Antinuclear antibodies as molecular and diagnostic probes

Molecular and Cellular Probes (1988) 2, 169-179

REVIEW

Antinuclear antibodies as molecular and diagnostic probes

Senga Whittingham and L . Jane McNeilage Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Post Office, The Royal Melbourne Hospital, Victoria 3050, Australia (Received 1 June 1988, Accepted 13 June 1988)

Much progress has been made in the past decade in defining the specificities of antinuclear antibodies (ANA) that are present in the blood of patients with multi-system autoimmune rheumatic diseases, and ANA now have an important place in diagnostic immunology. Disease-specific ANA have been defined and the intracellular autoantigens against which they react have been characterized . ANA have also established an important place in cell biology . Their use as probes has enabled molecular biologists to isolate, purify and assay the function of the highly conserved molecules with which they react, revealing new insights into the role these molecules play in gene transcription and translation . Cloning of the genes encoding these molecules in addition to providing information on primary structure has also provided human recombinant proteins for use as pure substrates for the development of simple and highly sensitive diagnostic assays in rheumatology . Studies on the epitopes with binding sites for ANA should provide new knowledge on how the immune response to these molecules evolves, how it is maintained and what role ANA play in the pathogenesis of disease .

KEYWORDS: antinuclear antibodies, diagnostic immunology, molecular probes, ribonucleoproteins, gene cloning, autoepitopes .

INTRODUCTION The discovery of the lupus erythematosus (LE) cell in 1948 1 heralded a new era in the diagnostic immunology of the multi-system autoimmune rheumatic diseases . The LE cell test is now considered an outmoded test for antinuclear antibodies (ANA) but, at that time, it strengthened the concept of autoimmunity and opened a Pandora's box whose objects of desire have continued to plague immunologists . It provided the challenge for developing more sensitive and specific tests for the diagnosis and classification of systemic lupus erythematosus (SLE) and related autoimmune rheumatic diseases, and it led to many questions that still defy definite answers .

Address correspondence to : Dr S . Whittingham . 0890-8508/88/030169+11 $03 .00/0

169

© 1988 Academic Press Limited



170

S. Whittingham and L. J . McNeilage

Why are highly conserved and functionally important nuclear molecules the targets of an autoimmune response in patients with multi-system autoimmune disease? Are these high-titre, disease-associated ANA merely an exaggerated natural response? How can the immune system 'see' and react to autoantigens that are in the protected environment of the cell? Hope, the saviour in the box, lies in the precise specification of ANA, the molecular characterization of the autoantigens, the identification of the autoepitopes with which B and T lymphocytes react, and the understanding of how immune responses to these epitopes are evoked and regulated .

Assays for ANA The specificities of ANA are currently established by several labour-intensive tests that utilize crude substrates because the target autoantigens are found in low abundance in the cell and are not readily isolated in pure form . Immunofluorescence is widely used as a screening test to detect ANA and for some ANA, for example, anticentromere antibodies, the specificity can be recognized by the pattern of fluorescence . However, there are multiple specificities of ANA giving many patterns that may range from subtle differences in uniform or homogeneous staining of the entire nucleus or the nucleolus to speckles varying from very fine grains to coarse clumps and discrete dots. The use of appropriately fixed continuous cell lines such as the human epithelial cell line, HEp .2, with its large nucleus and abundant cytoplasm, has greatly aided pattern recognition . Nevertheless, immunofluorescence, although a sensitive test, is relatively inaccurate for determining specificity and the patterns may become uninterpretable when more than one specificity is present . If the autoantigen is abundant and available in a relatively pure form, for example DNA, an enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay can be performed to confirm the specificity . However, most autoantigens are not readily available in this form, and in order to define ANA specificity reliance is placed on the use of non-human nuclear-enriched extracts such as the rabbit thymus extract available from Pel Freez (Arkansas, USA), and the relatively insensitive Ouchterlony or counter-immmunoelectrophoresis assays which require reference sera to identify precipitin lines . Western blots of nuclear-enriched extracts or immunoprecipitation of ANA with sonicates of biosynthetically labelled cells followed by gel electrophoresis can serve as confirmatory tests and provide additional information on the molecular nature of the autoantigens, but these methodologies are sophisticated and are not widely available in diagnostic laboratories .

