Autoantibodies in the Diagnosis of Systemic Rheumatic Diseases

Transcription

1 I Autoantibodies in the Diagnosis of Systemic Rheumatic Diseases Carlos A, von Mfihlen and Eng M. Tan Distinct profiles of autoantibodies directed to intracellular antigens can be detected in the systemic connective tissue diseases. They aid in establishing the correct diagnosis and are included in many sets of diagnostic criteria, such as the ones developed for systemic lupus erythematosus (anti-smith antigen and anti-double-strand DNA antibodies), mixed connective tissue disease (anti-u1- nuclear ribonucleoprotein antibodies), and Sj6gren's syndrome (SS) (anti-ss- A/Ro and anti-ss-b/la antibodies). They are useful prognostic markers in some situations and facilitate clinical and treatment follow-up. Autoantibodies have also been used as probes to gain insights into cell biology, helping to isolate and purify intracellular proteins involved in key cellular functions. We give detailed information on two of the most useful techniques for the detection of autoantibodies in the clinical and research laboratory settings, indirect immunofluorescence and immunoblotting. We also discuss several of the antigen-autoantibody systems found in systemic lupus erythematosus (Smith antigen, Ul-nuclear ribonucleoprotein, SS-A/Ro, SS-B/La, proliferating cell nuclear antigen ribosomal ribonucleoprotein, double-strand DNA, histones, antiphospholipids, Ku, Ki/SL), systemic sclerosis (centromere, topo i, RNA polymerases, fibrillarin, polymyositis-scl, Th/To), polymyositis/dermatomyositis (transferrna synthetases, signal recognition particle, and others), and SS (SS-A/Ro, SS-B/La, nucleolar organizing region-90, ps0-coilin), addressing their clinical significance, common detection methods, immunogenetic associations, and the molecular and cellular biology of the cognate antigens. Copyright 1995 by W.B. Saunders Company INDEX WORDS: autoantibodies; autoimmunity; systemic rheumatic diseases; antinuclear antibodies. n most of the connective tissue diseases, a humoral immune response is characteristically seen, with autoantibodies directed to distinct intracellular antigens. 1 This phenomenon can be shown in systemic sclerosis, systemic lupus erythematosus (SLE), Sj6gren's syndrome (SS), mixed connective tissue disease (MCTD), and polymyositis or dermatomyositis (PM/DM). Because a characteristic profile of autoantibodies is found in each of the diseases above, the determination of specificities involved can be very useful to the rheumatologist. They aid in establishing the correct diagnosis and prognosis and in facilitating the clinical and treatment follow-up in many cases. The presence of anti- Smith antigen (anti-sin) and anti-native DNA antibodies are important items to be considered in establishing a diagnosis of SLE. 2 Elevated anti-double-strand DNA (anti-dsdna) anti- body titers, especially when associated with falling complement levels, signal an imminent flare of acute SLE, determining changes in treatment strategies. 3 In scleroderma, anti- DNA topoisomerase 1 (Scl-70), anti-u3-ribonucleoprotein (anti-u3-rnp), and anti-rna poly- From The Scripps Research Institute, La Jolla, CA. Carlos A. von Muhlen, MD: Research Fellow, W.M. Keck Autoimmune Disease Center, The Scripps Research Institute, Eng M. Tan, MD: Director, W.M. Keck Autoimmune Disease Center, The Scripps Research Institute. This is publication 8606-MEM of The Scripps Research Institute. Supported by National Institutes of Health grants AR and A Dr. von Miihlen is the recipient of ~ant no ~86-O from CNPq, Brazil. Address reprint requests to Eng M. Tan, M.D., W.M. Keck Autoimmune Disease Centel, The Scripps Research Institute, North Torrey Pines Rd, La Jolla, CA Copyright 1995 by W.B. Saunders Company /95/ /0 Seminars in Arthritis and Rheumatism, Vor 24, No 5 (April), 1995: pp

2 324 VON MOHLEN AND TAN merase I, II, or III antibodies are associated with diffuse cutaneous involvement and more severe systemic disease, 4-1 dictating the necessity for a more aggressive approach to treatment. Jo-1 and other anti-transfer RNA (antitrna) synthetase autoantibodies characterize a subgroup of PM patients with interstitial lung involvement and poor prognosis, u,12 On the other hand, autoantibodies have been used extensively as probes to gain insights into cell biology (recently reviewed in1,13,14). Spontaneously occurring autoantibodies have helped isolate and purify intracellular proteins that, although present in low copy number, are involved in key cellular functions. The Sm and the nuclear ribonucleoprotein (nuclear RNP [nrnp], UI-nRNP) antigen-antibody systems, seen in SLE and MCTD, served as powerful probes for the understanding of precursor messenger RNA (mrna) splicing. 15 trna synthetases, targeted by antibodies in polymyositis, function in mrna translation, u In systemic sclerosis, some autoantibodies are directed to proteins and nucleic acids taking active part in ribosomal RNA (rrna) processing. 4 These and other systems were discovered in recent years, mainly by complementary DNA (cdna) library screening and cloning. 16 By studying the nucleotide and deduced aminoacid sequences, molecular biologists and immunologists acquired new insights into the genomic and molecular structure of many antigens involved in autoimmunity, unravelling novel molecules with important biological functions. This review discusses the most important autoantibodies seen in rheumatic diseases, the molecular characteristics of their cognate antigens, how they aid in the differential diagnosis, and the diverse prognosis with which they are associated. Some insights will also be given into two of the most common techniques used for the determination of autoantibodies in the clinical and research laboratory setting: the indirect immunofluorescence (IIF) assay and the Western blot (WB) procedure. THE IIF ASSAY The IIF assay is a powerful, sensitive, and comprehensive test for screening autoantibodies. It is probably the single immunological test most ordered by rheumatologists after the rheumatoid factor test. Figure 1 depicts the 4-step A B I t, I C D I I J Fig 1: Four steps in the indirect immunofluorescence assay (cross-sectional view). Monolayer cells grown on a glass slide (A) are fixed and permeabilized (B) with chemicals such as acetone, methanol, ethanol, or formaldehyde. After a first incubation with patient's serum containing autoantibodies (C), cells are washed to get rid of unbound antibodies, and the second incubation takes place with fluorescent-labeled anti-human antibody (D). The slides are again washed, mounted with coverslips, and read on a fluorescence microscope. Nu, nucleoplasm; Cy, cytoplasm.

3 AUTOANTIBODIES IN RHEUMATIC DISEASES 325 procedure involved in the IIF. The experiment, introduced in 1954 by Weller and Coons, 17 uses a monolayer of fixed and permeabilized cells. Routinely used in many laboratories is the HEp-2 human larynx epithelioma cancer cell line, 18,19 which is allowed to interact with immunoglobulins (autoantibodies) in the test serum. To detect a positive reaction, a fluorescentlabeled antibody is used next, and the slide is read using a fluorescence microscope usually at 400 x magnification. Specific staining of nuclear or cytoplasmic structures can be visualized as apple-green fluorescence when using fluorescein isothiocyanate (FITC), or as red fluorescence when tetramethylrhodamine isothiocyanate is the fluorochrome. 2 Six main patterns of fluorescence are recognized that are indicative of the different autoantibodies that may be present2>24: (1) homogeneous nuclear patterns suggest the presence of antihistone and/or anti-dsdna autoantibodies (Fig 2A); (2) peripheral nuclear patterns (Fig 2B) are seen with autoantibodies to proteins that are an integral part of the nuclear membrane, like lamins, 24-3 or targeting integral proteins of the nuclear pore complex, like gp ,32 Previous reports relating the rim or peripheral pattern to the presence of anti-dsdna autoantibodies were from studies using highly differentiated cells of organ sections, or organ imprints, as substrate. In those cells, most of the chromatin is inactive and localized in the nuclear periphery, as opposed to the highly active chromatin observed in tissue culture cell lines; (3) speckled nuclear patterns are indicative of autoantibodies targeting a large family of nonhistone antigens (Figs 2C-E). Several distinct speckled patterns are observed and are related to different antigens being targeted. With anti-sm and anti-ul-nrnp, the speckles are coarse. With anti-ss-a/ro or anti-ss-b/la, fine speckles are commonly observed. Distinct speckles varying in number are seen with anti-p80-coilin (Fig 2G) or anti-p95, an antigen commonly seen in primary biliary cirrhosis (PBC)33; (4) nucleolar patterns have specificities mostly seen in scleroderma patients or overlap syndromes with scleroderma features, with a few reported in other nonrelated diseases: DNA topoisomerase I (Scl-70, Fig 3A), showing nucleolar speckles; PM-Scl (Fig 3B), homogeneously decorating the nucleoli; fibrillarin (U3-RNP, Fig 3C), giving a clumpy nucleolar staining; and NOR-90 and RNA polymerase I (Fig 3D), also showing nucleolar speckles, among others; (5) centromere patterns, which are seen with antibodies to the kinetochore in the CREST syndrome (limited systemic sclerosis) and in PBC (Fig 2H); (6) cytoplasmic patterns reveal different patterns (Fig 4) and are indicative of the distinct antigens involved. 22,23,34-38 It is important to emphasize that the main patterns of IIF are neither specific nor diagnostic of any autoantibody or disease entity. They are indicative of the presence of an autoantibody gmng that specific pattern in a patient with an autoimmune disease or other conditions, and its presence should be verified using more specific tests such as the double-immunodiffusion assays, the immunoblot test, enzymelinked immunosorbent assay (ELISA), or even the immunoprecipitation assay with radiolabelled cells, using appropriate reference sera. At present, organizations such as the Centers for Disease Control in Atlanta, GA, and the World Health Organization Centers in Geneva and Amsterdam have established standards for sera containing autoantibodies to some specificities like native DNA, Sin, UI-nRNP, SS-A/Ro, SS-B/La, topoi, and Jo-1 to be used in immunodiffusion. Other sera have been established as standards for the different IIF patterns. They can be obtained by writing to: Arthritis Foundation/CDC ANA Reference Laboratory, Immunology Branch, A25; Centers for Disease Control; Atlanta, GA Great care should be exercised not to over interpret positive results. Normal persons and patients with many other nonrheumatic conditions (see references 1 and 45 for reviews)--chronic liver diseases, neoplasms, or active infections such as tuberculosis, malaria, and subacute bacterial endocarditis--may show similar antibodies. They are generally in lower titers than the ones detected in autoimmune diseases, but frequently precede the onset of disease in normal and asymptomatic individuals by many years. 46 The diagnostic value of any autoantibody determination depends heavily on the clinical presentation of any patient and should be considered carefully in the context of the clinical information.

