Diversity and Origin of Rheumatologic Autoantibodies

Transcription

1 CLINICAL MICROBIOLOGY REVIEWS, JUIY 1991, p Vol. 4, No /91/ $02.00/0 Copyright 1991, American Society for Microbiology Diversity and Origin of Rheumatologic Autoantibodies MARVIN J. FRITZLER* AND MARIO SALAZAR Rheumatic Diseases Unit, University of Calgary, Calgary, Alberta, Canada T2N 1N4 INTRODUCTION DIVERSITY OF ANTINUCLEAR ANTIBODIES NEWLY IDENTIFIED ANTIGENIC MOTIFS Unique DNA Conformations RNP Consensus Sequences and RNA Recognition Motifs Protein Motifs HTH Zinc fingers Leucine zipper Other protein motifs RELEVANCE OF NEW MOTIFS TO AUTOIMMUNITY DO RHEUMATOLOGIC AUTOANTIBODIES PARTICIPATE IN PATHOGENESIS OF DISEASE? SOURCE OF AUTOANTIGEN ORIGIN OF AUTOANTIBODIES MOLECULAR MIMICRY: A KEY TO UNDERSTANDING THE ORIGIN OF ANTINUCLEAR ANTIBODIES? IS THE AUTOANTIBODY RESPONSE INITIATED AND DRIVEN BY AUTOANTIGEN REFERENCES INTRODUCTION The sera of patients with systemic rheumatic diseases (SRD) are characterized by the presence of antibodies directed against a wide variety of tissue antigens. These include those that react with the cell membrane, hormones, cell receptors, immunoglobulins, platelets, plasma proteins, intercellular matrix, and cytoplasmic and nuclear components. Although most of the target antigens are not tissue specific, certain antigens are variably expressed in differentiated and undifferentiated tissues and in different phases of the cell cycle. This review is limited to a discussion of autoantibodies directed against intracellular antigens, although this is only one facet in a spectrum of autoantibodies seen in SRD. The use of these naturally occurring autoantibodies as probes of cellular structure and function has had an important impact on the disciplines of molecular biology and immunology. Cell biologists have used autoantibodies from SRD sera to study gene expression, cell division, and other intracellular processes. Studies of autoantibodies and their respective antigens have provided the immunologist with clues to the pathogenesis of SRD and the clinician with diagnostic tools that have improved diagnostic accuracy. DIVERSITY OF ANTINUCLEAR ANTIBODIES The list of autoantibodies related to SRD that react with intracellular antigens has increased over the past decade from approximately one dozen to several dozen (37, 44, 60, 122, 124) (Table 1). One of the key approaches to the identification and study of autoantigens has been the application of immunoblotting techniques, the results of which are schematized in Fig. 1. The importance of autoantibodies * Corresponding author. is underscored by an appreciation of the growing number of autoantigens that have been cloned (Table 2) (21, 138). These cloned antigens are being applied to studies of cell biology (138) and utilized in diagnostic immunology. Antibodies to DNA and histones were among the first to be extensively studied, and their specificity (43, 49, 127) and disease associations (37, 122) are well characterized. In recent years attention has focused on a group of nuclear proteins, collectively known as nonhistone proteins. One of the earliest described nonhistone antigens was called Sm (125). This antigen is present on proteins bound to small nuclear RNAs and is believed to participate in the processing of heterogeneous nuclear RNAs, the precursors to mrna (76, 93, 144). Another antibody, found in scleroderma patients, is directed against topoisomerase I, a 90- to 100-kDa nonhistone protein identified previously as scleroderma-70 (Scl-70), a 70-kDa portion of the native enzyme (27, 57, 126). The determinants recognized by antibodies from patients with Sjogren's syndrome are found on at least two polypeptides with molecular weights of 60,000 and 52,000. Both of these antigens, referred to as Sjogren's syndrome antigen A (SS-A/Ro), are part of a macromolecular complex which includes a class of low-molecular-weight cytoplasmic RNA molecules referred to as hyl, hy3, hy4, and hy5 RNA (144). While the biological role of the SS-A/Ro complex is not precisely known, it has been proposed that it regulates the conversion of cytoplasmic mrna into translationally active and inactive forms (139). Although studies of the molecular biology of some autoantigens has rapidly advanced, the molecular biology of most intracellular antigens is less clear. These autoantibodies and their respective targets can be recognized on the basis of the cellular organelles with which they react (Fig. 2). For example, certain SRD patients have autoantibodies that react with the nuclear matrix (Fig. 2a) (38, 58), the relatively insoluble "skeleton" of the nucleus (12). In recent years, the proposed role of the nuclear matrix has been expanded from 256

2 VOL. 4, 1991 RHEUMATOLOGIC AUTOANTIBODIES 257 TABLE 1. Autoantibodies in SRD: clinical association and biochemical identitya Autoantigen % Prevalence Molecular characteristic(s) Systemic lupus dsdna Histones Sm Nuclear RNP (Ul RNP) protein SS-A/Ro SS-B/La transcripts Ku Ribosomal RNP PCNA/cyclin Systemic sclerosis Scl-70 Topoisomerase I Centromere/kinetochore RNA polymerase I Pm-Scl Fibrillarin Nor-90 Th/To Alu RNA-protein Lamins Dermato/polymyositis Jo-1 PL-7 PL-12 Mi-2 Pm-Scl Sjogren's syndrome SS-A/Ro SS-B/La Mixed connective tissue disease Nuclear RNP (Ul RNP) 68/70-kDa protein Drug (procainamide)-induced lupus Histones Juvenile chronic arthritis Histones High-mobility-group proteins <5 70 (PSS) >75 CREST 4 38 Rare Rare Rare Rare 25 4Rare >95 > dsdna Hi, H2, H3, H4, H5 Proteins 28, 29, 16, & 13 kda complexed to Ul, U2, U4, U5, U6 RNAs Proteins 33 & 22 kda complexed to Ul RNA, 70 kda Proteins 60 & 52 kda complexed to Y1-Y5 RNA Phosphoprotein 48 kda complexed to RNA polymerase