Humoral Autoimmune Responses Against Nuclear Proteins
Patients with systemic rheumatic diseases such as systemic lupus erythematosus (SLE), scleroderma, and Sjögren’s syndrome characteristically produce antinuclear antibodies (ANAs) that recognize intracellular nucleoprotein complexes. Systemic rheumatic diseases constitute chronic, life-threatening, multiorgan autoimmune dis- orders (Reichlin 1998). Their etiology is unknown, but genetic, hormonal, and envi- ronmental factors are involved. SLE is best characterized as a systemic immune com- plex vasculitis due to continued production of autoantibodies directed against nucleoplasmic antigens (e. g., DNA, histones, spliceosomal components, ribonucleo- proteins), whereas scleroderma-associated autoantibodies primarily detect nucleo- lus-associated proteins such as DNA topoisomerase I (Scl-70) and centromeres.
Autoantibodies target evolutionary, conserved epitopes of nucleoprotein complexes, which often constitute the functional regions (Tan 1989). Thus, the immunofluores- cence staining patterns observed with autoantibody-containing serum samples can be regarded as an autoantigenic map that provides information on the subcellular distribution and function of the target antigens (Hemmerich and von Mikecz 2000, Tan 1991). Humoral autoimmune responses seem to be antigen driven and T-cell dependent (Lanzavecchia 1995). However, the molecular mechanisms responsible for stimulating autoreactive B and T cells, as well as for the generation of a systemic autoimmune response against the cell nucleus, remain unclear and represent an intensively investigated topic of current immunology.
ANAs in Cutaneous Lupus Erythematosus
SLE is a multisystem disorder with symptoms spanning from relatively benign cuta- neous eruption to a severe, in some cases fatal, systemic disease. Although SLE can affect virtually every organ, abnormalities of the skin or mucous membranes are very frequent, being the second most common manifestation of SLE. Cutaneous lesions occur in approximately 85% of patients with SLE and are associated with photosen- sitivity, which may be linked to the pathogenesis of the underlying autoimmune response. The idea is that ultraviolet (UV) irradiation of the skin may expose previ- ously cryptic antigens on the cell surface of epidermal cells, which, in turn, may be recognized by autoantibodies and T cells as foreign components, leading to (a) a sys- temic autoimmune response and (b) production of lupus skin lesions.
Antinuclear Autoantibodies in Cutaneous Lupus Erythematosus: Biochemical
and Cell Biological Characterization of Ro/SSA
Anna von Mikecz
24
Four subsets of cutaneous LE (CLE) have been defined: acute CLE (ACLE), sub- acute CLE (SCLE), chronic CLE (CCLE), and neonatal LE (NLE). In SCLE and NLE, the occurrence of specific autoantibodies against components of the cell nucleus has been observed. SCLE is defined by a nonscarring skin eruption in association with autoantibodies against Ro/SSA (Fig. 24.1) (Gilliam 1977, Sontheimer et al. 1979). Fifty percent of these patients fulfill the criteria of the American Rheumatism Association for SLE established in 1982 (Tan et al. 1982). Depending on the detection method, 60%
to 95% of patients with SCLE have anti-Ro/SSA antibodies in their serum.