4 326 VON MUHLEN AND TAN g o tl,

5 AUTOANTIBODIES IN RHEUMATIC DISEASES 327 IMMUNOBLOTTING The WB assay is an essential tool for the characterization of many autoantibodies. Proteins from an extract of cultured cells are separated by polyacrylamide gel electrophoresis under denaturing conditions with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). 47,48 Proteins in the gel are then electrotransferred to nitrocellulose (NC) paper, which is a solid support for antigens. The NC paper is cut into strips, and each is incubated with a serum to be tested. Several methods are in use for detecting a specific antigenantibody reaction: (1) radiolabeled probe, most commonly 125I-protein A, exploits the high affini.ty of staphylococcal protein A for human immunoglobulin (Ig)G. 49 In this procedure, the strips are exposed to x-ray film at -70 C and then developed in a process known as autoradiographys ; (2) the colorimetric method uses enzymes (most commonly alkaline phosphatase or horseradish peroxidase) that act on their chromogen substrates and give visible colors; (3) chemiluminescence reaction, a more recently developed procedure, takes advantage of the light emitted from luminol oxidation when the compound is exposed to the enzyme horseradish peroxidase. 5~ The oxidative degradation of luminol results in light emission that can be seen by autoradiography. Molecular mass markers and positive and negative controls are run together in each WB assay. They provide for the necessary comparisons among bands seen with sera being probed, those in the positive controls, and the ones from the protein standards. Figure 5 shows the WB technique, and Fig 6 details results with positive controls from our laboratory. SYSTEMIC LUPUS ERYTHEMATOSUS One of the mainstays in the diagnosis of SLE and clinical follow-up of treatment is the autoantibody determination. 2,3,52 Characteristically, more than one antigen-antibody system is seen in every serum tested. 1 Table 1 presents the most common autoantibodies found in SLE, their clinical associations, IIF pattern, and molecular weight of the cognate antigens as determined by WB. Anti-dsDNA Antibodies Anti-DNA antibodies are of two general populations, one directed to dsdna, also called native DNA, the other targeting single-strand DNA (ssdna). Anti-ssDNA antibodies are not specific for SLE, because they occur in patients with different autoimmune rheumatic diseases. 1 They recognize purine and pyrimidine bases originally not exposed in the native DNA molecule. Antibodies to dsdna, on the other hand, target base-paired DNA and are therefore capable of reacting either with native or with ssdna, which has looped back to form basepaired configurations. Caution needs to be exercised when testing for the presence of antibodies to dsdna, because some degraded DNA (ssdna) may be present in commercial kits using popular techniques such as ELISA. Another possibility would be the occurrence of anti-dna/dna immunocomplexes in the sera to be tested, because some DNA released from dying cells may be contaminating the system Fig 2: Nuclear patterns of indirect immunofluorescence in HEp-2 cells. (a) Nucleoplasmic staining, as well as staining of chromosomes in dividing cells (arrowheads), characteristic of autoantibodies targeting histones or dsdna, Autoantibodies to lamin proteins A, B, and C of the nuclear envelope also give nuclear staining pattern in HEp-2 cells, but associated with continuous nucleoplasmic rim staining (b}. The speckled nucleoplasmic patterns seen in figures c, d, and e are characteristic of autoantibodies directed to Sm, Ul-nRNP, and SS-A/Ro or SS-B/La respectively. PCNA-autoantibodies give a mosaic of nucleoplasmic patterns (f), ranging from homogeneous to distinct speckled, according to the phases of cellular division (details in the text). (g) Pattern obtained with anti-p80-coilin autoantibodies. Normally I to 4 isolated nucleoplasmic dots are seen, round and bright, and not to be taken as nucleoli. Anticentromere autoantibodies (h) stain distinct speckles in the interphase nuclei and are associated with each chromosome pair. They aggregate in the metaphase plates during cell division (arrowheads, two cells in telophase).

6 328 VON MUHLEN AND TAN a C b~ b Fig 3: Indirect immunofluorescence of HEp-2 cells with autoantibodies to distinct nucleolar antigens. Autoantibodies targeting DNA topoisomerase I (Scl-70) give weak homogeneous to dense fine speckled nucleoplasmic staining, brighter speckled nucleolar staining, and homogeneous staining of chromosomes in metaphase cells (arrowheads in a). PM-Scl autoantibodies do not stain chromosomes of dividing cells (arrowheads, b), but give bright homogeneous nucleolar pattern. Autoantibodies targeting fibrillarin (U3-nRNP) give a clumpy to homogeneous staining of all nucleoli (c). Autoantibodies to NOR-90 and RNA polymerase I give the same pattern in HEp-2 cells (d), with nucleolar speckles corresponding to the NOR (dividing cell with NOR pointed by arrowhead). Ku antibodies stain the nucleoli in a more homogeneous fashion (e) over a dense fine speckled to homogeneous nucleoplasmic pattern. Cells in division are designated with arrowheads. (assays for anti-dsdna were recently reviewed). 53 Nonspecific antibodies to ssdna or the presence of anti-dna/dna complexes will give positive and negative reactions respectively when testing for dsdna antibodies, bringing confusion to the clinician involved in diagnosing and treating a specific patient. When tested with the less sensitive but very specific Crithidia luciliae IIF test, many sera that tested positive using other assays turn out to be negative for dsdna antibodies. Anti-dsDNA antibodies occur in as much as

7 AUTOANTIBODIES IN RHEUMATIC DISEASES 329 Fig 4: Cytoplasmic patterns in indirect immunofluorescence of HEp-2 cells. Antimitochondriai autoantibodies display a coarse granular pattern involving the whole cytoplasm (a). Distinct dots in the nucleoplasm elicited by the serum used are related to a second population of autoantibodies targeting an antigen known as p95. (b) The characteristic pattern of autoantibodies to the Golgi organelle is seen, with granules arranged in clusters at one or both cell poles, sparing the nuclei. A dense granular, almost homogeneous cytoplasmic pattern is obtained with anti-rrnp antibodies (c), which target ribosomal phosphoproteins. As can be seen, autoantibodies targeting rrnp also commonly stain the nucleoli in a homogeneous pattern, although they stain weakly (arrowheads). Autoantibodies to nonmuscle myosin (d) decorate the stress fibers of the cytoskeleton similarly to antibodies targeting actin. Anti-keratin antibodies (e) decorate the cytoplasm with a fibrous network spreading out from the perinuclear regions. 7(1% of patients with active lupus, tending to fluctuate with disease activity. 3,54 The specificity for a positive test is 95%, s2 making this antibody a mainstay for the diagnosis. Of major importance is the fact that this is the only autoantibody system so far clearly implicated in the pathogenesis of SLE, 55 because the availability of extracellular DNA lead to the formation of immunocomplexes and kidney deposition, where they incite local inflammatory events with serious organ damage. Antibodies to Z-DNA constitute a topic that is still developing.56, 57 Their clinical relevance is not definitely established. Antibodies to the small nrnps Sm and UI-nRNP The U1 small nuclear RNP (snrnp) antigen was so named because the antigen-antibody

8 330 VON MUHLEN AND TAN C extract NC strips B D 23 II Fig 5: Four steps in the immunoblotting technique. In step 1, cultured cells or tissue extracts are loaded on the top of an acrylamide gel and are separated by electrophoresis (SDS-PAGE). Molecular weight standards (MW) are run in a separate lane. In step 2, proteins in the gel are electrically transferred to a nitrocellulose (NC) membrane. The latter is then cut into strips, which are individually incubated with normal human serum (negative control), sera containing known antibodies (positive controls), and sera to be probed (step 3). Step 4 depicts the detection procedure in which bands of diverse reactivities are seen on strips 2 to 5. Strip number 1 has no bands (negative control). reaction in immunodiffusion was sensitive to both nucleases and proteases. 5s,59 An essential participation of snrnps in the cell splicing machinery of pre-mrna is now well established.60, 61 Anti-Sm antibodies in SLE patients were identified in They are considered specific for the disease (specificity = 99%), 52,63 and were shown to exhibit a relationship with disease activity independent of fluctuations in dsdna autoantibody titers Sensitivity is rather low (around 20%). 1,52 Clinical correlations are not strong, but were shown for central nervous system involvement, kidney disease, lung fibrosis, and pericarditis. 63 Major targets of Sm autoantibodies are the B (28 kd), B' (29 kd), and D (16 kd) polypeptides by WB, although all core proteins of snrnps, from A to G, may be recognized. 66 Sera positive for anti-sin immunoprecipitate U1, U2, U4, U5, and U6 snrnas. 1,67 In the B polypeptide, three different epitopes were described, 6s but the D antigen 23 is targeted by two distinct populations of anti-sm D antibodies, one recognizing only the full length antigen, the other reacting with the carboxyl terminus containing a supercharged structure with homology to Epstein-Barr virus nuclear antigen type 1 (EBNA-1). 69,7 The presence of Sm autoantibodies in childhood-onset and adult SLE was related to the histocompatibility antigens HLA-DQw671,72 or HLA-DR7. 73 Racial differences were also noted, black children having higher frequency of Sm and UI-nRNP autoantibodies than white children with juvenile SLE. 71 Anti-nRNP-positive sera immunoprecipitate only UI-snRNA, and by WB recognize preferentially the 70 kd, A (33 kd) and C (20 kd) polypeptides (reviewed by GuldnerV4). The autoantibodies were later identified as reacting with the uridine-rich snrnp. Often seen in conjunction with Sm autoantibodies, anti-ul-nrnp are detected in 30% to 40% of SLE patients. 1,75 The clinical correlations are with less severe disease, lower frequency of renal involvement, presence of Raynaud's phenomenon, swollen hands, esophageal hypomotility, and with overlap symptoms of nonerosive arthritis, sicca syndrome, and scleroderma skin manifestations. 1,7s'79 Described as marker antibodies for these overlap syndrome features, UI-nRNP autoantibodies were primarily associated with MCTD. 7s They are also one of the major diagnostic criteria in the disease, 1,8,sl although some literature still disputes the existence of MCTD as a separate disease entity, s2-s6 Pediatric patients with MCTD showed a high prevalence of arthritis, swollen hands, Raynaud's phenomenon, abnormal lung tests, and sclerodactyly, s7 In the same study, 11 children were followed for a mean of 9.8 years, with favorable clinical outcomes and mild to moderate illness severity, even in the presence of high UI-nRNP autoantibody titers. There is a higher frequency of HLA-DR4 and HLA-DRw53 in adults s8-91 and children with MCTDy although in this system racial differences could also be observed. 7s The association in Japanese patients, for example, is with HLA-DQw3. 92 There is a correlation of SLE disease activity and titers of anti-ul-nrnp/sm. 64,93,94 Antibodies of the IgM isotype to B/B' by WB in one study appeared before increase in disease activ-