III Proteins 66 & 86 kda Phosphoproteins 38, 16, & 15 kda Protein 36 kda Protein 70 kda; native protein, 90 kda Proteins 17, 19, 50, & 140 kda Proteins 11 to 210 kda (subunits) Proteins 20 to 110 kda Protein 34 kda complexed to U3 RNA Protein 90 kda Protein complexed to 7-2 & 8-2 RNA Protein 68 kda complexed to Alu RNA Proteins 70 & 60 kda Histidyl-tRNA synthetase protein 55/60 kda Threonyl-tRNA synthetase protein 80 kda Alanyl-tRNA synthetase protein 110 kda Proteins 53 & 61 kda Proteins 20 to 110 kda Proteins 60 & 52 kda complexed with Y1-Y5 RNA Protein 48 kda complexed with RNA polymerase III transcripts Proteins 33 & 22 kda complexed with Ul RNA 68/70-kDa nuclear matrix protein Hi, H2A, H2B, H3, H4 H5, Hi, H2B HMG-1 (29 kda), HMG-2 (28 kda) proteins a Adapted from Clinical Immunology and Immunopathology (124) with permission of the publisher. CREST, Acronym for calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia; HMG, high-mobility group; SS, systemic sclerosis; SS-A, Sjogren's syndrome antigen A; SS-B, Sjogren's syndrome antigen B. a structure that was part of the intracellular network responsible for directing molecules bound for the cytoplasm to include a role in DNA replication, binding unprocessed heterogeneous nuclear RNA, assembly of heterogeneous nuclear ribonucleoprotein (RNP) particles, and splicing reactions for a variety of specific gene products (12, 144). Earlier studies showed that one of the reactive antigens was heterogeneous nuclear RNA (38) and a 68-kDa protein (40). This 68-kDa protein is identical to a 70-kDa antigen believed to be a component of the nuclear matrix (131). Sera containing antibodies directed to the nuclear membrane are recognized by indirect immunofluorescence as having a rim pattern of staining (Fig. 2b). In the decades after the introduction of indirect immunofluorescence for the identification of antinuclear antibodies, it was thought that the rim pattern of staining represented antibodies to doublestranded (ds) DNA (44). While these sera often contained antibodies that bound DNA, it is likely that the rim pattern of staining represents antibody systems directed against other components of the nuclear membrane. Advances in light and electron microscopic techniques have provided new information about the nuclear membrane. It is now recognized that the nucleus is surrounded by a double membrane consisting of an inner laminar layer, an outer layer that is continuous with the endoplasmic reticulum, and nuclear pores where the outer layer abuts the inner layer. The interlaminar "space" is rich in proteins such as the lamins (47). Some investigators have identified nuclear membrane autoantigens bound by the sera of a patient with linear scleroderma (82) and other diseases (75, 100, 121, 136) as the 74-, 68-, and 60-kDa nuclear lamins. The identification of major autoantigens in the nucleolus has been markedly advanced through the studies of Tan, Busch, and their colleagues (89, ) (Fig. 2c). One of the most interesting autoantibodies binds the nucleolar organizer region of the nucleolus, a 90-kDa antigen designated

3 258 FRITZLER AND SALAZAR CLIN. MICROBIOL. REV. 90 SS-A/ SS-B/ Sm RNP crnp Histone PCNA Ro La Ku ScI-70I 70 - FIG. 1. Schematic immunoblot of autoantigens: a schematized visualization of a Western immunoblot, using human antisera of defined specificity (i.e., Sm, Ul RNP, cytoplasmic RNP, etc.). The approximate relative mobility (Mr) shown on the ordinate may vary depending on the electrophoretic conditions of the assay. Sm, Smith antigen; RNP, Ul ribonucleoprotein; crnp, cytoplasmic ribonucleoprotein; SS-A, Sjogren's syndrome antigen A (Ro); SS-B, Sjogren's syndrome antigen B (La); Scl-70, scleroderma 70. Shaded rectangle represents 90-kDa parent molecule. NOR-90 (106). Although many of the common nucleolar antigens have been described (61, 102, 122), other nucleolar antigens await characterization. Sera from some patients with SRD react with the Golgi apparatus (39) (Fig. 2d). Other studies have observed Golgi antibodies in the sera of patients with Sjogren's syndrome Antigen La... Ro... RNP... TABLE 2. Cloned human autoantigensa La 60 kda 52 kda p70 A Sm... B" B D E Centromere... C CENP-B Polypeptide Jo-1... Histidyl trna synthetase Scl Topoisomerase I PCNA... Auxiliary protein DNA polymerase 8 Ku... Ribosome... co 0' 50- A - H1 30 SMM - 10 C -~D - - E p70 P protein H~IR - 4 a Adapted from the Annals of the Rheumatic Diseases (138) with permission of the publisher. (105) and in a patient with idiopathic late-onset cerebellar ataxia (46). The Golgi apparatus plays a pivotal role in the intracellular transport and concomitant processing of newly synthesized secretory, lysosomal, and plasma membrane proteins (31). The foregoing are examples of the antibody diversity represented in SRD sera. Despite rapid advances in the identification of antinuclear antibodies (ANA), a large number of autoantibodies cannot be identified by conventional techniques. For example, in a service laboratory that analyzes 1,500 ANA-positive serum samples annually, >40% react with antigens that have yet to be identified (Table 3). On the basis of staining pattern analysis, some of these antigens are apparently associated with chromatin and may be directed to high-motility-group proteins, enzymes involved in higher-ordered chromatin structure, or other previously uncharacterized antigens. NEWLY IDENTIFIED ANTIGENIC MOTIFS Studies of antigens described above have relied on techniques that have become standard research tools. More sophisticated tools, such as molecular modeling and crystallography, are now providing a more detailed understanding of the secondary and tertiary structures that comprise unique conformational epitopes on proteins and nucleic acids. In addition, these studies provide insight into the