NLE is a rare disease with skin manifestations resembling those seen in SCLE (Lee 1993). Approximately one half of the NLE cases exhibit skin disease, the other half
Fig. 24.1. Subcellular distribution of Ro/SSA.AIndirect immunofluorescence and confocal microscopy of HEp-2 cells with subacute cutaneous lupus erythematosus (SCLE) autoanti- bodies against Ro/SSA revealed bright speckles embedded in a weaker homogeneous staining pattern (green) within the nucleoplasm. Confocal sectioning was conducted by recording focal planes from the bottom to the top of one cell (intervals, 1 µm). Note that single Ro speckles occur in three to four focal planes (red arrows).BFocal sections displayed inAwere fused to one stack (left micrograph, green). The morphology of the cell was recorded simultaneously by differential interference contrast (right micrograph, gray). Nu, nucleoplasm; No, nucleolus;
Cy, cytoplasm. Bar, 5 µm.CImmunoblotting of patient sera against Ro60 and Ro52. Patient serum 1 recognizes both, Ro60 and Ro52, whereas serum 2 is exclusively directed against Ro52. Confocal microscopy was conducted with serum 2. Molecular weight (MW) is given in kilodaltons
have congenital heart block (Michaelsson and Engle 1972), and approximately 10%
have both skin disease and congenital heart block. The hallmark of NLE is that in approximately 95% of the cases, the serum of both the baby and the mother contains autoantibodies directed against Ro/SSA (Franco et al 1980, Lee and Weston 1988). In contrast, other ANA specificities are rarely found (Provost et al. 1987). Anti-Ro/SSA autoantibodies are so characteristically prevalent in SCLE and NLE that clinicians usually use these ANA specificities as a confirmatory diagnostic test in patients who present with symptoms of the disorders.
However, the presence of these antibodies is not simply a matter of diagnostic or academic interest. Anti-Ro/SSA antibodies have been eluted from kidneys of patients with SLE (Maddison and Reichlin 1979) and are commonly associated with photo- sensitive rashes. Antibodies to Ro/SSA can cross the placenta and induce in approxi- mately 1 of 20 cases NLE. Therefore, many investigators have explored the structure, origins, and precise targets of anti-Ro/SSA autoantibodies.
Discovery of Ro/SSA Antigens
In 1961, two precipitating autoantibody specificities known as SjD and SjT were described in patients with Sjögren’s syndrome (Anderson et al. 1961). SjD antigen was reported to be insensitive to trypsin or heat treatment, whereas SjT antigen could be destroyed by the same treatment. In 1969, Clark et al. (Clark et al. 1969) reported a novel antibody specificity known as Ro in 40% of patients with SLE. The Ro antigen was described as a protease-, RNase-, and DNase-resistant cytoplasmic antigen observed in various tissue extracts. Six years later, in 1975, Alspaugh and Tan (Alspaugh and Tan 1975) described precipitin lines for two distinct autoantibody specificities termed “SSA” and “SSB” occurring predominantly in serum from patients with Sjögren’s syndrome. It was not until 1979 that SSA and Ro were shown to be identical (Alspaugh and Maddison 1979); since then, this antigen system has been referred to as Ro/SSA. However, in the research areas of molecular biology and cell biology, this system is exclusively referred to as Ro ribonucleoprotein (RoRNP), since Ro antigens were shown to be associated with small RNAs (Lerner et al. 1981). Within the RoRNP complex, human serum containing anti-Ro/SSA antibodies recognizes two proteins with the molecular weight of 52 and 60 kDa (Fig. 24.1C). The 60-kDa Ro/SSA protein binds to a subpopulation of small RNAs, termed “hY RNAs” (Wolin and Steitz 1984). Antibodies to the Ro-associated RNA molecule hY5 RNA have also been detected (Granger et al. 1996), supporting the importance of the whole RoRNP particle in the autoimmune response.
Biochemical/Cell Biological Properties of Ro/SSA
The RoRNP complex in mammalian cells was first described as being composed of
one of at least four hY RNAs associated with two proteins of 52 kDa (Ro/SSA52) and
60 kDa (Ro/SSA60) (Ben-Chetrit et al 1988, Rader et al. 1989). hY RNAs are small
RNAs known as hY1, hY2, hY3, hY4, and hY5 RNA ranging from 84 to 112 nucleotides
and are present in about 1 to 5 ×10
5copies per cell (Wolin and Steitz 1984). All Y RNAs
can be folded into a structure containing a large internal loop and a long stem formed by base pairing the 5' and 3' ends (Van Horn et al. 1995). Within the stem is a highly conserved bulged helix that is the binding site for the Ro/SSA protein (Green et al.