ANA as diagnostic and molecular probes Enzyme digestion of the crude cell substrates used in the early assays yielded sufficient information to suggest that in addition to DNA and DNA-associated proteins such as histones, ribonucleoproteins and other nucleoproteins behave as autoantigens . These included a group of soluble autoantigens which, because they were readily extracted from tissues, became known as extractable nuclear antigens



ANA as molecular and diagnostic probes

1 71

(ENA) . As more and more ANA specificities were defined, it became clear that particular autoimmune rheumatic diseases or their subsets were associated with either one or a profile of ANA of different specificities . Thus, the laboratory demonstration of these autoantibodies became important for the diagnosis and classification of the autoimmune rheumatic diseases . Although the term ANA applies to antibodies reactive with molecules in the cell nucleus, it must be borne in mind that events in gene transcription and translation, and in cell division, result in the movement of molecules, thus altering their cellular distribution . Such changes will be represented in the immunofluorescence test with a rapidly dividing cell line like HEp .2 . With the recognition of different specificities of ANA came the need to provide names for these antibodies . A confusing nomenclature evolved which nominated the antibody either by the name of the patient in whom it was first detected, the disease in which it occurred or the cellular component recognized as the autoantigen . Worse still, subsequent demonstration of immunological identity between two previously designated specificities has resulted in some autoantigens having two names . It was fortuitous that, in the early 1980s, human sera with ANA known to react with cellular polypeptides complexed to ribonucleic acids (RNA) were used as probes for novel classes of small nuclear and cytoplasmic ribonucleoproteins (RNP) . 2,3 These experiments showed that antibodies to (U1)RNP, Sm, Ro (SS-A) and La(SS-B) reacted with discrete polypeptides complexed with small RNAs and that the RNP complex was different for each of the four specificities . 2,3 This union of molecular biology and diagnostic immunology proved a fruitful association . On the one hand, it showed that human sera with ANA could be used to probe the nature and function of intracellular molecules and, on the other hand, it showed that techniques in molecular biology could be used to identify the targets of the autoantibody response . Knowledge of the target of the ANA response gave meaning to the pattern of fluorescence, the precipitin line in the Ouchterlony or counterimmunoelectrophoresis tests and the polypeptides identified on Western blots and led to the exploitation of other techniques developing in molecular biology including those for gene cloning .

ANA as markers of disease The combined efforts of immunologists and clinicians to improve assays for the specification of ANA and develop criteria for diagnosis of disease have helped clarify associations between ANA of particular specificities and certain diseases (Table 1) . There are three scenarios : (1) a disease associated with a particular specificity of ANA, (2) a disease associated with a specific profile of ANAs, and (3) coexistent diseases associated with specific ANAs or specific profiles of ANAs . In the first scenario, exemplified by SLE and DNA-specific antibodies, the association appears to be close to 100%, suggesting that the ANA is a reliable serological marker of this disease . In the second scenario, there is a disease-specific profile of ANA reactive with seemingly unrelated molecules, for example anti-DNA and anti-Sm in SLE . In the third scenario, two or more diseases and/or two or more autoantibodies coexist, for example SLE with anti-DNA and Sjögren's syndrome with

172

S . Whittingham and L . J. McNeilage

Table 1 .

Antinuclear antibodies (ANA)* associated with autoimmune rheumatic diseases

Disease

Systemic lupus erythematosus Lupus nephritis

Autoantigen

ds/ss DNA histones Sm

Frequency in Caucasians (%)

100 50 5

Sub-acute cutaneous lupus

Ro

70

Lupus psychosis

ribosomes

90

Drug-induced lupus Mixed connective tissue disease Scleroderma CREST j' Diffuse Primary Sjögren's syndrome Polymyositis

Polymyositis/scleroderma overlap Rheumatoid arthritis

histones

100

U1 RNP

100

centromeres topoisomerase 1 (Scl-70)

70 35

La(SS-B) Ro(SS-B) tRNA synthetases

90 90 35

PM/Scl granulocyte nuclei histones

8 70 20

Comment

30% in Blacks and Asians Antigen is predominantly cytoplasmic Antigen is predominantly cytoplasmic

30% are anti-)o-1 ; antigens are cytoplasmic

"ANA of other specificities are known but these occur in low frequencies in this group of diseases . tCREST calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly, telangiectasia .

anti-La(SS-B) and anti-Ro(SS-A), or SLE with anti-DNA, anti-La(SS-B) and anti-Ro (SSA) . In the former case, both diseases and ANA are fully expressed clinically and serologically, and in the latter SLE, but not Sjögren's syndrome, is expressed clinically although the full profile of ANA is present . This suggests that Sjögren's syndrome is in the preclinical stage of disease, and when other markers of this disease are examined some will be present .