4 VOL. 4, 1991 RHEUMATOLOGIC AUTOANTIBODIES 259 I I Downloaded from FIG. 2. Indirect immunofluorescence of autoantibodies reacting with uncharacterized antigens in a human epithelial cell line (HEp-2). (a) Nuclear matrix antibodies are identified as large nuclear speckles that are outside of the nucleolar region and absent staining of metaphase cells (arrow). (b) Nuclear membrane staining is characterized by bright staining of the nuclear membrane and lack of staining of metaphase cells (arrow). This antibody is representative of nuclear lamin staining. (c) Nucleolar antibodies. (d) Golgi apparatus antibodies represented as cytoplasmic staining in an asymmetric perinuclear pattern. Magnification, x400. on April 16, 2018 by guest possibility that abnormalities in the autoimmune response directed against nuclear antigens observed at the cellular and humoral levels are more closely linked than previously anticipated. Primary among the structural motifs that may be autoantibody targets are several newly described DNA conformations, RNA recognition structures, helix-turn-helices (HTH), zinc finger structures, and leucine zippers (Fig. 3). Extensive reviews concerning the biochemistry and crystallography of these structures have been published (1, 30, 117). Unique DNA Conformations One of the more recently described forms of DNA, left-handed or Z DNA, has been shown to be an autoantigen that reacts with both SRD and murine systemic lupus erythematosus (SLE) sera (73, 114, 128). Studies of other nucleic acid conformations believed to be important in cell division, in the control of gene expression, and in the incorporation of viral DNA into the mammalian genome are still required. These structures include "hairpin" forms of

5 260 FRITZLER AND SALAZAR TABLE 3. Autoantibodies in 2,500 consecutive serum samples referred to a clinical laboratory Laboratory samples Red Cross' Autoantibody No. of serum %b No. of serum samples samples dsdna RNP <0.5 SS-A/Ro SS-B/La <0.5 Nucleolar Centromere Other Unidentified' 1, a Data from reference 41. b Some sera have more than one autoantibody. ' Unidentified, Positive ANA but identity not related to known antigens. Heix-turn-Helix DNA with the general structure poly(dg-dc)5dt4, "dumbbell" structures with the general structure dt4(dg-dc)5dt4, and cruciform DNA. The importance and relevance of these studies are based on observations that these unique DNA structures are relatively nuclease resistant and similar nuclease-resistant nucleic acids are highly immunogenic (13). As more information unfolds on the distribution and functional significance of these DNA structures in various cell systems (e.g., virus replication), research on the presence of antibodies to these structures in patients with SRD might add important information to the underlying cause of rheumatic diseases. It would also add supporting evidence for the concept that mammalian autoantigens share functional and structural identity with neoantigens expressed during viral infection and replication processes. RNP Consensus Sequences and RNA Recognition Motifs Molecular biologists interested in the control of gene expression have identified conserved sequences found in all examples of a particular type of regulatory region (i.e., promoter genes) in DNA. These regions of conserved nucleic acid sequences are referred to as consensus sequences. A study of the structure of the cloned RNP autoantigens has shown that they bear RNP consensus sequences and RNA recognition motifs (Table 4). Since these motifs are highly conserved, it suggests that the immune system could respond to similar antigens from a wide range of organisms. Although there appears to be little autoantibody reactivity to the motif structures themselves (60, 97), it has been suggested that they serve as T-cell epitopes (138). Protein Motifs The more newly discovered protein motifs (Fig. 3) have several common, but not exclusive, characteristics. First, they represent several classes of proteins referred to as DNA-binding proteins (DBP). Second, although these motifs are highly conserved, many of them appear to bind specific sequences in promoter and enhancer regions of genes (i.e., consensus sequences). Third, their affinity for binding nucleic acids is related in large part to domains of basic amino acids. In some of these proteins, these domains are represented as highly amphipathic structures that could be epitopes for T-cell recognition (25). Even more interesting are observations that proteins bearing these motifs are the targets of the autoimmune response as well. HTH. HTH (Fig. 3a) is the best-characterized DBP motif (140). Proteins with this motif are found in a number of procaryotic (109) and eucaryotic (111) gene expression systems. As the name implies, the HTH consists of two alpha helices separated by a sharp beta turn. Like histones, HTH proteins contain predominantly basic amino acids (15). Proteins with established HTH structures include the lambda Cro and the 434 repressor proteins in Escherichia coli bacteriophage (3, 72). A DBP that binds to the enhancer region of immunoglobulin-secreting B cells contains an HTH structure (85, 118). The presence of a beta turn in the HTH motif allows the peptide to change direction, resulting in potentially unique conformational epitopes. Although most observations have been made in procaryotic systems, eua. b. C. Zinc Finger COOH COOH Leucine Repeat Leucine Repeat Basic Region Leucine Zipper CLIN. MICROBIOL. REV. FIG. 3. Protein motifs seen as part of some autoantigen structures (Table 4). (a) HTH; (b) zinc finger; (c) leucine zipper.