1998). In human cells, RoRNPs consist of one of the four hY RNAs and two core pro- teins: Ro/SSA60 and La/SSB. Physicochemical studies on native RoRNPs indicate that the particles segregate into three discrete subpopulations, one containing hY5, another containing hY4, and a third containing hY1, hY3, and hY4 (Boire and Craft 1989). Initially, it seemed that the 60-kDa Ro/SSA polypeptide was the exclusive pro- tein component in the complex, but Boire and Craft (Boire and Craft 1990) were able to biochemically isolate RoRNPs in which the autoantigen La/SSB was a stable com- ponent.
Taken together, biochemical characterization of the RoRNP complex lead to the following model: the hY RNA is noncovalently bound to the 60-kDa Ro/SSA via the lower stem of the RNA formed by base pairing their 5' and 3' ends (Wolin and Steitz 1984), whereas La/SSB binds to the 3' oligo uridylate residues of hY RNAs (Fig. 24.2) (van Venrooij et al. 1993). In addition to the two core proteins, conditional association of other proteins such as calreticulin (Rokeach et al. 1991), Ro/SSA52 (Slobbe et al.
1992), RoBPI, and the RNA-binding protein nucleolin (Fournaux et al. 2002) to the RoRNP complex has been reported. However, these associations seem to be depend- ent on the structure of the hY RNA.
Although the evolutionarily conserved RoRNPs have been the subject of investi- gation in a number of laboratories for many years, their precise molecular definition, function, and subcellular localization remained unclear. The cellular distribution of Ro/SSA has been a matter of controversy since the early description of the autoanti- gen, because the Ro/SSA antigen seems to be present in both the cytoplasm and the nucleus. Although reports on association of Ro with cytoplasmic hY RNAs suggested
Fig. 24.2. The structure of the Ro ribonucleoprotein (RoRNP) particle (schematic diagram). In most higher eukaryotic cells, the Ro60 kDa protein is complexed with one of several small cyto- plasmic RNAs known as Y RNAs. The Y RNA is folded into a structure containing internal loops and a long stem formed by base pairing the 5’ and 3’ ends. The 3’ end of Y RNA is stably associ- ated with the La protein, whereas the association of Ro52, nucleolin, RoBPI, and calreticulin is conditional
that RoRNPs are cytoplasmic RNP particles (Lerner et al. 1981, Peek et al. 1993), the study of Harmon et al. (Harmon et al. 1984) (see also Fig. 24.1) clearly showed that specific Ro/SSA antibodies predominantly detect nucleoplasmic speckles. Corrobo- rating the idea of nuclear Ro/SSA location, rabbit anti-Ro/SSA60 antibodies displayed a speckled nucleoplasmic immunofluorescence staining pattern, which could be completely abolished by addition of purified Ro/SSA60 protein in serologic competi- tion experiments (Mamula et al. 1989). Moreover, Ro/SSA52 contains transcription factor-like motifs, for example, leucine zippers, which have been shown to promote protein dimer formation and to contribute to inhibition of transcriptional activity in vitro (Wang et al. 2001).
Cloning of complementary DNAs (cDNAs) for Ro/SSA allowed further character- ization of the proteins. The complete cDNA and protein sequence of Ro/SSA52 (Gen- Bank/EBML accession numbers M35041) revealed that the open reading frame con- sists of 475 amino acid residues with a predicted molecular mass of 54 Kda and pI of 6.35. Based on the amino acid sequence, Ro/SSA52 is expected to contain three domains: (a) an N-terminal zinc finger domain, (b) a central coiled-coil (leucine zip- per) domain, and (c) a C-terminal rfp-like domain. The N-terminal zinc finger domain is a member of the previously described ‘Ring’ finger proteins (Reddy et al.