ANA as molecular probes in cell biology Whilst immunologists have puzzled over why some ANA should recognize epitopes on autoantigens that are functional sites on conserved molecules, cell biologists have used human sera containing ANA as tools for identifying, assaying the function of intracellular molecules and for cloning the genes encoding these molecules . The classical study illustrating what powerful tools ANA are for identifying little-



ANA as molecular and diagnostic probes

173

known, functionally essential, intracellular molecules was performed by Lerner & Steitz . 2 They used sera containing ANA to (U1)RNP and Sm to identify small ribonucleoprotein (snRNP) particles . Antibodies to (U1)RNP, the ANA of patients with mixed connective tissue disease, precipitated the most abundant of the small nuclear (sn) RNPs, U1, whilst antibodies to Sm, the ANA of SLE, precipitated the U1 RNP and four other RNPs containing the snRNAs U2, U4, U5 and U6 . 2 Since then, many molecules have been identified in this manner, including components of subcellular structures such as ribosomes, 4' 5 coated vesicles, 6 the nucleolar organiz8,9 the kinetochore,' 0-12 the non-kinetochore region ing region,' the mitotic spindle 14,15 and the 5S RNP complex ." of the chromosome, 13 the signal recognition particle ANA to RNA have also been identified as potentially useful reagents for the analysis of higher order RNA structure and RNA-protein interactions . 11,18 In addition to identifying the components of intracellular particles, ANA have been of value for the investigation of the function of these molecules . Examples of ANA that have been used as probes for function include the (U1)RNP-specific antibodies of mixed connective tissue disease, the anti-Sm and anti-proliferating cell nuclear antigen (PCNA) antibodies of SLE, anti-La(SS-B) antibodies of primary Sjögren's syndrome, anti-RNA-polymerase 1 and anti-DNA topoisomerase 1 (Scl-70) antibodies of scleroderma, and the anti-tRNA synthetases (including anti-Jo-1) of autoimmune polymyositis . (U1)RNP-specific and Sm-specific antibodies have been shown to inhibit mRNA splicing in vitro 19,20 and in vivo, 21 providing evidence that U1 and U2 snRNPs are required for splicing . The contribution of these ANA to the understanding of the splicing of precursor mRNA is reflected in the reference by molecular biologists to 'Sm' proteins as components of the spliceosome . La(SS-B)-specific antibodies have been used to show that the La antigen is an RNA polymerase 111 transcription factor required for the synthesis of full-length transcripts 22 (E .Gottlieb & J .Steitz, pers . comm .) . In addition, La(SS-B)-specific antibodies have also been used to identify RNPs containing virus-encoded RNA polymerase 111 transcripts including the Epstein-Barr virus-encoded small RNAs EBER 1 and EBER 2,3,23,24 the adenovirus small RNAs VA1 and VA23,24,2' and the vesicular stomatitis virus leader RNA . 26 Anti-RNA polymerase 1 antibodies inhibit transcription of rRNA both in vitro 27 and in vivo 2' and prevent the formation of nucleoli if injected into mitotic celis . 29 This implies that transcriptional activation of rRNA genes during and after mitosis is essential for normal nucleologenesis .29 PCNA-specific antibodies inhibit DNA synthesis, suggesting that PCNA influences, either directly or indirectly, the events leading to DNA synthesis . 30 This example of an ANA as a probe for analysing the function of fundamental nuclear molecules is an interesting one . The ANA, anti-PCNA, owes its discovery to the observation by Tan and colleagues 31,32 that the serum reacted only with nuclei of proliferating cells . The autoantigen, PCNA, was subsequently shown to be identical with cyclin, 33,34 which has recently been shown to be an auxiliary protein of DNA polymerase 6 . 35,36 Autoantibodies to DNA topoisomerase 1 (Scl-70) inhibit the supercoiling of DNA in vitro . 37 These antibodies have also been used to define the cellular distribution of DNA-topoisomerase 1 . 38,39 Other ANA known to inhibit the function of their antigenic targets include those found in the sera of patients with polymyositis that react with a variety of tRNA



17 4

S. Whittingham and L. J . McNeilage

synthetases 40-44 and antibodies to the DNA repair enzyme poly(ADP)-ribose (C . A . Penning, pers . comm .) . ANA have also proven to be useful as probes for the screening of cDNA expression libraries to isolate cDNAs encoding several of the nuclear autoantigens including La, 45,46 the 70 kDa U1 RNA-associated protein, 47 49 the 80 kDa centromere-associated protein, 50 the B"antigen associated with U2 RNA, 51 three acidic ribosomal phosphoproteins P0, P1 and P2, 52 poly(ADP-ribose) polymerase S3 and DNA topoisomerase 1 . 54 In other examples, anti-Sm sera have been used in hybridarrested translation to select a cDNA clone encoding the 11 kDa protein of the Sm snRNP, 55 and sera reactive with the mitotic spindle have been used to isolate a 59 kDa yeast spindle pole protein .9