6 VOL. 4, 1991 TABLE 4. Motif Protein motifsa: potential relationship to autoimmunity Relation Reference(s) RNP consensus se- SS-B/La antigen quences and RNA 60-kDa SS-A/Ro antigen 26, 70 recognition motifs 70-kDa UlRNP antigen 87 A protein of Ul RNP antigen 88 B" protein of U2 RNP antigen 88 HTH B-cell-specific enhancer kDa SS-AIRo antigen 26 Zinc finger 52-kDa SS-A/Ro antigen kDa SS-A/Ro antigen 20 Steroid receptors 65 ADP-ribose polymerase 55 Leucine zipper 52-kDa SS-A/Ro antigen 123 Ku antigen 143 c-myc, c-fos, c-jun oncogenes 74, 130 Oct-2/NFA 110 Highly amphipathic 74 a See reviews in references 1, 30, 111, 117, and 138. caryotic HTH proteins continue to be described. The first such eucaryotic structures described were in the homeodomain proteins of Drosophila spp., which play a role in embryonic differentiation (18). Zinc fingers. The zinc finger (Fig. 3b) was originally described as a part of the transcriptional factor TFIIIa of Xenopus laevis (15, 111) and has recently been described in yeast transcription factors (67), steroid receptors (65), and the 52-kDa SS-A/Ro autoantigen (123). The zinc finger is characterized by a highly conserved repeating unit of approximately 30 amino acids with a loop of 12 amino acids (24, 56, 118). There are at least two classes of zinc finger proteins that are distinguished on the basis of their ability to bind zinc ions at an intrachain cysteine-histidine or cysteinecysteine complex. Zinc is essential for the DNA binding property of the protein, and it also protects the protein from degradation, a factor that might be responsible for antigen persistence in autoimmunity. These proteins interact with approximately five nucleotides of DNA by coiling in the major groove at up to nine contact points by virtue of specific sequences within the finger region (24, 56, 118). Leucine zipper. The newest member of the unique protein motifs is the leucine zipper (Fig. 3c) (1, 74). This motif has been described in a number of procaryotic and eucaryotic proteins, most notably, products of nuclear oncogenes including c-fos and c-jun (130). The c-jun protein has been shown to be a member of the AP-1 family of transcription factors (132). Octamer 2 (Oct-2), a lymphocyte-specific protein believed to bind the enhancer region of immunoglobulin genes, has been shown to contain a leucine zipper structure (71). As with the other motifs, proteins with leucine zippers are able to bind DNA and are predominantly regulatory in their function. The leucine zipper contains a domain of 30 to 35 amino acids with a high ratio of the basic amino acids arginine and lysine, which are immediately adjacent to the putative zipper structure (74). The most striking feature of this structure is the four or five repeats of leucine residues at every seventh position. The observation that this structure does not contain proline lends itself to a RHEUMATOLOGIC AUTOANTIBODIES 261 model of an alpha-helical peptide with a highly amphipathic moiety (25, 113). It has recently been shown that the Ku autoantigen, the target of antibody responses in certain patients with SLE and a scleroderma-polymyositis overlap syndrome, bears a leucine zipper motif (143), as does the 52-kDa SS-A/Ro antigen (20, 123). Other protein motifs. Although the preceding examples represent the best-described motifs, others described recently include the transcription factor Ap-2, which contains a proline-rich domain, and a protein from the Myc family characterized by a helix-loop-helix structure (85). RELEVANCE OF NEW MOTIFS TO AUTOIMMUNITY As stated above, it is interesting to note that an increasing number of autoantigens bear these DNA and protein motifs (Table 4). It is not clear whether the autoimmune response involves processing or targeting motifs themselves or whether these motifs are only common structural features of a family of autoantigens that participate in key cellular functions such as gene regulation. On a functional basis, these protein motifs hold promise for further understanding of the mechanisms that underlie the immune response and the phenomenon of autoimmunity. For example, DBP that participate in the immune response include the lymphocytespecific Oct-2 or nuclear factor A2 (NFA2), which may bind to the enhancer regions of immunoglobulin genes and thereby activate transcription (1, 71, 110). A recurrent theme in the study of the origin of ANA is that virtually all ANA systems in SRD involve processes central to gene regulation and gene expression. The universality of these motifs, particularly their presence in oncogene products, is an exciting area that warrants further study. As more information on the sequence of nuclear antigens is known, it will be interesting to see whether more possess these unique DBP motifs. DO RHEUMATOLOGIC AUTOANTIBODIES PARTICIPATE IN PATHOGENESIS OF DISEASE? A question that arises from the study of autoantibodies found in the sera of patients with SRD is, How do these antibodies participate (if they do) in disease processes? For the most part, autoantibodies in SRD tend to be considered epiphenomena. Unlike a clearer role for autoantibodies to the acetylcholine receptor in myasthenia gravis or parietal cell antibodies in pernicious anemia (137), it has been difficult to see how antibodies directed against DNA, RNP, or histones are involved in the pathogenesis of SRD. The inference that there is a relationship between the in vitro effects of these antibodies on the function or activity of the antigen and an in vivo pathological effect has been untenable. One reason is that the evidence showing that these antibodies gain entry into living cells (2, 48, 79) is still controversial. As discussed in other parts of this review, an alternative is that the epitopes of intracellular autoantigens might have homologs or molecular mimics on the cell surface or in other compartments of lymphocytes or other cells. Antibodies directed against native DNA have been thought to participate in the pathogenesis of renal disease (see review in reference 116). While this view has been challenged (6), the mechanisms by which DNA/anti-DNA complexes are deposited in the renal glomerulus are being more clearly established (22). It has been shown that the binding of antibody to the glomerular basement membrane