9 AUTOANTtBODIES IN RHEUMATIC DISEASES 33i ~ii 45 -/ii b!-t0 P ~ -70 +, ~ -52!i /ii ,oo+_,,o+ ;? i, i/ ~-80 [, -200 t ='200 ii I ii I+ ~i!!ii~ i !ili'i~ iili! ~-16 -Is + Fig 6: Immunoblotting analysis of common autoantibodies seen in rheumatic diseases. MOLT-4 cells (human T lymphocyte cell line) were solubilized in appropriate buffer before separating the proteins in a 15% SDS-PAGE. After transfer to NC paper, strips were cut and probed with different human sera. As detecting reagent, 1251-protein A was used. Results are shown after autoradiography. Left, migration of molecular mass standards can be seen. Lane 1, normal human serum (negative control). The next lanes show the following reactivities: lane 2, the 70-kD band of Ul-nRNP; lane 3, a strong 48-kD band of SS-B/La and the 60-kD band of SS-A/Ro; lane 4, two bands of 70 and 80 kd corresponding to Ku autoantibodies; lane 5, the isolated 52-kD band characteristic of Jo-1; lane 6, three main antigens recognized by rrnp antisera, with 15, 16, and 37 kd; lane 7, DNA-topoisomerase I (Scl-70) migrating in the 100-kD region; lane 8, the centromere proteins CENP-A (18 kd) and CENP-B (80 kd), as well as faint anti-topo I reactivity corresponding to a second population of antibodies present in this scleroderma serum; lane 9, p80-coilin; lane 10, the major 200-kD band corresponding to the heavy chain of nenmuscle myosin, and some fainter bands resulting from its degradation products; lane 11, the 200-kD NuMA antigen, as well as other unidentified bands of lower molecular mass; lane 12, PCNA (36 kd). Note that antibodies to p80-coilin and CENP-B have been reported to have a molecular mass of approximately 80 kd, but they can be clearly separated in our gel system. Thus, migration patterns may vary according to the experimental conditions and gel concentration, and appropriate positive controls should always be used as reference for observed reactivities. ity:, and the ones of the IgG isotype targeting the 70--kD protein were positive during clinical relapses. 93 Such findings were, however, not confirmed by another laboratory in a prospective study with an ELISA using recombinant antigens. 95 Earlier studies using hemagglutination or immunodiffusion techniques also found stable titers of anti-sin and anti-ul-nrnp antibodies in SLE, without fluctuations related to disease activity.79, 96 Antibodies targeting the snrna backbone of snrnps itself were described in 1986 in patients with autoimmune disorders. 97 They were de- tected by other investigators in MCTD, overlap syndromes, and SLE. 98,99 Anti-Ul-snRNA antibodies were seen by ELISA and immunoprecipitation in 49% of patients with SLE. 1 The clinical associations reported are with diminished lung diffusing capacity, restrictive lung function impairment, Raynaud's phenomenon, and lower density of capillaries by nailfold capillary microscopy examination, w -1 2 suggestive of SLE with scleroderma features. Almost 40% of the patients with UI-nRNP autoantibodies in one study had anti-ul-snrna, but no sera contained the Sm serotype. ]m Oscillating

10 332 VON MUHLEN AND TAN Table 1: Autoantibodies in Systemic Lupus Erythematosus Autoantibody Clinical Associations IIF Pattern in to and Prevalence HEp-2 Cells dsdna Sm 40%-70% in active disease, nephropathy, CNS involvement; marker antibody 15%-30% of all cases; CNS symptoms, nephropathy (?); marker antibody Homogeneous, nucleoplasmic Coarse speckled; nucleoplasmic UI-nRNP SS-A/Ro SS-B/La Histones Nibosomal RNP 30%-40% of all cases; Raynaud's phenomenon; MCTD 24%-60% of all cases; subacute cutaneous lupus (70-90%); photosensitivity; neonatal lupus (>90%); C2 and C4 deficiencies (90%) 9%-35% of all cases; neonatal lupus syndromes (75%) > 95% in drug-induced lupus; SLE (50-70%); RA (5-14%, in low titers); pulmonary fibrosis in systemic sclerosis; rare in UCTD 56%-90% in cerebritis with psychosis; 10-20% of all cases Coarse speckled, nucleoptasmic Fine speckled nuclear; ANA negative in some substrates Fine speckled; nucleoplasmic Homogeneous, nucleoplasmic Dense cytoplasmic; homogeneous nucleoli PCNA 3% of all patients Polymorphic, cell-cycle related phospholipids 24%-26% in unselected SLE patients; throm- Negative Ku bosis, recurrent fetal loss, thrombocytopenia, livedo reticularis, hemolytic anemia 1%-19% of all patients; marker antibody for PM/scleroderma overlap (26%) in orientals Homogeneous nuclear and nucleolar ssdna 70% in active disease Negative HMG-17 34%-70% in active disease, nephropathy Not described hnrnp protein A1 hsp 90 31%-37% of all cases; Raynaud's phenomenon, esophageal dysmotility 5%-50% of all cases Distinct coarse speckled nuclear Not described Abbreviations: CNS, central nervous system; CTD, connective tissue disease; HMG, high mobility group; hnrnp, heteronuclear ribonucleoprotein; hsp, heat shock protein; IIF, indirect immunofluorescence; JRA, juvenile rheumatoid arthritis; MCTD, mixed connective tissue disease; MW, molecular weight; PCNA, proliferating cell nuclear antigen; PM, polymyositis; RA, rheumatoid arthritis; RNP, ribonucleoprotein; SS, Sj6gren's syndrome; UCTD, undifferentiated CTD; WB, Western blot, immunoblot. titers according to the disease activity were also observed in nine patients. 1 3 Antibodies to SS-A/Ro and SS-B/La Four small RNAs (by1, hy2, hy3, and by5), associated with either of three proteins, 60 kd SS-A/Ro, 52-kD SS-A/Ro, or 48-kD SS-B/La, form the antigenic target in this system. 1 4,1 5 SS-B/La seems to work as a termination factor for RNA polymerase III transcription, but the function of SS-A/Ro is unknown. 1 6 Originally described as having only the 60-kD component, 1 7 with a putative zinc finger and a RNAbinding consensus motif, 1 8,1 9 the SS-A/Ro autoantibodies also recognize a different component of 52 kd described in our laboratories. 1 4 The 52-kD protein sequence has putative zincfinger domains, which are regions that bind

11 AUTOANTIBODIES IN RHEUMATIC DISEASES 333 Table 1: Autoantibodies in Systemic Lupus Erythematosus (Cont'd) MW in Comments WB {kd) References High avidity Abs related to nephritis; low avidity Abs correlate with CNS involvement Core proteins B (28), B' (29), D (16), E (13), F and G of 13/16/28/29 U1, U2, U4, U5, and U6 snrnps; active in processing of pre-mrna transcripts Proteins 33(A) and 22(C)kD, unique to UI-nRNP; pro- 22/33/70 tein 70 kd associated with UI-nRNP; pre-mrna processing In most cases associated with anti-ssb/la; also occurs 52/60 in 3%-10% of RA patients and in the majority of patients with SS!,53 1,60-64,75 60,61,64,74-77,99 75,113,118,314 In most cases associated with anti-ssa/ro 48 1,75,118,315 H1, H2A, H2B, H3, H4, H5, [H2A-H2B]-DNA dimer are main antigens; role in the organization of nucleosomes Phosphoproteins P0, P1, and P2, active in the elongation step of protein synthesis; no association with disease activity, cognitive impairment or other psychiatric symptoms Auxiliary protein of DNA-polymerase Highly associated with the anticardiolipin syndrome (primary or secondary) and antiphospholipid antibodies Known as p70/p80; bind directly to DNA in vivo and in vitro; also occurs in primary pulmonary hypertension (23%) Nonspecific (>80% in drug-induced lupus and other CTDs) Also found in primary SS (11%), MCTD (10%), RA, and normals (4%); antigens associated with active chromatin Also occurs in MCTD, RA, and in 7% of normals Also found in PM (33%) and SS /16/38 1, ,126,162,164,165,316 1,128,129, , ,121,137, ,145,150,317, /80-86!,8,121, ,165, , , ,175, ,185,320,321 zinc, and a leucine zipper motif, which is a region containing repeating leucine motifs apposed to each other, in the amino terminal half. The sequence is highly similar to human ret transforming protein, as well as to mouse T cell rpt-1 protein, l~ The SS-B/La peptide, on the other hand, does not share epitopes with the SS-A/Ro proteins, and the cdna sequences have no homology, m-h5 Main clinical associations of the SS-A/Ro system in SLE are with subacute cutaneous lupus erythematosus, lupus of homozygous complement C2 and C4 deficiencies, and the neonatal lupus syndrome. ~,1 7,116 Transient or permanent manifestations of the neonatal lupus syndromes include congenital heart block, photosensitive skin lesions, cytopenias, and liver inflammation. Hv The risk of having a child with the syndrome is higher if the mother has both antibodies to SS-A and SS-B, and if the SS- A/Ro reaction is also directed to the 52-kD antigen by WB. The risk is lower if the mother has isolated anti-ss-a/ro in low titer detected by EL1SA and not by WB. 118 It is important to emphasize that commercially available ELISAs for these snrnps do not distinguish between