ANA at the interface of molecular biology and medicine The growing confidence in the success of the marriage of molecular biology and medicine forecasts a glowing future for both fields of endeavour . The cloning of genes coding for human intracellular autoantigens is providing recombinant proteins for use as pure substrates for the development of simple and highly sensitive assays such as ELISA for the detection of ANA . 56,57 These assays will circumvent the labour-intensive tests presently required for specification of ANA and this improved precision will lead to better classification of disease . Knowledge of the sequence of the autoantigen provides a means of defining the epitopes recognized by the B and T lymphocyte arms of the immune response . One approach to studying epitopes recognized by autoantibodies has been the construction of a panel of recombinant proteins representing various regions of the gene encoding the autoantigen . Studies of the autoepitopes on a centromereassociated antigen recognized by highly disease-specific ANA present in patients with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangectasia) variant of scleroderma, and on the La(SS-B) autoantigen recognized by the anti-La(SS-B) ANA of primary Sjögren's syndrome have used this technique . An analysis by Earnshaw and colleagues58 of epitopes on the 80 kDa centromeric autoantigen (CENP-B), one of three autoantigens recognized by centromere-specific antibodies (ACA), has defined at least five independent epitopes designated CE,-, . CE,, the major autoepitope recognized by sera with ACA, is located in the carboxyl-terminal of CENP-B ; CE 2 , recognized by approximately 20% of ACA-positive sera, is also located in the carboxyl-terminal of CENP-B and CE 3 is within the amino-terminal 60% of CENP-B . Approximately 90% of ACA-positive sera bind to CE 3 , although the majority of ACA do so only weakly . Epitopes CE 4 and CE, have not been localized to specific regions of CENP-B and are shared with the 140 kDa (CENP-A) and the 17 kDa (CENP-C) centromeric polypeptides, respectively . Binding studies utilizing affinity-purified antibodies to CE, CE 3, CE 4 and CE, have shown that these epitopes are structurally independent and two of these were recognized concurrently by over 90°10 of patients ." This strongly implies that the autoantibody response is polyclonal, an important point to note in theoretical discussions on 'causes' of autoimmunity . A similar study has been undertaken on La(SS-B) . Sturgess and colleagues" subcloned three non-overlapping fragments of La cDNA generating subclones La I



ANA as molecular and diagnostic probes

1 75

(bases 1-251), La Il (bases 252-755) and La III (bases 756-3' terminus) . La I produced a stable fusion protein that was not reactive with anti-La(SS-B) sera . La II failed to produce a stable fusion protein . However, La III produced a stable fusion protein which was strongly reactive with anti-La(SS-B) sera, demonstrating that the 103 carboxyl-terminal amino acids contained an epitope which reacted with all sera with anti-La(SS-B) . An alternative approach to defining epitopes on La(SS-B) that did not make use of a cloned molecule was taken by Chan and colleagues ." Using a crude HeLa cell extract, they defined two relatively protease-resistant domains : domain X, which was a methionine-containing, non-phosphorylated, 28 kDa polypeptide and domain Y, a phosphorylated, methonine-poor, 23 kDa polypeptide . Extending this analysis further, Chan & Tan 60 have compared the targets on La recognized by human anti-La(SS-B) sera with those recognized by murine monoclonal La(SS-B)specific antibodies induced by immunization . The human anti-La(SS-B), in contrast to the murine monoclonal antibodies, recognized highly conserved epitopes . Another important advance in the methodology for defining epitopes recognized by ANA has been the development of synthetic peptides . Elkon and colleagues 61 used this approach to define an autoepitope on the three ribosomal proteins P0, P1 and P2, which are the target antigens of ANA found in patients with SLE and psychosis .62,63 Using a synthetic peptide, they defined a carboxyl-terminal autoepitope present on all three of these ribosomal proteins .

ANA as probes for disease mechanisms One of the puzzling features of ANA has been the large number of seemingly unrelated specificities that have been observed . There has been a tendency to dismiss this diversity by attributing it to non-specific polyclonal B lymphocyte stimulation when clearly there are disease-specific ANA which constitute profiles of ANA . Some of these serologically associated ANA are presumed to be 'linked', and to explain how linkages might occur Hardin' has suggested that ANA arise from antigenic stimulation of the immune system by highly selected hierarchical sets of epitopes . These epitopes are on individual particles that may be structurally related or form part of a complex . For example, Sm-specific antibodies are normally associated with (U1)RNP-specific antibodies and La(SS-B)-specific antibodies with anti-Ro (SS-A) . In both cases, the two autoantigens are found associated in a ribonucleoprotein complex even though the predominant antibody has a specific disease association . Migliorini and colleagues 65 have suggested that profiles of ANA may arise through antibodies which have recurrent idiotypes or are parallel sets of antibodies ." They prepared hybridomas from MRL-Ipr/Ipr mice with lupus and antibodies to DNA . They then selected a monoclonal antibody which did not bind to DNA, but shared an idiotype with the antibodies that did bind to DNA, and they showed that one such antibody was anti-Sm-specific and bound to DNA-specific antibodies . Binding of this antibody to the DNA-specific antibodies was inhibited by DNA, indicating that the idiotype it defined was the antibody-binding region . These antibodies appeared to constitute a network of autoantibodies which Migliorini 65 speculated arose by mutation of germ line-encoded DNA-specific antibodies . The mechanism(s) by which ANA induce disease remains obscure . In SLE, the