7 262 FRITZLER AND SALAZAR may be related to the nature of the antigen or the antibody (22, 29, 98). Added to the background of the passive participation of autoantibodies in disease processes is more recent evidence that many autoantibodies bind to and inhibit the functional or physiological role of intracellular antigens (7, 77, 122). Although the significance of these observations in the pathophysiology of SRD remains to be elucidated, it is unlikely that these antibodies exert a pathogenic effect through inhibition of intracellular protein or enzyme function in vivo. Studies of the SS-A/Ro autoantigen system have provided some insight into the pathogenic role of autoantibodies in a subset of SLE patients. Antibodies to SS-A/Ro were first found in high frequency in patients with primary Sjogren's syndrome and SLE and in lower frequency in other rheumatic diseases (37, 44, 122). A more recent association has been with the neonatal lupus erythematosus syndrome characterized clinically by cutaneous lupus and/or congenital heart block (34, 96). A pathogenic role for anti-ss-a/ro in the production of the cutaneous and cardiac lesions has been suggested by the observation that, as the cutaneous lesions resolve, maternally derived SS-A/Ro disappears from the infant's circulation. An attractive hypothesis is that anti-ss- A/Ro antibodies participate in the pathogenesis of congenital heart block. Evidence showing that the SS-A/Ro antigen is present in cardiac tissue (134) and that cardiac and brain tissues have high copy numbers of the mrna for SS-A/Ro (139) supports this hypothesis. Of interest, certain SS-A/Ro sera bind to calreticulin, a calcium-binding protein found in the nucleus and the cytoplasm (81). The pathogenesis of the characteristic heart block and nervous system disease of some patients bearing this autoantibody may be related to alterations of the function of this protein. Studies in animal models, using monoclonal antibodies directed against viral peptides that cross-react with uninfected human cells, have provided some evidence that certain antibodies have pathogenic effects. The observations here have been limited to a few polypeptides, including myelin basic protein. The approach was to select the encephalitogenic domain on myelin basic protein which shared a high degree of sequence similarity with hepatitis B virus polymerase (28, 92). Both humoral and cellular responses against myelin basic protein were observed in rabbits immunized with octamers and decamers of the viral peptide. In addition, rabbits inoculated with hepatitis B virus polymerase demonstrated vascular lesions similar to those induced after inoculation with the encephalitogenic domain of myelin basic protein. Thus, in this experimental system it was shown that a peptide from an unrelated species that shares sequence similarity with a homologous peptide is able to induce both autoimmune responses and a pathological lesion. SOURCE OF AUTOANTIGEN One of the difficulties in understanding the significance of ANA is the lack of knowledge about the source of the antigen that initiates and drives autoantibody responses (Fig. 4). Recent studies of the Jo-1 antigen (histidyl-trna synthetase) have shown that the autoantibody shows affinity maturation, the sine qua non of an immune response to a specific antigen (84). Earlier studies have shown that highaffinity DNA antibodies characterize some SLE sera, particularly those implicated in the pathogenesis of glomerulonephritis (129). These studies provide evidence that the origin of the triggering antigen is endogenous and is part of 21- CLIN. MICROBIOL. REV. ~rmm Angen FIG. 4. Pathways to molecular mimicry in SRD. Antigenic epitopes may be shared by macromolecules from diverse species (virus, bacteria, yeasts, insects, and mammals), between tissues in the same species (brain, thymus, liver, kidney, and muscle), or between compartments in a human cell (nucleolus, ribosome, mitochondria, and cell membrane). Another potential source of antigenic stimulus is food (i.e., meat, vegetables, eggs, or milk) peptides absorbed from the gastrointestinal tract. an intracellular macromolecular complex. How do these intracellular antigens become available for recognition and processing by the immune system? Some evidence suggests that the antigens are not all localized to intracellular compartments. For example, it has been shown that DNA (94) and a DBP (receptor) (10, 11, 45) are present on the surface of splenic lymphocytes and other circulating lymphocytes (i.e., B lymphocytes). Recently published investigations have substantiated these observations by showing that a monoclonal antibody that binds dsdna also binds to cell surface proteins that bear a striking resemblance to nucleosomes (69). There has been a long-held notion that diet is an important factor in the induction and/or maintenance of autoimmunity (32, 54). Certainly, humans are exposed to a wide range of foreign proteins through diet (Fig. 4) and to contact at other mucosal surfaces. It is possible that the induction of autoimmunity is related to antigens absorbed from gut and other mucosal surfaces. The oral intake of foreign protein and nucleic acids provides a constant stimulation and challenge of the gut-associated lymphoid tissue (133). Clearly, intact peptides can traverse and are transferred across mucosal barriers (54, 133) and, if they have the appropriate moieties, could induce an autoimmune response through the mecha-

8 VOL. 4, 1991 nism of molecular mimicry. In animal studies, mice fed bovine gamma globulin or casein normally develop tolerance to the protein, but strains of autoimmune mice demonstrate defective tolerance to the ingested protein (16, 17). Interestingly, SLE patients have increased levels of circulating bovine gamma globulin, anti-bovine gamma globulin, and anti-bovine casein (17). It has been postulated that these antibodies cross-react with self-antigens (17). These examples represent studies of autoantigens that are not located in intracellular compartments. In addition, there is an interesting and rapidly growing body of evidence showing that macromolecules located in diverse intracellular structures share significant structural identity and thus have the potential to initiate the autoimmune response. For example, it is assumed that the target of the autoimmune response in SLE patients with DNA antibodies is nuclear DNA. However, it is possible that DNA in mitochondria, centrioles, or basal bodies is the immunogen. Indeed, it has been observed that SLE patients have antibodies directed against mitochondrial DNA (104). As noted above, it has been shown that the membranes of cells, notably lymphocytes, bear DNA and DNA receptors (5, 9, 10, 69, 94). Although the origin of this DNA is not known, it is most likely from the host and may arise via the process of programmed cell death, or apoptosis (4, 19, 141). This is an attractive possibility in drug or virus induction of autoimmunity because it has been suggested that accelerated programmed cell "death" may occur in this setting. Apoptosis has been described in vitro and in vivo in a variety of tissues, including lymphoid tissues. The role of apoptosis in a number of physiological processes, including the immune response, has been investigated. Recent evidence (8) has shown that nuclear material resembling nucleosomes extruded during apoptosis of splenocytes has mitogenic effects when added to lymphocytes. Therefore, it is possible that the cell surface DNA and histonelike material are released into the extracellular environment, bind to receptors on the cell surface, and thereby serve as immunogens and also