12 334 the 52- and the 60-kD components. In a recent retrospective study from Finland, mothers of children with isolated congenital heart block had more subclinical primary SS rather than SLE features.~19 Some comments are necessary with regard to ANA-negative SLE, found in 1% of patients with active disease. Many of the cases could display a limited repertoire of antibodies targeting phospholipids or ssdna, with negative IIF. But the association with SS-A/Ro autoantibodies in the majority of patients, as determined by double immunodiffusion or WB, in the context of negative antinuclear antibodies (ANA) in IIF, seems to reflect a washout of the antigen during the cell fixation and permeabilization steps for IIF. The consequence is a negative IIF assay. Additionally, some cell lines do not express the antigen in sufficient amount to be detected by the IIF technique, ~2 as can be the case with liver cell imprints, which are still widely used in many clinical laboratories. Antibodies to Histones Antibodies to all classes of mammalian histones occur in SLE and in drug-induced lupus syndrome. 52Jzv124 Common lupus inducing drugs are hydralazine, procainamide, isoniazid, and chlorpromazine, although many others have been implicated. For detailed lists see references 122, 123. One main target of the histone antibodies is the [H2A-H2B]-DNA complex, but different drugs may induce antibodies targeting different histones (Table 2). Another interesting feature of these antibodies is their probable role in the LE cell phenomenon induction in vitro, as previously shown with antibodies to DNA (DNA-histone complexes) Probably the most reliable laboratory test to detect antihistone antibodies is the ELISA technique using purified antigens. 124 The IIF test in the presence of antihistone antibodies shows the same pattern as with antibodies to dsdna (eg, homogeneous nucleoplasmic staining of interphase cells and homogeneous chromosomal staining in dividing cells). There is no particular clinical association of antihistone antibodies in SLE or juvenile SLE, with the possible exception of a correlation between disease activity and high antibody levels. ~24 VON MOHLEN AND TAN Table 2: Distinct Antihistone Antibodies in Drug-Induced Lupus Inducing Drug Associated Antibody Specificity (Prevalence) Procainamide ~ (95%), H1 and other individual histones Hydralazine [H2A-H2B]-DNA complex (35%), individual histones c~-methyldopa H1, [H2A-H2B]-DNA complex Isoniazid [H2A-H2B]-DNA complex D-Penicillamine [H2A-H2B]-DNA complex Quinidine [H2A-H2B]-DNA complex (58%), individual histones Acebutolol [H2A-H2B]-DNA complex Carbamazepine Total histones Timolol eye drops [H2A-H2B]-DNA complex Data from Fritzler et al, 123 Rubin, TM and Rubin eta[. 322 Anti-rRNP and Other Autoantibodies in Neuropsychiatric Lupus Some 10% to 20% of unselected SLE sera show a dense fine speckled or homogeneous cytoplasmic pattern in IIF ~ (Fig 4C), sometimes in high titers, corresponding to three ribosomal phosphoproteins (rrnp) of 15, 16, and 37 kd in WB assays. The same C-terminal peptide is antigenic in the three proteins, and any affinity purified antibody from each of the immunoblotting bands was able to react with the other two antigens in one study. 12s Association of antirrnps with major central nervous system disorders in SLE, mainly psychotic symptoms, was reported some years ago in a retrospective study. 129 The antibody levels increased before and during periods of active psychosis, independently of dsdna antibodies, sepsis, or any other disease manifestation. This study was later confirmed in other series, 13 J31 where IgG and IgM anti-rrnp were significantly associated with neuropsychiatric disorders, but not with cognitive impairment or disease activity./32'133 Previous associations with depression were not confirmed, because the antibodies did not differentiate the groups with neurological and psychiatric manifestations. ~32 Matsunaga et al t34 recently reported patients with SLE and central nervous system manifestations as typically having low IgM serum levels and low titer of antimyosin antibodies, as seen by ELISA. Significant differences in the prevalence of anti-

13 AUTOANTIBODIES IN RHEUMATIC DISEASES 335 rrnp is found among three races: 38% of Malaysian Chinese, 13% of English, and 20% of Afro-Caribbean patients with SLE were positive in a recent survey. 135 Many discrepancies still exist in the literature regarding the clinical associations of anti-ribosomal rrnp antibodies in SLE. 136 Autoantibodies to surface neuronal antigens are seen in SLE patients with signs of diffuse central nervous system involvement, but are also detected in rheumatoid arthritis (RA) and in lupus patients without central nervous system disease531 Other antibodies considered important in central nervous system SLE are the antiphospholipids discussed below. PCNA Autoantibodies Previously also called cyclin and involved in cell cycle regulation, this intranuclear system blots in the region of 36 kd 137,138 and is detected in 3% of unselected SLE patients. 1 The antigen, DNA-polymerase delta auxiliary protein, is essential for leading strand DNA replication. 139,14 It gives a polymorphic nuclear pattern in IIF studies, corresponding to different phases of the cell cycle, because it associates with different sites of the cell nucleus where DNA is being replicated. ~21,137 The expression of this highly conserved protein may be increased by a 3-fold factor in late G and early S phase. Accordingly, there is a strong and variable staining in S-phase and weak or negative staining in Go or G1 phases in IlF. 137,138 Proliferating cell nuclear antigen (PCNA) autoantibodies are rarely found in other diseases. Other cell-cycle related antigens were shown to be targets of human autoantibodies, but with no predominant disease or disease subgroup associations; these antigens are anti-dividing cell antigen (anti-dca), 141 nuclear mitotic-associated protein (NuMA), 142 and a 55-kD protein. 143,144 Antiphospholipid Antibodies Antiphospholipid antibodies are found by ELISA in 26% of unselected SLE patients 145 and in 41% of those seen in a tertiary center? 46 Cardiolipin is the preferred antigen. The lupus anticoagulant consists of a different population of autoantibodies, H7 detected by activated partial thromboplastin time prolongation, a functional assay. Both systems are clinically associ- ated with multiple thrombotic events. Antiphospholipid antibodies, most commonly of the IgG2 or IgG4 subclasses, target all negatively charged phospholipids, with an unknown mechanism of action. 148 The binding is dependent on the presence of a serum cofactor identified as 132- glycoprotein-i, 149 giving rise to the speculation that the antibodies are targeting a complex of anionic phospholipid/132-glycoprotein or a "hidden" epitope exposed by the interaction. Repetitive spontaneous abortions, thrombocytopenia, Coomb's positive hemolytic anemia, livedo reticularis, and many types of venous or arterial thrombosis are described in association with antiphospholipid antibodies and the lupus anticoagulant, a46,~48, whether or not a diagnosis of SLE is made. The clinical associations hold true for antibodies of the IgG isotype and not for IgM. In the central nervous system, strokes, dementia due to multiinfarcts, and chorea were reported. 155 General psychiatric disorders had no association with antiphospholipid antibodies in an unselected and consecutive group of 164 lupus patients. 145 On the other hand, patients with Sneddon's syndrome (characterized by extensive livedo reticularis with multiple ischemic cerebrovascular episodes) had antiphospholipid antibodies. 156 Many patients with antiphospholipid antibodies do not show clinical criteria for SLE or any other autoimmune disease, even after years of follow-up; therefore a primary antiphospholipid syndrome was described by three different groups of investigators in Patients are negative or have low titers of ANA, display high titers of antiphospholipid antibodies, and present with the same spectrum of thrombotic events as SLE patients. On the other hand, patients with antiphospholipid syndrome secondary to SLE have higher frequencies of heart valve disease, hemolytic anemia, low serum complement, and neutropenia. 16 One report cited a strong association between titers of anti-dsdna, as detected by the Farr assay, and a positive direct Coomb's test with raised anticardiolipin antibodies.151 Antibodies to Ku The anti-ku system was described in 1981 in Japan. 161 The antibodies occur most typically in the PM-scleroderma overlap (55% in Japanese

14 336 VON MOHLEN AND TAN patients), in overlap syndrome of SLE-PMscleroderma, in SLE (1% to 19%), and in scleroderma alone (1% to 14%) Racial differences seem to be responsible for the fact that North-American patients do not display the association of Ku antibodies with overlap syndromes, but do have an association with the HLA-DQwl allele (89% of anti-ku positive patients) and with the presence of Sm autoantibodies. 163q65 No specific clinical association was reported in SLE. There was an earlier confusion in the literature regarding the Ku and Ki systems because of the serum used as prototype, a,~64 This issue was clarified lately, confirming that both systems are distinct. Anti-Ku antibodies immunoprecipitate two proteins of 66 and 86 kd, with different structure and function. Western blots in our laboratory consistently display the reactivities in the regions of 60 to 66 and 80 to 86 kd (Fig 6). The antigens can be localized in the nucleus and nucleoli of interphase cells, giving a homogeneous pattern in the IIF of HEp-2 cells. The two noncovalently linked proteins form an heterodimer that binds DNA, with a possible role in transcriptional activation, DNA replication, and cell proliferation. Such cellular functions and B cell epitopes of Ku were recently reviewed by Chou et al. 121 Griffith et al ~66 cloned the p70 protein demonstrating several copies of homologous DNA sequences in the human genome and bringing new evidence that multiple genes are responsible for a family of Ku(p70) proteins. Interestingly, Ku antibodies were recently detected in 23% of patients with primary pulmonary hypertension, a disease often associated with Raynaud's phenomenon, ANAs, and pulmonary vasculitis. In the control population, comprised of patients with secondary pulmonary hypertension, the prevalence was 4%. 267 High Mobility Group Antibodies The high mobility group (HMG) chromosomal nonhistone proteins were well characterized in the early 1980s, 168 being a component of nucleosomes from actively transcribed genes with unknown function. A recent study investigated the associations between the presence of antibodies to HMG-17 and clinical and serological SLE features. 169 Greek patients with active disease had a higher prevalence of the antibodies, showing good correlation with anti-dsdna levels and a negative correlation with the complement component C4. The usefulness of HMG-17 antibody determinations for the differential diagnosis of the rheumatic diseases is limited, because some of them display closely similar prevalence: 11% of primary SS patients, 4% of RA patients, and 4% of normal blood donors in Greece based on a newly developed ELISA assay. 169 In another study, 39% of ANApositive children with juvenile RA displayed high titers of anti-hmg-1/2.17 In SLE and MCTD, antibodies to HMG-17 can be found in 34% and 10% of the cases respectively. 169,171 Antibodies to hnrnp-a1 Montecucco et a1172 recently showed that antibodies to heteronuclear RNP (hnrnp) protein A1 are associated with Raynaud's phenomenon and esophageal dysmotility in SLE patients, as determined by an ELISA using recombinant A1 protein. The findings were independent of UI-nRNP antibodies, because patients positive or negative for anti-hnrnp-a1 showed the same frequency of UI-snRNP autoantibodies. No correlation was found with antidsdna antibodies or complement levels, but patients presenting with anti-hnrnp-a1 tended to be older at the time of diagnosis in that small series of patients, lv2 The same investigators previously reported that in SLE the antibodies react with the A1 protein N-terminal domain containing an RNA-binding region, 173 but that in RA many of the reactivities are due to anti-keratin antibodies cross-reacting with homology region in the A1 protein C-terminal portion. The RA33 nuclear antigen in RA, seen in 35% of the patients in one series, was reported to be identical to the A2 hnrnp, pointing to the fact that hnrnp proteins may be highly antigenic. 174 In another study, 37% of SLE patients produced anti-a1 antibodies, as opposed to 7% of normal controls. 175 These interesting findings, to be confirmed in larger series of patients, seem to point to a subgroup of SLE patients with scleroderma features characterized by anti-a1 antibodies. Fritzler et a1176 had already found that all 32 patients with MCTD and UI-nRNP autoantibodies in their