1 76

S . Whittingham and L. J . McNeilage

multi-system features of the disease have been attributed to immune complexes, particularly in the skin and renal glomerulus . However, it has been suggested that DNA-specific antibodies in SLE may be pathogenic because they cross-react with proteoglycans in the renal glomerular basement membrane .67 Matsiota et al . 68 suggested that there are two distinct kinds of DNA-specific antibodies in SLE : those that are polyspecific and those that are monospecific, and that possibly the monospecific antibodies were the more pathogenic of the two since the majority of the DNA-specific antibodies eluted from glomeruli were monospecific . How and whether the specificities of ANA induce damage is unclear . So far, there is no evidence that the inhibitory effect on cell metabolism demonstrated in vitro occurs in vivo, and if ANA 'see' their autoantigens these would need to be exteriorized to the cell membrane . Finally, what induces the ANA response is also obscure . Retroviruses are potential candidates but the evidence for implicating retroviruses is circumstantial . Of interest is the observation that the four murine monoclonal antibodies which bound to the Sm-specific antibody from the MRL-Ipr/lpr lupus mouse in the experiments of Migliorini et al ." all recognized retroviral proteins . Other evidence includes that from Query & Keene, 69 who showed that a cDNA-derived amino acid sequence encoding the 70 kDa protein associated with U1 RNP contained 23 residues homologous to a region of the murine leukaemia virus group-specific antigen and that immunization with the retroviral antigen elicited (U1)RNP-specific antibodies . Other viruses may also be candidates, but again most of the evidence for this is circumstantial and the cases documenting evidence have been isolated .70 Many questions remain unanswered, but clearly ANA have now become recognized as diagnostic markers and have an established place as molecular probes in cell biology .

ACKNOWLEDGEMENTS We acknowledge grant support from the National Health and Medical Research Council, Canberra, Australia .

REFERENCES 1 . Hargraves, M . M., Richmond, H . & Morton, R . (1948) . Presentation of two bone marrow elements : the 'tart' cell and the 'L . E.' cell . Proceedings of the Mayo Clinic 24, 25-8 . 2 . Lerner, M . R . & Steitz, J . A . (1979) . Antibodies to small nuclear RNAs complexed with proteins are produced by patients with systemic lupus erythematosus . Proceedings of the National Academy of Sciences, USA 76, 5495-9 . 3 . Lerner, M. R ., Boyle, J . A ., Hardin, J . A . & Steitz, J . A . (1981) . Two novel classes of small ribonucleoproteins detected by antibodies associated with lupus erythematosus . Science 211, 400-2 . 4 . Elkon, K . B., Parnassa, A . O . & Foster, C . L . (1985) . Lupus antibodies target ribosomal P proteins . Journal of Experimental Medicine 162, 459-71 . 5 . Francoeur, A . M ., Peebles, C . L., Heckman, K . J ., Lee, J . C . & Tan, E . M . (1985) . Identification of ribosomal protein autoantigens. Journal of Immunology 135, 2378-84 . 6. Bower, D . J . & Jeppesen, P . (1986) . Characterization of a polypeptide associated with coated vesicles and the cytoskeleton which is recognized by a CREST serum . Experimental Cell Research 167,166-76 . 7 . Rodrigues-Sanchez, J . L ., Gelpi, C ., Juarez, C . & Hardin, J . A . (1987) . Anti-NOR 90 . A new