induce the proliferation of specific lymphocytes. This possibility is supported by the observation that circulating plasma DNA from SLE patients is most likely human (78). Key observations from the study of autoantibodies and the molecular biology of autoantigens can be summarized in the following statements. First, the majority of autoantibodies bind to highly conserved determinants on ubiquitous cellular proteins (122). Second, in antigen/antibody systems amenable to testing, autoantibodies can inhibit the cellular functions served by the antigens. Taken together, these observations suggest that the conserved epitopes recognized by autoantibodies in patients with SRD are often the functional or active sites of these intracellular proteins (122). It is less clear why autoantibodies are directed to a relatively restricted set of intracellular proteins. Third, studies of certain autoantibodies in SRD (i.e., Sm, Scl-70, and SS-A/Ro antibodies) show that they are sequestered in one or a few rheumatic diseases and therefore have important diagnostic implications (122). Fourth, patterns of end organ involvement might be related to the presence of certain autoantibodies that possess unique phlogistic characteristics. Fifth, the origin or source of autoantigens, thought previously to be intracellular, might be functional components of the cell surface or they may arise via necrotic cell death or by apoptosis. RHEUMATOLOGIC AUTOANTIBODIES 263 ORIGIN OF AUTOANTIBODIES An understanding of the origin of autoantibodies in general, and antinuclear antibodies in particular, is based on three main concepts. The first is that autoantibody production is due to a process referred to as molecular mimicry. The second is that autoantibody responses are generated and driven by true autoantigens. And third, autoantibody production is not driven by self-antigen but is the outcome of an aberration in the control of the immune network. The latter theory has credibility in light of the observation that normal individuals and the relatives of patients with autoimmune disease have circulating autoantibodies (41, 44) and that in autoimmune animal models the primary defect lies in the bone marrow stem cells (68). The latter concept will not be discussed further but the reader is referred to discussions on this topic elsewhere (37, 59, 112, 122). On a superficial level, the first two views might appear to be incongruent but, as will be discussed later, this is not necessarily so. MOLECULAR MIMICRY: A KEY TO UNDERSTANDING THE ORIGIN OF ANTINUCLEAR ANTIBODIES? Molecular mimicry can be defined as structures shared by molecules or protein products from dissimilar genes (28, 92). More specifically, linear or conformational epitopes on macromolecules from different organisms (i.e., human and viral protein) or from different cellular compartments (i.e., nucleoli and mitochondria) that bear striking similarity can be said to demonstrate molecular mimicry. The macromolecules might be nucleic acids, proteins, glycoproteins, lipoproteins, or phospholipids. Similarities between proteins have been described on the basis of immunologic reactions at the humoral or cellular level or by identifying similar amino acid sequences of antigens stored in computer data banks. The rapid expansion of DNA and protein sequence data banks is providing a constantly changing and growing resource to study molecular mimicry and the possible role of viruses and other microorganisms in the etiology of SRD. Molecular mimicry is more common than would be expected by chance alone. It is widely accepted that a minimum of six or seven peptides are required for recognition by antibodies (28). By using mathematical analysis, the probability that the 20 amino acids occur in six identical sequences is 206 or 1 in 128,000,000 (92). This calculation, which assumes that all amino acids are represented equally and at random, might be an overestimation because certain protein sequences and conformations depend on nonrandom alignments. For example, many proteins such as histones have hydrophobic domains composed predominantly of nonpolar amino acids. Nevertheless, the probability that protein antigens would share high degrees of similarity with microorganisms on the basis of chance alone is low. An analysis of the potential for molecular mimicry was carried out by Srinisvappa and his collaborators (115), who tested over 600 monoclonal antibodies raised against viral polypeptides for cross-reactions with a panel of normal tissues. Their analysis showed that over 4% of monoclonal antibodies raised against DNA and RNA viruses reacted with determinants on uninfected tissues. Further, some of the monoclonal antibodies reacted with cells from more than one organ, and none of the reactivities could be assigned to a single virus or class of viruses. One approach to a study of molecular mimicry in autoimmunity is to study the epitopes on specific antigens (for a review, see reference 138). For example, histone antibodies

9 264 FRITZLER AND SALAZAR are found in over 50% of patients with SLE (37, 42, 108, 122). Analysis of the determinants on H5, a nonmammalian variant of histone Hi, that react with antibodies from SLE sera demonstrated that the determinants are primarily located in the carboxyl terminus (50, 127). When computer analysis of the sequences in the Protein Identification Research Databank was performed, high degrees of similarity with a variety of mammalian proteins were observed (95). As expected, the greatest similarity was with other histone variants. However, a scan for sequence identity to the C terminus of H5 demonstrates that goose H5 has a 27.2% similarity, but scores for all other histones and proteins were <10%. Only three regions of similarity to other Hi sequences are noted. Of these sequences, amino acids 148 to 155 (SPKKAKKP), 183 to 189 (RKSPKKK), and 167 to 172 (KAKKVK) are identical. All three of these regions are similar only to nonmammalian (chicken, goose, sea urchin, and annelid) Hi variants. Although this analysis does not provide evidence for epitope similarity with viral or bacterial peptides, it challenges the concept that the autoimmune response is completely driven by autoantigen. One of the obstacles to an understanding of the mechanisms by which viruses or other microorganisms induce or perpetuate autoantibody production is that of viral persistence. Careful searches for offending viruses in a number of autoimmune diseases, including SLE, have proven unrewarding. The concept of molecular mimicry, however, does not depend solely on viral persistence, although it does not exclude it either. Various pathways leading to autoimmunity after viral infection have been proposed (28, 92). One of these is the "hit and run" concept. In this hypothetical scenario, a virus initiates the autoimmune event but is rapidly cleared or enters a cryptic state by the time the disease is clinically manifest. The disease is then perpetuated through