15 AUTOANTIBODIES IN RHEUMATIC DISEASES 337 series also had antibodies targeting hnrnp/ RNA. Other Autoantibodies, Ki, Lamins, hsp 90, Adenosine Diphosphate-Ribose Polymerase, and Others Less Frequently Seen In 1981 Tojo et a1177 described Ki-antibodies in 10% of SLE patients associated with synovitis, pericarditis, and pulmonary hypertension. The system was more recently found by ELISA in 19% of SLE patients and was thought to be a useful marker for disease activity. 17s Another group reported a 21% prevalence by ELISA using highly purified antigen, with a higher frequency of lupus cerebritis in positive patients. 179 The autoantibody could be shown in 8% of the patients by double immunodiffusion. Ki is a 32-kD protein that may exist in a polymer of identical subunits reaching 224 kd. The sicca lupus (SL) system, described by Harmon et al in 1981,18 is probably identical to the Ki system, ~8~ but its characteristics were not yet clearly defined in the literature. The name came from the frequent clinical association found with SLE and with sicca features sometimes associated with SS. Autoantibodies to Ki are nonspecific, being present in many rheumatic diseases (7% in scleroderma, 7% to 19% in PM/scleroderma overlap syndrome), Hashimoto's thyroiditis, isolated autoimmune thrombocytopenia, and idiopathic interstitial lung disease. 8,18 Lamins A, B, and C, with molecular weights of 70, 68, and 60 kd respectively, are major components of the inner nuclear membrane, acting as anchoring sites for chromosomes in interphase cells, and providing an architectural framework for the nuclear envelope. 121,182 High titers of antibodies to lamins A, B, or C were reported in patients with some lupus features, but that did not fulfill diagnostic criteria for SLE. 2,25-27 Patients had autoimmune hepatitis with cutaneous or brain vasculitis, circulating lupus anticoagulant or antiphospholipid antibodies, and thrombocytopenia or hemolytic anemia responsive to corticosteroids in one series from France. 26 Thirty-one patients from Denmark positive for antilamin antibodies also showed a correlation with antiphospholipid antibodies and nonerosive arthritis. 25 Another study in which patient selection was made on the basis of a positive peripheral fluorescent ANA pattern on HEp-2 ceils, did not show any relationship with specific clinical or laboratory signs, a7 There are descriptions of the presence of antibodies to glycoprotein 210, the major glycoprotein of the nuclear pore, and to different lamins in PBC and in autoimmune hepatitis. ~s,29,32a83 It seems that associated chronic liver conditions are a common denominator when lamin autoantibodies are detected in many diseases. 3 Only one patient is reported in the literature with autoantibodies to lamins and a diagnosis of linear scleroderma.184 Antibodies to the heat-shock protein 90 are present in 5% to 50% of unselected SLE patients, but also were described in 33% of patients with idiopathic PM in one study. 185 Antibodies to adenosine diphosphate (ADP)-ribose polymerase were identified in a heterogeneous group of patients, including some connective tissue diseases. 1 The antigen of 116 kd is involved in the ADP ribosylation of nuclear proteins and is a DNA-binding protein. Antibodies to Alu RNA protein recognize a 68-kD antigen, and were seen in 14% of SLE patients in the original series, ls6 A small ribosomal subunit protein of 20 kd, called S10, may be found in 11% of SLE patients, as characterized by two-dimensional gel electrophoresis and WB. ls7 A direct correlation with the presence of anti-sin and anti-rrnp autoantibodies was shown. The system seems to be specific, because only 1 patient with primary SS was also positive in this particular study. Prothymosin-c~ from goat spleen extracts was purified by highpressure liquid chromatography and used in an ELISA assay, lss The investigators could show antiprothymosin-a autoantibodies in 18% of 44 SLE sera compared with 1.8% in controls, correlation with the presence of dsdna autoantibodies, and no association with disease activity. SYSTEMIC SCLEROSIS With the help of modern immunological assays, autoantibodies can be detected in more than 95% of patients with scleroderma],4 although those autoantibodies seen in SLE are rarely detected. For unknown reasons, the nucleolus seems to play a very important role in driving the antibody response in scleroderma, because all major autoantigens in the disease,

16 338 VON MOHLEN AND TAN Table 3: Autoantibodies in Systemic Sclerosis Clinical Associations IIF Pattern in Autoantibody to and Prevalence HEp-2 Cells DNA topoisomerase I (Scl-70) Centromere PM-Scl Fibrillarin (U3-RNP) RNA-Pol I RNA-Pol II RNA-pol III UI-nRNP HMG-17 Ku 20%-59% of all patients; 70%-76% in diffuse form; 13% in CREST; 12% in PM/scleroderma overlap; severe systemic disease, poor prognosis; acroosteolysis; less prevalent in Caucasians 57%-82% in limited scleroderma (CREST); 25% in primary Raynaud's phenomenon; 8% in diffuse form; better prognosis 2%-5% in scleroderma; 24% in PM/scleroderma overlap; DM-like skin changes, sclerodactyly, Raynaud's phenomenon, machinist's hand, calcinosis, telangiectasia 6%-8% of all patients; 5% in diffuse form (mainly in blacks); 10% in CREST; skeletal muscle and small bowel involvement; pulmonary hypertension 4%-20% in scleroderma; 13% in diffuse form 4% in scleroderma; diffuse form 23% of all patients; 6% in limited; 45% in diffuse form 2%-5% in scleroderma; 24% in PM/scleroderma overlap; 95%-100% in MCTD Limited scleroderma (CREST); correlated with anticentromere antibodies 1%-14% in scleroderma; 26%-55% in PM/scleroderma overlap Homogeneous nuclear and speckled nucleolar, homogenous chrom in metaphase Diffuse speckled nuclear; centromeres in metaphase Homogeneous nucleolar and nuclear Clumpy nucleolar Speckled nucleolar Speckled nuclear, chromosomes homog in metaphase Nuclear staining Speckled nuclear Not described Homogeneous nuclear and nucleolar Th/To (7-2 RNP) 4%-10% of all patients; 8%-19% in limited, 1%-11% in diffuse form; 0%-3% in PM/scleroderma overlap; 3% in primary Raynaud's phenomenon; rare in SLE Nucleolar staining NOR-90 Rare Speckled nucleolar Alu RNA-protein Rare Not described Wa 3% in scleroderma; associated with sec- Not described ondary SS Abbreviations: CREST, calcinosis, Raynaud's phenomenon, esophageal motor disturbances, sclerodactyly, and telangiectasias. DM, derrnatomyositis; IP, immunoprecipitation; kd, kilodaltons; PBC, primary biliary cirrhosis; RNP, ribonucleoprotein; SS, Sj6gren's syndrome; SLE, systemic lupus erythematosus; WB, Western blot, immunoblot; RNA-Pol, RNA polymerase. NOTE. The IIF pattern observed with mouse anti-rna-polymerase II monoclonal antibodies is not usually the same as the one seen with human sera, because the later also decorate the nucleoli because of the presence of RNA-polymerase I antibodies. 247 even centromere-associated proteins) ~9 were shown to be associated with the nucleolus. No specific subset of autoantibodies seems to identify patients with either morphea, linear scleroderma, or scleroderma symptoms associated with silicone breast implants, s, but autoantibodies against histones H1 and H3 were major antigens in a recent study of Japanese patients with localized forms of scleroderma, as shown by ELISA and WB5 96 In primary Raynaud's phenomenon, the absence of ANA is also of diagnostic significance3 97 Table 3 shows the main autoantibodies detected in systemic sclerosis, recently reviewed by Fritzler. s

17 AUTOANTIBODtES IN RHEUMATIC DISEASES 339 Table 3: Autoantibodies in Systemic Sclerosis (Cont'd) MW in Comments WB (kd) References May be associated with cancer developing in scleroderma patients; the enzyme unwinds DNA; marker antibody ,8, ,202,204,205, , 213,216,224 Major centromere proteins A (15-16 kd), B (80 kd), and C (120 kd); also occurs in PBC; less prevalent in blacks 8% in PM; HLA-DR3, -DQw2 associated 15-16/80/120 75/100; 20to 110 in IP 8,30,204, ,218,221~,224 8,210,216,228,230, Protein of the U3-RNP particle; the antibodies rarely coexist with anticentromere and anti-topo I in the same patient 34 5,6,8,9,210,216,239,240 Transcripts rrna precursors Transcripts mrna (hnrna) Transcripts 5S rrna, trna, other small RNAs Proteins 33 (A), 22 (C) kd, 70 kd Also found in primary SS, MCTD, SLE, RA, and normals Also in primary (23%) or secondary (4%) pulmonary hypertension; SLE (1%-19%) 40 kd protein complexed with 7-2 and 8-2 RNAs; in limited scleroderma associated with reduced survival Complex ofllto 210 kdinip / / inlP 80/135/160 kdinlp 22/33/ / kd, detected in IP 6,8,196,210,216 5,7,249 7,10 8,76,77 8, ,319 1,8, ,167,216 6,8,10,216, Also in SS (4%) and other diseases; dou- 89/93 btet in WB Protein complexed with Alu RNA 68 Protein associated with trna ,253, ,323 Antibodies Targeting DNA- Topoisomerase I Antibodies to DNA topoisomerase I (antitopo I), also known as Scl-70,198 are generally associated with diffuse cutaneous systemic sclerosis. 8J99,2 Besides presenting widespread skin sclerosis, patients have more important and earlier organic manifestations, such as renal crisis, interstitial lung disease, and intestinal involvement. Racial differences in the prevalence of topo I autoantibodies were recently emphasized, because they were detected in 26% of Australians and in 76% of a Thai population, all with widespread skin involvement characteristic of diffuse cutaneous systemic sclerosis. 201 As was shown for anticentromere antibodies, topo I autoantibodies may be present in patients with Raynaud's phenomenon before systemic sclerosis development, =02 constituting a useful tool for the clinician in early diagnosis. Titers tend to remain stable, and in one particular study they remained so for up to 16 years of follow-up. = 3 By WB, the specificity of topo I