ANA as molecular and diagnostic probes

177

autoantibody in scleroderma that recognizes a 90-kDa component of the nucleolus-organizing region of chromatin. Journal of Immunology 139, 2579-84 . 8 . Sager, P . R ., Rothfield, N . L ., Oliver, J . M . & Berlin, R . D. (1986) . A novel mitotic spindle pole component that originates from the cytoplasm during prophase . Journal of Cell Biology 103,186371 9 . Snyder, M. & Davis, R . W . (1986) . Molecular analysis of chromosome segration in yeast . Yeast 2, S363 . 10 . Cox, J . V ., Schenk, E . A. & Olmsted, J. B . (1983) . Human anticentromere antibodies : distribution, characterization of antigens, and effect on microtubule organization . Cell 35, 331-9 . 11 . Guldner, H . H ., Lakomek, H .-J . & Bautz, F . A . (1984) . Human anticentromere sera recognize a 19 . 5 kD non-histone chromosomal protein from HeLa cells . Clinical and Experimental Immunology 58, 13-20 . 12 . Earnshaw, W . C . & Rothfueld, N . (1985) . The identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma . Chromosoma 91, 313-19. 13 . Jeppesen, P. & Nicol, L. (1986) . Non-kinetochore directed autoantibodies in scleroderma/CREST . Identification of an activity recognising metaphase chromosome core non-histone protein . Molecular Biology and Medicine 3, 369-84 . 14. Okada, N ., Mimori, T., Mukai, R ., Kashiwagi, H . & Hardin, J . A . (1987). Characterization of human autoantibodies that selectively precipitate the 7SL RNA component of the signal recognition particle . Journal of Immunology 138, 3219-23 . 15 . Reeves, W. H ., Nigam, S . K . & Blobel, G . (1986) . Human autoantibodies reactive with the signal recognition particle . Proceedings of the National Academy of Sciences, USA 83, 9507-11 . 16 . Steitz, J . A ., Berg, C ., Hendrick, ) . P . et al. (1988) . A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells . The Journal of Cell Biology 106, 454-556 . 17 . Wilusz, J . & Keene, J . D . (1986). Autoantibodies specific for U1 RNA and initiator methionine tRNA . Journal of Biology and Chemistry 261, 5467-72 . 18 . Deutscher, A . L . & Keene, J . D. (1988) . A sequence-specific conformational epitope on U1 RNA recognised by a unique autoantibody . Proceedings of The National Academy of Sciences, USA 85, 3299-303 . 19 . Yang, V . W ., Lerner, M . R ., Steitz, J . A . & Flint, S . J . (1981) . A small nuclear ribonucleoprotein is required for splicing of adenovirus early RNA sequences. Proceedings of The National Academy of Sciences, USA 78, 1371-5 . 20 . Padgett, R. A ., Mount, S . M., Steitz, J . A . & Sharp, P. A . (1983) . Splicing of messenger RNA precursors is inhibited by antisera to small nuclear ribonucleoprotein . Cell 35, 101-7. 21 . Bozzoni, I ., Annesi, F.,Becczari, E . et al. (1984) . Splicing of Xenopus laevis ribosomal protein RNAs is inhibited in vivo by antisera to ribonucleoproteins containing U1 small nuclear RNA . Journal of Molecular Biology 180,1173-8 . 22 . Gottlieb, E . & Steitz, J . A . (1987). The mammalian La protein associated with known transcription components. In RNA Polymerase and the Regulation of Transcription . (Keznikoff, W . S . et al., eds) pp . 465-8. Cambridge : Elsevier Science Publishing Co ., Inc . 23 . Lerner, M . R ., Andrews, N . C ., Miller, G. & Steitz, J . A . (1981) . Two small RNAs encoded by EpsteinBarr virus and complexed with protein and precipitated by antibodies from patients with systemic lupus erythematosus . Proceedings of The National Academy of Sciences, USA 78, 805-9 . 24 . Rosa, M . D ., Gottlieb, E ., Lerner, M . R . & Steitz, J . A . (1981) . Striking similarities are exhibited by two small Epstein-Barr virus-encoded ribonucleic acids and the adenovirus-associated ribonucleic acids VA1 and VA11 . Molecular and Cellular Biology 1, 785-96 . 25 . Matthews, M . B. & Francoeur, A . M . (1984) . La antigen recognises and binds to the 3'oligouridylate tail of a small RNA . Molecular and Cellular Biology 4,1134-40 . 26 . Kurilla, M . G . & Keene, J . D . (1983) . The leader RNA of vesicular stomatitis virus is bound by a cellular protein reactive with anti-La lupus antibodies . Cell 34, 837-45 . 27 . Stetler, D . A . & Jacobs, S . T . (1984). Phosphorylation of RNA polymerase I augments its interaction with autoantibodies of systemic lupus erythematosus patients . Journal of Biology and Chemistry 259,13629-32 . 28 . Reimer, G ., Rose, K . M ., Scheer, U . & Tan, E . M. (1987) . Antibody to RNA polymerase in scleroderma sera . Journal of Clinical Investigation 79, 65-72 . 29 . Benavente, R ., Rose, K . M ., Reimer, G., Hügle-Dörr, B . & Scheer, U . (1987) . Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase I into mitotic cells . Journal of Cell Biology 105, 1483-91 .