the autoimmune assault on normal tissues with the release of antigen, the production of additional autoantibodies, and so on. In some cases of viral persistence, the virus might periodically re-express antigens that again become the target of the immune response, thus initiating flares or relapses. Recent observations that the BK virus terminates tolerance to dsdna and histone antigens (36) support this concept. In the past, viruses and other microorganisms have been the favorite topic of research aimed at identifying the cause of autoimmune disease through the mechanism of molecular mimicry (see reference 135). However, it is not necessary to restrict one's view to the viral or bacterial pathogenesis of autoimmunity. The probability that molecular mimicry operates at a higher evolutionary level (i.e., between humans and other mammalian species or between macromolecules in different cellular compartments) is as likely as the notion that it operates between widely divergent organisms, such as viruses and humans (Fig. 4). An example that illustrates this concept is provided by studies of the Th antigen that reacts with some scleroderma antibodies (70, 91). Th antibodies were first described in 1982 (61); later, the anti-to system (99) in sera with nucleolar antibodies was shown to be identical (64, 99). The reactive determinants were found on 7-2 RNP and a cytoplasmic 8-2 RNP. A relationship between between the 7-2 and 8-2 RNPs was suggested by observations that both of these molecules were bound by antibodies in the same sera (52). In 1988, the RNA component of the 8-2 RNA was identified as RNase P, the enzyme involved in processing trna (52). Later, it was CLIN. MICROBIOL. REV. shown that the 7-2 RNA was identical to a mitochondrial RNase MRP particle (53). Further, evidence that the mitochondrial RNase MRP particle shared RNA and protein determinants with the Th nucleolar antigen was provided when Th antisera absorbed RNase MRP activity from cell extracts. Thus, an autoantigenic epitope on Th is distributed in different cellular compartments (nucleus, nucleolus, cytoplasm, or mitochondria) and on different proteins. This provides an example that supports the concept of intracellular molecular mimicry (Fig. 4). As discussed elsewhere in this review, other macromolecules are widely distributed in cellular organelles and compartments and thus share the theme illustrated by the studies of the Th antigen. Important insight into the role of molecular mimicry and self-antigen in the induction and perpetuation of autoimmunity might come from the study of drug-induced autoimmunity. There are a number of clear examples in which certain drugs are responsible for triggering an autoimmune response. One of the most interesting is the induction of a lupuslike syndrome by procainamide. In procainamide-induced lupus (PIL), the patient develops fever, pleuropericarditis, arthritis, and an autoantibody response that overlaps with idiopathic SLE (37, 49, 50). The predominant autoantibody in PIL, directed against nuclear histones, is also seen in over 50% of patients with SLE (50, 127). Of further interest, the hierarchical reaction of PIL and SLE histone antibodies with histone classes is identical (H1>H2B>>H3-H4) (49, 50), as are the histone determinants identified by autoantibodies in both syndromes (49, 50, 127). Thus, an idiopathic syndrome (SLE) and a syndrome with a defined etiology (PIL) are identical in their autoantibody responses to at least one autoantigen. The key question is how the drug "induces" this autoantibody response because the answer would give clearer insight into the pathogenesis of the idiopathic form of the disease. The observations in PIL are seminal in a number of contexts, some of which will be alluded to later. But germane to the topic of molecular mimicry is the observation that, like the events in the viral etiology hypothesis, the inciting agent need not be present for the persistent or continued production of autoantibodies. This parallels the concept of the hit-and-run virus theory, which has become a theme of molecular mimicry (28, 92). In this setting the virus or drug is considered to be responsible for "breaking tolerance" to self-antigens, with the only remaining fingerprint being an antibody that binds to an epitope bearing identity or similarity to the infecting agent. That a drug is related to the induction of autoantibodies against a self-protein such as histone suggests, among other theories, that on exposure to drugs changes occur to the protein that render them immunogenic. In support of this concept, it has been shown that hydralazine alters the physical and physicochemical properties (e.g., decreased sensitivity to protease and nuclease digestion) of chromatin (120, 145). It is possible that similar drug-induced alterations of antigens might expose epitopes not present on the native protein or nucleic acid. Thus, unlike the notion that microorganisms themselves bear the cross-reacting epitopes, alteration of endogenous macromolecules may occur during drug treatment or exposure to certain environmental agents such as viruses. One of the reactions of cells to noxious agents, including drugs, is the ADP ribosylation of proteins, most notably, histones (142). It is appealing to consider that ADP-ribosylated proteins might be immunogenic and lead to a break in tolerance. Among the many lines of evidence supporting this concept is the observation that histones are the prime acceptors of ADP ribose (142) and that the hierarchical acceptance of this ribose diphosphate moiety, H1>

10 VOL. 4, 1991 H2B>H2A>H3=H4 (50, 93), is identical to the hierarchical representation of histone antibodies in SLE and PIL. In addition, it has been observed that histones are weakly immunogenic but, when coupled to ribose nucleic acids (i.e., trna), become highly immunogenic (83). The epitopes on nuclear antigens and most other autoantigens studied to date are primarily linear or continuous. This does not imply that conformational epitopes are unlikely targets of autoimmune responses, but rather that they are somewhat more difficult to study. There is evidence that conformational epitopes might be as important as (or more important than) linear conformations in the role of autoantigen recognition structures (84). IS THE AUTOANTIBODY RESPONSE INITIATED AND DRIVEN BY AUTOANTIGEN? The preceding discussion has provided evidence suggesting the potential of molecular mimicry as an integral event in the development of ANA. There is equally compelling evidence that the ANA response is initiated and driven by autoantigens (60, 84, 138). The evidence in favor of this conclusion is fourfold. First, many ANA responses are highly specific and restricted to specific disease (Table 1). Second, certain diseases are characterized by unique ANA profiles. For example, SLE is characterized by a wide diversity of autoantibody responses, whereas drug-induced lupus is characterized by a relative