18 340 VON MUHLEN AND TAN autoantibodies is 93%, inasmuch as they are rarely found in SLE and MCTD patients. 2 4 An association of topo I autoantibodies with malignancies in patients with scleroderma was suggested.205, 206 In 1986, three different groups independently reported that the antigen recognized by anti-scl 70 positive sera was DNA-topo I, an enzyme involved in relaxation of supercoiled DNA. It is also localized in the nucleoplasm, nucleolus and nucleolar organizing regions (NOR), 2 7 a fact that explains the common finding of speckled nucleolar decoration in IIF, which is superimposed on a densely speckled nucleoplasmic pattern. 2a The protein is recognized in WB assays at 95 or 100 kd, depending on the gel conditions used, with a degradation product reacting at 70 kd. 6,202,208 One immunodominant B cell epitope (epitope III) in the carboxytermihal region of topo I was recently described. 2n Anticentromere Antibodies The centromeric proteins are localized at the inner and outer kinetochore plates. They interact with the mitotic spindle apparatus via its microtubules when the cell enters division. In 1980, researchers in our laboratory showed the presence of antibodies targeting the kinetochore in sera of scleroderma patients with CREST syndrome, without reacting with the centromeric heterochromatin. 212,213 Since then, the main clinical association of the autoantibodies has been consistently shown with the limited form of systemic sclerosis, classically associated with a more benign and protracted eourse. 199,206, Nevertheless, a recent report described anticentromere antibodies and presence of sclerodactyly as the only predictors of severe digital ischemia. 217 The digital ischemic events were not related to disease duration, smoking habit, duration of Raynaud's phenomenon, or patient's age. Other clinical associations in the literature are with arthralgias, Raynaud's phenomenon, telangiectasias, lung involvement, and a significantly younger age at disease onset. 214 In IIF using HEp-2 cells as substrate, 50% to 70% of patients with limited scleroderma (CREST variant) may be anticentromere positive, 6,213 and 25% of those with primary Raynaud's phenomenon are positive. 218 This second group of patients most probably represent a subset destined to develop limited scleroderma or a forme fruste of the disease, because they also display definite changes in the nailfold capillaries by capillary microscopy examination. 218,219 Anticentromere antibodies are frequently seen in PBC, but other patients may rarely test positive in IIF and WB, such as patients with isolated pulmonary hypertension, patients with other connective tissue diseases (mainly SLE, primary SS, or undifferentiated connective tissue disease), and patients without apparent disease (0.08% of female blood donors not clinically evaluated).41,44,214,220 The antibody titers in such situations will usually be low. In WB assays of whole cell extracts, 3 bands are consistently recognized: 16 kd (centromere protein [CENP]-A), 80 kd (CENP-B), and 120 kd (CENP-C). 22~,222 In our experience, it is a rare serum that will present concomitant anticentromere and anti-topo I activities. The association of autoantibodies to centromere and total histones, on the other hand, was recently suggested as defining a subset of scleroderma patients with severe pulmonary or vascular disease in a retrospective study. 223 In any case, the prevalence seems dependent on racial differences, because a study showed that anticentromere antibodies could be detected in 36% of whites but in only 4% of American blacks. 224 The opposite was found for topo I autoantibodies, with American blacks displaying a higher frequency than whites. This interesting phenomenon holds true even among distinct ethnic subsets of whites. 225,226 PM-Scl Antibodies PM-Scl antibodies give a characteristic IIF pattern of strong homogeneous nucleolar fluorescence on a weak homogeneous nucleoplasmic staining. They typically occur in the PM/ scleroderma overlap syndrome, 227,228 but they also occur in PM or scleroderma alone. The specificity was known as PM-I when first described in 61% of PM/DM patients in In later studies, it became clear that the PM-1 system actually encompassed the anti-pm-scl and many other autoantibodies. The antigen is localized in the granular component of the nucleolus. 23 Using immunoprecipitation, the PM-Scl antigen comprises more than 10 pep-

19 AUTOANTIBODIES IN RHEUMATIC DISEASES 341 tides, with molecular weights ranging from 20 to 110 kd. 23 In immunoblots, two bands of 75 and 100 kd are the reactivities most commonly found. Recently, cdna clones were identified for both peptides, with the 100-kD peptide sharing some aminoacid sequence homologies to serine/threonine protein kinases in different species. 232 Nearly all patients with PM-Scl autoantibodies present the haplotype HLA-DR3, HLA- DQw2. 228' Oddis et al, 228 in a study of 617 patients with connective tissue diseases, found 4% of the cases positive for anti-pm-scl by IIF and Ouchterlony double immunodiffusion. The antibodies identified a subset of patients with myositis, limited scleroderma, or an overlap of both diseases. Calcinosis (39%), Raynaud's phenomenon (65%), and arthritis (82%) were frequently seen in the anti-pm-scl group of patients, but only calcinosis and arthritis were significantly more prevalent than in all other patients negative for PM-Scl. The survival rate was 100% in 10 years, and no serious visceral involvement was noted, conferring a prognostic significance to PM-Scl antibodies. The same good prognosis was attested by another study in which 50% of the anti-pm-scl positive patients were on minimal or no immunosuppression after 8 years of follow-up. 236 Although primarily associated with myositis and described as nonpruritic, hyperkeratotic eruption with scaling, fissuring, and hyperpigmentation, 237 the socalled machinist's hands have been increasingly described in patients with PM-Scl antibodies. 228,234 Whether renal involvement is more prevalent in the presence of PM-Scl autoantibodies is debatable. 2a,234 Antibodies to Other Nucleolar Antigens: Fibtillarin (U3-snRNP) and Th / To Fibrillarin is probably involved in the maturation of pre-rrna, formation of ribosomal subunits, and ribosome assembly. It is an important member of the nucleolar snrnps and a component of the coiled bodies, which are prominent intercromatin structures probably involved in the processing and transport of pre-mrna and pre-rrna. 6,23s The name fibrillarin emerged from the antigen localization in the dense fibrillar component of nucleoli by electronmicroscopy. 239 Anti-U3-snRNP autoantibodies were detected only in scleroderma patients in a recent large series of rheumatic patients 162 and are mostly seen in young men with systemic sclerosis, with lower frequency of joint involvement. 21 In only one report, 3 patients with the antibody had diffuse cutaneous systemic sclerosis and multiple organ involvement, with widespread skin telangiectasia, but two patients also had psoriasis. 24 Another study found fibrillarin autoantibodies to be frequent in black patients with diffuse cutaneous systemic sclerosis and skeletal muscle and small bowel involvement. Pulmonary hypertension was seen in patients of all races, but diffuse cutaneous systemic sclerosis was not an association in whites. 5 In the control group, just 1 patient with primary Raynaud's phenomenon had the antibody. Antifibrillarin antibodies are rarely seen in other diseases. 6,241,242 Patients with diffuse cutaneous systemic sclerosis have the same survival rate at 5 years (around 70%), independent of the presence or absence of U3-snRNP autoantibodies. 5 The Th/To systems, independently describ ed 243"245 but later shown to be the same autoantibodies, are present in 10% to 19% of patients with limited scleroderma, in 11% of patients with diffuse cutaneous systemic sclero.. sis, and in 3% of patients with primary Raynaud's disease. 6,216,246 The original description of Th was in association with anti-ss-a/ro autoantibodies, in a patient with SLE. 243 A greater prevalence of puffy fingers, small bowel involvement, and hypothyroidism was noted in one series involving 371 consecutive systemic sclerosis patients, together with a lower frequency of arthralgia or synovitis. 246 In the same series, the cumulative survival rate was lower at 10 years in the anti-th positive group because of 4 deaths from pulmonary hypertension. Hirakata et ap 62 examined 91 patients with inflammatory muscle diseases and 254 controls with other rheumatic diseases: only 6 patients were anti-th/to positive, 5 with systemic sclerosis alone and 1 with PM/scleroderma overlap. No patient with isolated PM/DM was positive. Immunolocalization data showed that the 7-2 RNP is probably involved in ribosome biogenesis (assembly, maturation, and/or transport of preribosomes), because the particles could be traced to the granular component of the nucleolus. 247

20 342 VON MUHLEN AND TAN Antibodies to the RNA Polymerases The three known RNA polymerases in eukaryotes catalyze the transcription of genes from a DNA template into RNA. 248 RNA polymerase I is localized in the nucleolus, and its transcripts consist of large rrna precursors. RNA polymerases II and III are in the nucleoplasm, with mrna precursors (hnrna) being transcribed by RNA polymerase II, and 5S rrna, trna, and other small RNAs being transcribed by RNA polymerase III. The antigenic epitope in RNA polymerase II could be traced to the highly charged, heavily phosphorylated, and repetitive carboxyl terminal domain of the large 220 kd subunit. 249 Autoantibodies to the RNA polymerases tend to occur together in the same patient, seem to be very specific for scleroderma, and mostly identify individuals with diffuse cutaneous systemic sclerosis. 1,21,2a6,249 There is a high prevalence of internal organ involvement, mainly heart and kidney, renal crisis, and a poor survival rate at 5 years of follow-up. 7 Other characteristics are male with older age onset, less peripheral vascular disease, and lower frequency of interstitial lung disease. Autoantibodies reactive with RNA polymerase I or II could be detected by immunoprecipitation in 66 and 13 of 278 scleroderma cases (24% and 5% respectively), but in none of 247 controls, including SLE, PM/DM, MCTD, and normal patients. 249 Of the 13 anti-rna polymerase IIpositive patients, 10 had diffuse cutaneous systemic sclerosis and 3 had a limited form. In another study, antibodies to RNA polymerase I, as detected by immunoprecipitation, were positive in 13% of patients with diffuse cutaneous systemic sclerosis. 2a6 Autoantibodies to RNA polymerase Ill were reported to be more prevalent than topo I autoantibodies in diffuse cutaneous systemic sclerosis in a series of 252 consecutive scleroderma patients from the same group of researchers. 1 RNA polymerase III autoantibodies were detected in 45% of diffuse cutaneous systemic sclerosis patients, in 6% of the ones with limited disease, and in no case with scleroderma overlap syndrome. The autoantibodies were related to more extensive skin involvement; however, they were related to lower prevalences of telangiectasias, inflamma- tory myopathy, restrictive lung disease, and severe cardiac abnormalities than patients with topo I autoantibodies. 1 Renal crisis was more frequent than in patients with RNA polymerase I autoantibodies. Anti-RNA polymerase II! autoantibodies were specific for systemic sclerosis in that study, because they were absent in 170 sera from patients with other connective tissue diseases tested as controls. The results need to be confirmed by other investigators from different centers. Ku Antibodies Anti-Ku (p70/p80) are considered marker antibodies for the PM/scleroderma overlap in Japanese patients, with a sensitivity of 26% and specificity of 99%. 162 Such association seems to be different in other populations, since in our laboratory autoantibody to Ku was found in 10% of SLE sera and in none of more than 100 scleroderma or overlap syndrome patients, a Another series from a tertiary center found 14% of anti-ku frequency in scleroderma patients, using immunoaffinity-purified Ku protein in a quantitative WB assay. 163 In that study, the prevalence of antibodies to Ku was the same in the diffuse cutaneous as well as in the limited form, indicating no special clinical association. A trend for better prognosis in the overlap patients displaying Ku autoantibodies has been reported. 8 Ki and UI-nRNP autoantibodies are other systems not infrequently seen in PM/ scleroderma overlap syndrome (7% to 19%, and 13%, respectively). 8 Antibodies Targeting the Nucleolar Organizing Region Work done in our laboratories identified nucleolus organizer region (NOR)-90 antibodies as recognizing the RNA polymerase I transcription factor human upstream binding factor (hubf) in the fibrillar center of the nucleolus. 25 NORs are regions where the nucleolus reforms after mitosis, with clusters of ribosomal RNA genes, and where Scl-70, U3-RNP/ fibrillarin, NOR-90, and RNA polymerase I antigens can be detected. 2sa NOR-90 autoantibodies selectively stain the NOR regions in chromosomes 13, 14, 15, and Rarely found in American patients with systemic sclerosis,

