MND-pattern seem to be a valuable marker for PBC, specially when a mitochondria-like cytoplasmic speckled fluorescent pattern is also present. However, isolated MND pattern per se is useful in supporting the diagnosis of PBC in the appropriate clinical setting [19]. There is no data suggesting the usefulness of this group of autoantibodies in monitoring disease activity in PBC patients, nor in predicting prognosis or response to treatment. There is no study on possible association of this autoantibody specificity and MHC alleles or other genetic feature. It has been reported in centers from North America, Europe, Asia, and South America. No racial or ethnic influence has been observed on the expression of this autoantibody. Antibodies depicting a MND-like pattern, designated “nuclear spot pattern” [17], were reported in the serum of eleven patients with a diverse array of clinical conditions, such as Sjögren’s syndrome (n = 1), Wegener’s granulomatosis (1), arterial hypertension (1), atherosclerosis (1), T cell lymphoma (1), PBC (1), and undifferentiated connective tissue disease (n = 5). The clinical dissimilarity of this group of patients in relation to the other MND-pattern reports may be due to differences in the screened population. Alternatively, the possibility exists that autoantibodies associated to the “nuclear spot pattern” represent a distinct autoantibody system.

2.4 Immunological detection The characteristic MND-pattern may be detected in routine laboratories through IIF on human cycling cells. Immunoblot with human cycling cell whole lysate or nuclear fraction provides confirmation when reactivity around 100 kDa is observed. The main isotype is IgG.

3. Autoantibodies associated with other discrete speckled patterns

A few reports deal with atypical discrete speckled patterns that do not match the ones just described. The best defined one refers to “variable large speckles” or VLS [22]. It consists of 3-10 speckles with variable shape and size, present in several different tissue substrates (all of them in quiescent state) and absent in lymphocytes and proliferating cell cultures. These autoantibodies are mostly IgM and have specificity to histone H3 [22]. Most patients reported to present these autoantibodies had ill-defined autoimmune conditions (myopathy, arthritis, sicca complex, esophageal dysmotility, and pleurisy), while some of them presented rheumatoid arthritis (n = 5), undifferentiated connective tissue disease (n = 5), mixed cryoglobulinemia (n = 1), chronic leukocytoclastic vasculitis (n = 1), and idiopathic pulmonary fibrosis (n = 1). AMAN-C8.2/8

An exquisite IIF pattern was obtained with apparently rare autoantibodies, designated NSp2, originated from three patients with Sjögren’s syndrome [4]. Mouse kidney tubular cells presented 5–10 discrete nuclear speckles. Interphase cycling cells were stained in a faint fine nuclear speckled pattern, but mitotic cells presented several large and bright speckles in the metaphase plate. No further characterization of the target antigen has been so far performed.

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