1 78

S . Whittingham and L . J . McNeilage

30. Wong, R. L ., Katz, M . E ., Ogata, K ., Tan, E . M . & Cohen, S. (1987) . Inhibition of nuclear DNA synthesis by an autoantibody to proliferating cell nuclear antigen/cyclin . Cellular Immunology 110, 443-8 . 31 . Miyachi, K ., Fritzler, M . J . & Tan, E . M . (1978) . Autoantibody to a nuclear antigen in proliferating cells . journal of Immunology 121, 2228-34 . 32 . Takasaki, Y ., Deng, J . A . & Tan, E . M. (1981) . A nuclear antigen associated with cell proliferation and blast transformation . Its distribution in synchronized cells . journal of Experimental Medicine 154, 1899-909 . 33 . Bravo, R., Fey, S . J ., Bellatin, J . et al. (1981) . Identification of a nuclear and of a cytoplasmic polypeptide whose relative proportions are sensitive to changes in the rate of cell proliferation . Experimental Cell Research 136, 311-19 . 34 . Mathews, M . B ., Bernstein, R . M ., Franze, B . B ., Jr & Carrels, J . I . (1984). Identity of the proliferating cell nuclear antigen and cyclin . Nature 309, 374-6 . 35 . Prelich, C ., Tan, C.-K ., Kostura, M . et al . (1987) . Functional identity of proliferating cell nuclear antigen and a DNA polymerase-S auxiliary protein . Nature 326, 517-20. 36 . Bravo, R ., Rainer, F., Blundell, P . A . & Macdonald-Bravo, H . (1987) . Cyclin/PCNA is the auxiliary protein of DNA polymerase delta . Nature 326, 515-17. 37 . Shero, J . H ., Bordwell, B., Rothfield, N . F . & Earnshaw, W. C . (1986) . Autoantibodies to topoisomerase I are found in sera from scleroderma patients . Science 231, 737-40. 38 . Guldner, H . H ., Szostecki, C ., Vesberg, H . P. et al . (1986) . Scl-70 autoantibodies from scleroderma patients recognise a 95 kD protein identified as DNA topoisomerase I . Chromosoma 94, 132-8 . 39 . Maul, G . G ., French, B . T., Van Venrooij, W . J . & Jimenez, S. A . (1986) . Topoisomerase I identified by scleroderma 70 antisera: enrichment of topoisomerase I at the centromere in mouse mitotic cells before anaphase . Proceedings of The National Academy of Sciences, USA 83, 5145-9 . 40 . Rosa, M . D ., Hendrick, J . P ., Lerner, M . R . & Steitz, J. A . (1983) . A mammalian tRNA His-containing antigen is recognised by the polymyositis specific antibody Jo-1 . Nucleic Acids Research 11, 85370 . 41 . Mathews, M . B . & Bernstein, R. M. (1983) . Myositis autoantibody inhibits histidyl-tRNA synthetase . Nature 304, 177-9 . 42 . Okada, N ., Mukai, R ., Harada, F . et al . (1984) . Isolation of a novel antibody which precipitates ribonucleoprotein complex containing threonine tRNA from a patient with polymyositis . European Journal of Biochemistry 139, 425-9 . 43 . Mathews, M . B ., Reichlin, M ., Hughes, G . V . & Bernstein, R. M. (1984) . Anti-threonyl tRNA synthetase, a second myositis relating antibody . Journal of Experimental Medicine 169, 420-34 . 44 . Bunn, C . C., Bernstein, R. M . & Mathews, M . B. (1986) . Autoantibodies against alanyl-tRNA synthetase and tRNA Ala coexist and are associated with myositis . journal of Experimental Medicine 163,1281-91 . 45 . Chambers, J . C . & Keene, J . D . (1985) . Isolation and analysis of cDNA clones expressing human lupus La antigen . Proceedings of The National Academy of Sciences, USA 82, 2115-9 . 46. Sturgess, A . D ., Peterson, M . G ., McNeilage, L . J ., Whittingham, S . & Coppel, R. L . (1988) . Characteristics and epitope mapping of a cloned human autoantigen La . Journal of Immunology 140, 3212-18 . 47 . Theissen, H ., Etzerodt, M ., Reuter, R . et al. (1986) . Cloning of the human cDNA for the U1 RNPassociated 70K protein . EMBO Journal 5, 3209-17 . 48 . Spritz, R . A ., Strunk, K ., Surowy, C . S . et al . (1987) . The human U1-70K snRNP protein : cDNA cloning, chromosomal localization, expression, alternative splicing and RNA-binding . Nucleic Acids Research 15, 10373-91 . 49 . Query, C . C . & Keene, J . D . (1987) . A human autoimmune protein associated with U1RNP contains a region of homology that is cross-reactive with retroviral p305a5 antigen . Cell 51, 211-20. 50 . Earnshaw, W. C ., Sullivan, K . F ., Machlin, P . S . et al. (1987) . Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen . Journal of Cell Biology 104, 817-29 . 51 . Habets, W . J ., Sillekens, P . T . G ., Hoet, M . H . et al. (1987) . Analysis of a cDNA clone expressing a human autoimmune antigen : full-length sequence of the U2 small nuclear RNA-associated B" antigen . Proceedings of The National Academy of Sciences, USA 84, 2421-5 . 52 . Rich, B . E . & Steitz, J . A . (1987) . Human acidic ribosomal phosphoproteins P0, P1 and P2 : analysis of cDNA clones. Molecular and Cellular Biology 7, 4065-73 . 53 . Yamanaka, H., Penning, C. A ., Willis, E . H ., Wasson, D . B . & Carson, D . A. (1987) . Characterization of human poly-ADP-ribose polymerase with autoantibodies . Journal of Biological Chemistry 263, 3879-83 .