21 AUTOANTIBODIES IN RHEUMATIC DISEASES 343 NOR-90 autoantibodies seem to be frequent in Spanish cases. In another example of divergent racial distribution of autoantibodies The characteristic IIF reading along the metaphase plate in dividing cells shows speckled nucleolar decoration and distinct speckles indistinguishable from the pattern seen with RNA polymerase I autoantibodies (Fig 3D). One recent series from our laboratory found four of eight anti- NOR-90 positive patients with Raynaud's phenomenon, the only frequent clinical association. 254 The specificity of this antigen-antibody system is otherwise still largely unknown. Mitochondrial Antibodies Antimitochondrial antibodies are detected in a significant proportion of patients with systemic sclerosis, although they are more characteristically seen in PBC. 3,255 In a recent study, Chou et a1256 showed that 43% of their scleroderma patients had reactivities to the 70- and/or 50-kD components of the mitochondrial antigens by WB, not recognizing the 39-kD protein that is also a target in sera from PBC patients. Because one third of the patients with PBC also displayed anticentromere antibodies, an association between the two disorders was speculated. Many other investigators had already identified a subset of limited scleroderma patients with concomitant PBC. 253 From 250 sera from systemic sclerosis patients tested by Fregeau et al, % recognized the 70-kD protein associated with the M2 mitochondrial complex, IgG3 being the predominant isotype. Antimitochondrial antibodies are rare in other connective tissue diseases. POLYMYOSITIS AND DERMATOMYOSITIS Almost 90% of patients with PM/DM may show autoantibodies when tested by conventional immunofluorescence in monolayer cells or by the Ouchterlony immunodiffusion assayy 7,258 The best defined specificities are the ones directed to trna-synthetases, a group of cytoplasmic enzymes responsible for the complexing of each aminoacid to its cognate trna1~: histidyl- (Jo-1), threonyl- (PL-7), alanyl- (PL- 12), isoleucyl- (OJ), and glycyl- (EJ), antisynthetases (Table 4). These autoantibodies define a subgroup of patients with PM, arthralgia, and interstitial lung disease, poor response to therapy, and higher death rates than PM patients without the autoantibodies. 12 In addition, some patients also display more synovitis and Raynaud's phenomenon than PM patients without antisynthetase antibodies The prevalence of antisynthetase antibodies may be as high as 37% in PM/DM patients. 16z Subsets of autoantibodies in PM/DM were suggested as a way of better classifying patients with idiopathic inflammatory myopathy in homogeneous groups. 12 Antibodies Targeting Jo-] Certainly the most studied system in the group of antisynthetases is the one with autoantibodies directed to Jo-1 (histidyl-trna-synthetase), formerly known as PL-1. It is present in 20% to 40% of PM patients, the majority also with interstitial lung disease and the markers HLA-DR3/-DRw52, a combination termed the "Jo-1 syndrome. '' IgG1 is the major isotype. 26s At least one study showed a clear correlation of disease activity and levels of anti-jo-1 antibodies, 259 another group reporting that lymphocyte activation CD markers fluctuate with disease activity in a much more marked way in PM patients with Jo-1 autoantibodies. 269 Poor response to therapy, probably in most part due to the associated lung disease, and significantly more flares when tapering drugs are clinical characteristics also observed.~2 A recent report correlated the presence of Jo-1 antibodies to the diagnosis of D-penicillamine-induced myositis in a patient with RA. 27 The antibodies disappeared after cessation of the inducing drug. Nevertheless, it is generally accepted that Jo-1 autoantibodies are highly specific (> 95%) for patients with myositis and interstitial pulmonary fibrosis, commoner in PM than in DM, and rare in children with PM or in other connective tissue diseases. 259,271 The Jo-1 system is thus clinically useful in the differential diagnosis and follow-up of PM patients, sometimes appearing even before the interstitial lung fibrosis onset. 259 PM-Scl Autoantibodies Other antibodies have also helped establish distinct subgroups of myositis patients, such as the anti-pm-scl, 227 which is found in patients

22 344 VON MOHLEN AND TAN Table 4: Autoantibodies in PM/DM Clinical Associations IIF Pattern in Autoantibody to and Prevalence HEp-2 Cells Jo-1 PM, ILD (18%-23% by Ouchterlony, Speckled cytoplasmic PM-Sc[ PL-7 PL-12 SRP EJ OJ Fer KJ Ku Mi-2 Mas SS-A/Ro Ul -nrnp U2-nRNP 36% by ELISA); DM (13%); overlap syndrome (43%); poor response to therapy; high mortality 8%-12% in PM alone; PM/scleroderma overlap (25%) 3%-5% in PM/DM; ILD 3% in PM/DM; ILD 4%-5% in PM/DM patients; occurs in PM, not in DM; severe myositis, resistant to corticosteroid treatment <3% in PM, ILD <3% in PM/DM; ILD <1% in PM/DM < 1% in PM; ILD, Raynaud's phenomenon 5%-12% in PM/DM; PM/scleroderma overlap in orientals 15%-35% in DM; 5%-9% in PM <1% in PM/DM 5%-10% in PM/DM 4%-17% in PM/DM; overlap with SLE and scleroderma (MCTD) 1% in PM/DM; PM/scleroderma Homogeneous nucleolar and nuclear Speckled cytoplasmic Dense speckled cytoplasmic Nucleolus and speckled cytoplasm Cytoplasmic Not described Not described Dense speckled cytoplasmic Homogeneous nucleoplasmic and nucleolar Fine speckled nucleoplasmic Not described Fine speckled nucleoplasmic Speckled nucleoplasmic Speckled nuclearoplasmic 56 kd overlap 87% in PM/DM; 10% in other CTD Not described Abbreviations: CTD, connective tissue disease; ILD, interstitial lung disease; IP, immunoprecipitation; kd, kilodaltons; MCTD, mixed connective tissue disease; PM, polymyositis; DM, dermatornyositis; PL, precipitin line; RNP, ribonucleoprotein; SRP, signal recognition particle; WB, Western blot, immunoblot. with polymyositis alone (8% to 12%) or in a subgroup of patients with overlapping symptoms of myositis and scleroderma (25%), 272 but not in Japanese patients. 1~2 Anti-U2-snRNP antibodies were also related to patients presenting similar overlap features. 162 Antibodies to Signal Recognition Particle A subgroup of adult PM patients reported by Targoff et a1273 and confirmed by Hirakata et al, 162 displayed severe myositis and/or rapid onset of PM, absence of cutaneous features, low frequency or absence of interstitial lung disease, arthritis, and Raynaud's phenomenon, as well as no clinical overlap with other connective tissue diseases. Characteristic of this subgroup and proposed as a marker system is the presence of anti-srp antibodies. Patients, usually black females, show resistance to treatment and higher mortality. 12 In the literature, from a total of 19 patients identified as anti-srp positive until 1992, 18 were diagnosed as PM/DM. 26~a7. 27~ The series of patients with SRP-antibodies are nevertheless too small for definitive conclusions to be drawn. These autoantibodies were earlier reported by Nakao et al, 276 Reeves et al, 274 and Okada et a1275 as occurring in myositis patients. These investigators showed by nucleic acid hybridization and immunoprecipitation that the target is a unique 7SL RNA complex, with no binding to RNA itself. In WB, the antibodies recognized the 54-kD protein, one of six polypeptides with molecular weights from 9 to 72 kd that form this special cytoplasmic particle. 274 Its function is to identify signal sequences of secretory,

23 AUTOANTIBODIES IN RHEUMATIC DISEASES 345 Table 4: Autoantibodies in PM/DM (Cont'd) MW in Comments WB (kd) References Antigen is histidyl-trna-synthetase; HLA-DR3, -DRw52 associated , 259, , 269, 271,324 Complex of 11 to 16 proteins 75/ ,228,272 Antigen is threonymrna-synthetase Antigen is alanyl-trna-synthetase Particle of 6 proteins and 7SL RNA; no ILD thr-trna and 80 kd in IP Multiple trnas in IP, mainly 110 kd ,257,260, ,257, ,261, Antigen is glycyl-trna-synthetase Antigen is isoleucyl-trna-synthetase Elongation factor I~x Unidentified translation factor SLE (1%-19%); scleroderma (1%-14%) Nuclear protein complex, found in calf thymus extract Unidentified trna Commonly associated with SS-B/La Proteins 33 (A), 22 (C) kd, 70 kd Recognizes A',B" snrnps; commonly associated with U1-nRNP RNP component; titers related to disease activity gly-trna and kdinlp 162,261,325, ,326, No WB reactivity 261, / ,163,260 No WB reactivity 261, /60 257,261 22/33/70 76,77, , ,328,329 lysosomal, and membrane proteins, attach to them through the 54-kD protein, and bind their nascent transcripts to specific receptors in the endoplasmic reticulum, regulating the translocation of proteins across the organelle KJ Autoantibodies KJ autoantibodies identify the same clinical group of patients as those with anti-synthetase antibodies, but the antigen is as yet unknown. 28 It is probably not a synthetase due to lack of inhibition of the enzyme activity with the original serum, a general finding when using anti- Jo-1 positive or other antisynthetase sera. Also, antisynthetase antibodies precipitate their cognate trna, a property not found with anti-kj positive sera. 28 Interestingly, the same researchers reported that several patients with antisynthetase antibodies, as well as those only two with anti-kj so far described, display weak anti-ss-a/ro activity. Other Autoantibodies Other antigen-antibody systems described in PM/DM patients are either exceedingly rare (anti-fer, anti-mas), or frequent but nonspecific, like antibodies targeting cytoskeletai components of nonmuscle cells (86% prevalence), skeletal muscle myosin (90%), or myoglobin (71%). 261 Mi-2 antibodies are found almost exclusively in DM patients in all clinical subgroups: adult DM (22%), juvenile DM (14%), overlap syndromes with DM, and malignancy with DM. 261, There is a reported low prevalence of interstitial lung disease 2s3 and an association with HLA-DR Overall, anti-mi-2 are considered the only marker antibodies for