homogeneity of antibodies. Third, even when certain diseases, such as SLE or scleroderma, appear to be associated with a wide range of autoantibody responses, careful analysis of the antigens shows that the diverse antibodies are directed to either physically associated or functionally related macromolecules. For example, the antigens involved in SLE (histones, DNA, RNP, and ribosomes) are related to gene expression. On the other hand, patients with scleroderma produce autoantibodies that are primarily associated with cytokinesis (i.e., centriole, kinetochore, mitotic spindle, and mid-body) and processing of ribosomal genes (i.e., nucleolus-related antigens). Fourth, purified or cloned nuclear antigens have multiple epitopes on a single molecule. For example, SLE autoantibodies recognize epitopes on both the carboxy and amino termini of Hi (35), proliferating cell nuclear antigen (PCNA) (90), and SS-B/La (14). Adding more weight to these observations is the demonstration that autoantibodies from patients with drug-induced lupus, in whom a single identifiable agent induces a relatively specific autoimmune response, bind to multiple determinants on different histone molecules, a response indistinguishable from that of idiopathic SLE (49, 50). Evidence at the cellular level supports these observations in that different proteolytic fragments of autoantigen are capable of inducing lymphoproliferative responses (122). These and other observations indicate that not only is the autoantibody response polyclonal in individual patients and in individual diseases but also it is directed against more than one determinant. These observations provide a challenge for the proponents of molecular mimicry, especially when widely divergent interspecies antigens (i.e., virus versus human) are thought to be the target of autoantibodies. It would be difficult to envision a process of interspecies molecular mimicry being exercised several times in one disease (122, 138), much less in one individual. Perhaps in single-organ diseases such as multiple sclerosis, myasthenia gravis, or Hashimoto's thyroiditis the single- or oligo-epitope concept of molecular mimicry is easier to accept than it is in diseases such as SLE, scleroderma, RHEUMATOLOGIC AUTOANTIBODIES 265 Sjogren's syndrome, or even drug-induced lupus. The obvious caveat held by the molecular mimicry proponent is the hit-and-run concept referred to earlier in this review. One facet of this concept suggests that determinants on the infecting agent shared by an endogenous macromolecule are sufficient to break tolerance (33, 36). Another important observation is that naturally occurring human autoantibodies recognize different epitopes or domains than do monoclonal antibodies or immunizationinduced antibodies. This is especially true when purified antigens (proteins and nucleic acids) rather than native macromolecules (nucleosomes and ribosomes) are used as immunogens. For example, human autoantibodies to PCNA/ cyclin recognize different epitopes than do murine monoclonal antibodies or polyclonal antibodies to the amino-terminal peptide of the protein (119). Only the human autoantibody directed against PCNA/cyclin inhibits the auxiliary proteindependent DNA polymerase delta function (119). Similar observations have been made with antibodies directed against histidyl-trna synthetase (84), threonyl-trna synthetase (23), and SS-B/La (122). These observations suggest that the induction of autoantibodies is different from the immunization response. With the argument supporting the autoantigen-driven hypothesis aside, it is important to point out some difficulties with the autoantigen-driven concept and some possible resolutions. As pointed out above, the autoantibody response in SLE, and some other SRD, appears to be directed against functionally or physiologically related cellular processes. Earlier reference was made to observations that DNA is located in the nucleus, the cytoplasm, organelles (mitochondria, centrioles, and basal bodies), and on the cell surface of certain cells. Histone or histonelike proteins almost certainly are bound to DNA in these sites (8). Likewise, ribosomal RNP is localized to the nucleolus and the cytoplasm (86); the Th autoantigen is represented in the nucleus, cytoplasm, and mitochondria (53); and the SS-A/Ro antigen is distributed in both the cytoplasm and the nucleus (63, 107). Although these observations are conceptually difficult for proponents of the reductionist view of autoantigen-driven autoantibody responses, they do provide some cause for reappraisal of both the autoantigen-driven and the molecular mimicry concepts. These observations challenge the concept that the autoantigen is nuclear in origin although, through the mechanism of apoptosis and necrotic cell death, there are ample means by which intracellular antigens are exposed to the immune system. The observation that some, and perhaps all, nuclear antigens are represented widely throughout intercellular and intracellular compartments gives support to the concept that molecular mimicry may operate at an intracellular rather than an interspecies level (Fig. 4). Perhaps this is the level at which both views will eventually come to a common ground. A consideration of the newer antigenic motifs described above as targets of the autoimmune response supports this view. An attractive part of this concept is that it may give more insight into the pathogenic role of autoantibodies, especially when nuclear proteins are represented or mimicked as cell surface or cytoplasmic structures with similar physiologic and functional properties. This concept raises the possibility that the pathophysiology of autoimmunity includes the recognition of cell surface proteins by antibodies, thus leading to an inflammatory response. Examples of such cell surface proteins (i.e., calregulin, DNA, and histones) have been alluded to earlier in this review. One of the major deficiencies in our understanding of the

11 266 FRITZLER AND SALAZAR pathologic and clinical relevance of autoantibodies in SRD is the lack of quality longitudinal studies that address the question of clinical presentation and outcome of patients with a defined clinical picture or a defined autoantibody profile. One of the difficulties for the clinician and clinical investigator is the wide spectrum of diseases represented by a clinical label such as SLE, scleroderma, or Sjogren's syndrome. Clearly, these labels are rapidly outliving their usefulness in the management of patients and in the understanding of the origin or significance of autoantibodies. For example, ideally, therapy of SLE patients with the anticardiolipin syndrome should differ from that of the SLE patient with diffuse proliferative glomerulonephritis. Perhaps no clear answers will come from such labor-intensive and time-consuming studies. Perhaps meaningful subsets of these diseases cannot be defined. 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