ANA as molecular and diagnostic probes

179

54 . D'Arpa, P ., Machlin, P. S ., Ratrie, H . Ill . et al. (1988) . cDNA cloning of human DNA topoisomerase I : catalytic activity of a 67 .7- kDa carboxyl-terminal fragment . Proceedings of The National Academy of Sciences, USA 85, 2543-7 . 55 ., Wieben, E . D ., Rohleder, A . M ., Nenninger, J . M . & Pederson, T . (1985) . cDNA cloning of a human autoimmune nuclear ribonucleoprotein antigen . Proceedings of The National Academy of Sciences, USA 82, 7914-18 . 56 . Whittingham, S ., McNeilage, L. J ., Naselli, G., Coppel, R. L . & Sturgess, A . D. (1987) . Serological diagnosis of primary Sjögren's syndrome by means of human recombinant La(SS-B) as nuclear antigens . Lancet 2, 1-3 . 57 . Rothfield, N ., Whitaker, D ., Bordwell, B ., Weiner, E., Senecal, J .-L. & Earnshaw, W . (1987) . Detection of anticentromere antibodies using cloned autoantigen CENP-B . Arthritis and Rheumatism 30, 1416-19 . 58 . Earnshaw, W . C., Machlin, P . S ., Bordwell, B . J ., Rothfield, N . F . & Cleveland, D . W. (1987). Analysis of anticentromere autoantibodies using cloned autoantigen CENP-B . Proceedings of The National Academy of Sciences, USA 84, 4979-83 . 59 . Chan, E . K . L ., Francoeur, A .-M . &'Tan, E . M. (1986). Epitopes, structural domains and asymmetry of amino acid residues in SS-B/La nuclear protein . Journal of Immunology 136, 3744-9. 60 . Chan, E . K . L. & Tan, E. M . (1987) . Human autoantibody-reactive epitopes of SS-B/La are highly conserved in comparison with epitopes recognised by murine monoclonal antibodies . Journal of Experimental Medicine 166, 1627-40 . 61 . Elkon, K ., Skelly, S., Parnassa, A . et al. (1986) . Identification and chemical synthesis of a ribosomal protein antigenic determinant in systemic lupus erythematosus . Proceedings of The National Academy of Sciences, USA 83, 7419-23 . 62 . Bonfa, E . & Elkon, K . B. (1986). Clinical and serologic associations of the anti-ribosomal P protein antibody. Arthritis and Rheumatism 29, 981-5 . 63 . Bonfa, E ., Golombek, S . J ., Kaufman, L . D . et al . (1987) . Association between lupus psychosis and anti-ribosomal P protein antibodies . New England Journal of Medicine 317, 265-71 . 64. Hardin, J . A . (1986) . The lupus autoantigens and the pathogenesis of systemic lupus erythematosus . Arthritis and Rheumatism 29, 457-60. 65 . Migliorini, P ., Ardman, B ., Kaburaki, J . & Schwartz, R . S. (1987) . Parallel sets of autoantibodies in MRL-Ipr/Ipr mice . An anti-DNA, anti-SmRNP, anti-gp70 network . Journal of Experimental Medicine 165,483-99 . 66. Jerne, N . K . (1975) . The immune system : a web of v-domain . Harvey Lecture 7, 93 . 67. Faaber, P., Capel, P . J . A., Rijke, G . P . M . et al. (1984) . Cross-reactivity of anti-DNA antibodies with proteoglycans . Clinical and Experimental Immunology 55, 502-8 . 68. Matsiota, P., Druet, P., Dosquet, P., Gilbert, B . & Aurameas, S . (1987) . Natural autoantibodies in systemic lupus erythematosus . Clinical and Experimental Immunology 69, 78-88. 69. Query, C. C . & Keen, J. D . (1987) . A human autoimmune protein associated with U1RNA contains a region of homology that is cross-reactive with retroviral p309' 9 antigen . Cell 51, 211-20 . 70. Whittingham, S ., McNeilage, L . J . & Mackay, I . R . (1987) . Epstein-Barr virus as an etiological agent iri primary Sjögren's syndrome . Medical Hypotheses 22, 373-86 .