24 346 VON MOHLEN AND TAN DM, but the results need to be confirmed by other investigators. SJOGREN'S SYNDROME Primary SS is characterized by autoantibodies to small nuclear/cytoplasmic ribonucleoprotein particles in more than 90% of the patients (Table 5), and their presence is suggested as one of the main criteria for disease classification. 285 Organ-specific autoantibodies, such as the ones binding to salivary duct epithelium, are also not uncommon. Autoantibodies targeting SS-A / Ro and SS-B / La The Ro system was described in 1969 by Clark et al in sera from patients with SLE. 287 In Table 5: Autoantibodies in SS Clinical Associa- Autoantibody tions IIF Pattern in MW in to and Prevalence HEp-2 Cells Comments WB (kd) References SS-A/Ro 88%-96% in primary Fine speckled In most cases associated 52/60 107, 113, 294, SS; vasculitis; nucleoplasmic; in with SS-B/La; also 296, 314 hypergamma- some substrates occurs in 3%-10% of globulinemia; RF ANA is negative RA patients; HLA-DR3, positive; leuco- -DRw52, -B8 associated penia SS-B/La 71%-87% Fine speckled In most cases associated 48 1,294, 315 nucleoplasmic with SS-A/Ro Ku 20% Homogeneous SLE (1%-19%); sclero / 163 nucleoplasmic derma (1%-14%) and nucleolar Ki/SL 2%-8% in primary SS, also occurs in SLE Speckled nucleo- Nonspecific, also found in , 180 plasmic scleroderma (7%), in PM/scleroderma overlap syndrome (7% to 19%), Hashimoto's thyroiditis, isolated autoimmune thrombocytopenia, idiopathic interstitial lung disease HMG-17 11% Not described Also found in SLE, MCTD, ,319 NOR-90 Speckled nucleolar RA, and normals; antigens associated with active chromatin hubf, active in the regu- 89/ Secondary SS with RA and pulmonary fibrosis in Japanese patients lation of rrna transcription doublet p80-coilin 4% Distinct speckled Antigen localizes in ,303 nucleoplasmic (1 nuclear coiled bodies to 8 dots/cell) with unknown function NuMA Rare Spindle poles in Nuclear protein associ , 304 metaphase cells ated with the mitotic apparatus during mitosis; proposed function is postmitotic chromosomal reorganization Abbreviations: ANA, antinuclear antibodies; HMG, high mobility group; hubf, human upstream binding factor; MCTD, mixed connective tissue disease; NOR, nucleolar organizing region; NuMA, nuclear mitotic-associated protein; PM, polymyositis; RA, rheumatoid arthritis; RF, rheumatoid factor; SS, Sj6gren's syndrome; SL, sicca lupus; SLE, systemic lupus erythematosus; WB, Western blot, immunoblot.

25 AUTOANTIBODIES IN RHEUMATIC DISEASES , in our laboratories, three different autoantibodies were detected in sera from patients with SS. They were named SS-A and SS-B (those found only in patients with primary SS) and SS-C (those found in patients with secondary SS associated with RA). 2s8 SS-C was later seen to be RA nuclear antigen (RANA), a nuclear antigen recognized by RA sera in cells infected by Epstein-Barr virus. 289 Thanks to a collaborative effort between the laboratories that had described the Ro and La systems at one side and our laboratories at the other side, Alspaugh and Maddison were able to show in 1979 that the antigens recognized by anti-ro and anti-la positive sera were, respectively, the same antigens as the ones targeted by anti-ss-a and anti-ss-b positive sera. 29 The immune response to SS-B/La appears linked to the one directed to SS-A/Ro, so that either one or the other antibody seldom appears alone in SS. 29~ It seems not to be the case in SI_,E, where autoantibodies to SS-A/Ro can be seen without the ones to SS-B/La in the same patient, and where the patients are generally younger. 286 The SS-A/Ro and SS-B/La autoantibodies in SS are clinically associated with increased frequency of purpura, hypergammaglobulinemia, severe salivary gland dysfunction, parotid swelling, presence, and titer of rheumatoid factor, lymphopenia, and leukopenia. ~ 7, Normal older adults with the antibodies displayed impaired exocrine gland function in a recent study, with symptoms of dry eyes and dry mouth. 39 When patients with primary SS are analyzed for the presence of isolated SS-A/Ro autoantibodies, they are found to have extraglandular manifestations much more frequently than those negative for the antibodies, with symptoms of vasculitis, lymphadenopathy, and anemia, as well as laboratory findings of cryoglobulinemia and positive rheumatoid factor.~ 7 Another interesting clinical aspect is that marly patients with primary SS and autoantibodies to SS-A/Ro or SS-B/La will also fulfill criteria for SLE, because of the higher prevalence of vasculitis, serositis, and cytopenias. 293 Patients without detectable amounts of anti-ss-b/la in the serum may test positive in the saliva, reflecting the findings of salivary gland periductal depositions of the autoantibody. 295 Although these autoanti- bodies are seen in the majority of patients with primary SS, their contribution to the disease pathogenesis is unknown. A characteristic immunogenetic feature of white and black patients with SS-A/Ro/SS- B/La autoantibodies and primary SS is the high prevalence of the antigens HLA-B8, -DR3, and _ DRw52.90,286,293,294, Other Autoantibodies (NOR-90, Ku, p80-coilin) Our observations in 14 patients with NOR-90 autoantibodies did not show any correlation with specific diseases, 241,254,299 (unpublished results). The antibodies were positive in scleroderma, SLE, juvenile SLE, RA, SS, undifferentiated connective tissue disease, PBC, IgA nephropathy associated with hypothyroidism, malignant melanoma, and hepatocellular carcinoma patients. In a recent Japanese study, Fujii et al 3 reported the prevalence of anti-nor-90 positive sera as 4.7% among 85 sera giving nucleolar staining in IIF. All 4 positive patients in this series, 3 with RA and 1 with interstitial lung disease, had SS and no scleroderma features. In the 10 patients seen by Rodriguez- Sanchez et al, 252 only 1 had SS. The others were diagnosed as scleroderma (n = 5), RA (n = 1), angioedema (n = 1), and unknown (n = 2). Only one study reported 20% of SS patients with Ku autoantibodies in WB, a seemingly high prevalence. ~63 Antibodies to p80-coilin were described in our laboratory, 3 v3 3 giving a characteristic staining pattern in IIF of HEp-2 cells (Fig 2G). The antigen is localized in nuclear coiled bodies, which sometimes segregate to the nucleolus. Its function is still unknown, but a partial cdna clone has been isolated? ~ A small number of patients with primary SS display p80-coilin autoantibodies (4%), a specificity that is also infrequently found in SLE and PBC patients. 3m,3 4 In this review, we decided not to discuss anti-neutrophil cytoplasmic antibodies (ANCA), rheumatoid factor, and the venereal disease research laboratory (VDRL) test. ANCA is under extensive investigation and would merit a review of its own. Rheumatoid factor is closely associated with RA, a disease not discussed here in depth. Whether RA should be called an autoimmune disease is debatable; we decided to exclude RA in this review. The VDRL test has

26 348 VON MUHLEN AND TAN been available for many years and there is no new work in this area. The reader is referred to the section on SEE to get insights on other methods of antiphospholipid antibodies testing. AUTOANTIBODIES AND THE PATHOGENESIS OF CONNECTIVE TISSUE DISEASES It is improbable that most of the autoantibodies described are involved in direct tissue injury in different connective tissue diseases, 1,13 with the exception of dsdna and histone autoantibodies. Even considering that many autoantibodies were shown to inhibit the function of their cognate antigens in vitro 3 5,3 6 (Table 6), autoantibodies would have to gain access through cell membranes to bind to intracytoplasmic or intranuclear/intranucleolar antigens. Normally these autoantibodies, which are normal Igs in structure and function, would not be expected to enter living cells. Because DNA and histones were shown to exist in the extracellular fluids, where they are accessible to antibodies, complexes of DNA-anti-DNA or histone-antihistone antibodies could be generated. Such complexes can activate complement and the coagulation and kinin systems, leading to local Table 6: Autoantibodies That Were Shown to Inhibit Cellular Functions Type of Autoantibody DNA topoisomerase I Proliferating cell nuclear antigen Pyruvate dehydrogenase E2 rrnp phosphoproteins RNA polymerase I RNA polymerase II RNA polymerase Ill Sm/UI-nRNP trna synthetases (Jo-1, PL-7, PL-12) Function Inhibited Modification of DNA topology Polymerase S-regulated DNA replication Lipoic acid binding to mitochondrial enzymes mrna translation 28S and 18S rrna synthesis Transcription of mrna precursors 5S rrna, trna, other small RNAs synthesis Splicing of precursor mrna Charging of trnas with aminoacid residues Abbreviations: rrnp, ribosomal ribonucleoprotein; Sm, Smith antigen; mrna, messenger RNA; trna, transfer RNA. Data from Tan and Chan 3 5 and Tan. 3 6 inflammatory reactions with clinical manifestations such as vasculitis or glomerulonephritis. On the other hand, the fact that many autoantibodies can be shown months or years before the clinical expression of disease, like anti-topo I or anticentromere autoantibodies in scleroderma, s speaks against the hypothesis that they are just epiphenomena emerging through inflammatory events leading to exposure of enclosed or sequestered antigens after cell death. Another very interesting aspect is the targeting by autoantibodies of epitopes, which represent the active sites of the polypeptides involved in each antigen-autoantibody system. 1,13,299,3 5 Unlike antibodies raised in experimental animals that often recognize denatured epitopes, the binding of autoantibodies occurs commonly at conformational determinants that are often functional domains of the cognate antigens. Hence, the suggestion that activated forms of intracellular particles participating in biosynthetic functions are driving the immune response. 3 7 An antigen-driven mechanism is also supported by studies on immunoglobulin gene usage in autoantibodies to DNA, where somatic mutations in V region genes are consistent with antigen-driven maturation. 3 8 Such antigendriven responses would be facilitated depending on whether HLA molecules are capable of presenting the correct antigens to immunocompetent lymphocytes. 3 9,3~ In other words, in a genetically susceptible individual, the way peptides are presented to immunocompetent cells may bypass tolerance mechanisms and autoantibodies would be formed. For more comprehensive analyses of those questions, the reader is referred to recent published reviews Synthesis of the available data suggest that the autoimmune response is antigen driven and that the antigen is a subcellular particle or a multimolecular complex involved in important and sometimes essential cellular function. Most probably, the immunogen is in its activated form, because functional active sites are very frequently targeted by autoantibodies.l,3 7 ACKNOWLEDGMENTS The authors thank E.K.L. Chan for reviewing the manuscript and R. Rubin for providing an update of Table 2.

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