1992), and the C-terminal rfp-like domain belongs to a second protein domain super- family named after the rfp protein (Takahashi et al. 1988). It is important to note that all sequence motifs of Ro/SSA52 represent DNA and/or RNA-binding motifs. The full-length cDNA sequences of the 60-kDa Ro/SSA protein has been published by Deutscher et al. (Deutscher et al. 1988) and Ben-Chetrit et al. (Ben-Chetrit et al. 1989) and showed identical sequences except the C-terminal nucleotides. Ro/SSA60, like Ro/SSA52, contains nucleic acid binding motifs. The N-terminal RNA binding domain is highly conserved between Xenopus and human Ro/SSA60 sequences (O’Brian et al. 1993) and may account for the binding of the 60-kDa protein to hY RNAs. In contrast, the function of the Ro/SSA60 zinc finger domain has not yet been characterized.
The genes for Ro/SSA52 and Ro/SSA60 have not been published yet. However, using cDNA probes it was demonstrated that the human Ro/SSA52 gene is located on chromosome 11 (Frank et al. 1993), and the human Ro/SSA60 gene has been localized to gene locus 1q31 by fluorescence in situ hybridization (Chan et al. 1994).
Detection of Anti-Ro/SSA Antibodies
A hallmark of eukaryotic cells is their separation into subcellular compartments. The nucleus contains many internal nuclear domains, including the nuclear envelope, the nucleolus (Carmo-Fonseca et al. 2000), splicing speckles (Spector 1993), and a variety of nuclear bodies (Gall 2000, Zhong et al. 2000). This spatial organization reflects the requirement for spatial and temporal coordination of nuclear processes: Nuclear pro- teins with related functions, such as DNA replication, transcription of RNA, or sub- sequent RNA splicing, are assembled in multiprotein/nucleic acid complexes and thus colocalize at the cytological level.
During the past 25 years, characterization of ANAs has helped to identify many
nuclear proteins by their subcellular localization. The commonly used technique for
detection of ANA, immunofluorescence microscopy, represents a valuable tool for both the clinician to identify certain autoantibody specificities to diagnose subsets of systemic autoimmune diseases and the biomedical researcher to analyze the archi- tecture and function of the cell nucleus. Immunofluorescence enables the visualiza- tion of antigens and the identification of structures in cells and tissues. The emitted signal of excited fluorochromes is viewed against a black background, thus providing high contrast. In addition, fluorescence imaging delivers superb specificity. These advantages of immunofluorescence techniques can be further enhanced by confocal laser scanning microscopy. Confocal microscopes differ from conventional (wide- field) microscopes because they fade out out-of-focus signals. In a confocal micro- scope, most of the out-of-focus light is excluded from the final image, which greatly increases the contrast and the visibility of fine details in the specimen. Thus, confo- cal microscopy may lead to refined analyses of nuclear autoantigens as well as new opportunities for differential diagnosis of systemic rheumatic diseases and identi- fication of (new) autoantigens, which might have been missed by conventional epi- fluorescence microscopy (Hemmerich and von Mikecz 2000).
Figure 24.1 shows confocal imaging of an autoantibody that is specifically directed against Ro52 (Fig. 24.1C, lane 2). Confocal sectioning of a human epidermal cell line (HEp-2) reveals a unique staining pattern of bright speckles embedded in a weak homogeneous staining of the nucleoplasm (Fig. 24.1A, B, green color), whereas nucle- oli as well as the cytoplasm are not labeled. The typical feature of a confocal Ro/SSA image is the visibility of one speckle in three to four focal planes (Fig. 24.1A, arrows), suggesting that nucleoplasmic Ro/SSA speckles have a size of approximately 2 µm.
Thus, Ro/SSA speckles can be easily and unmistakably distinguished from splicing speckles, which occupy a larger subnuclear area, and from other nuclear structures, such as nucleoli, centromeres, and nuclear bodies; the latter display a staining pattern of smaller dots.
Many clinical laboratories still use immunodiffusion or counterimmunoelectro- phoresis for the detection of anti-Ro/SSA antibodies, whereas biomedical research laboratories apply immunofluorescence, immunoblotting, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) techniques. The early work leading to the discovery of anti-Ro/SSA antibodies was based on immunodiffusion using extracts from human thyroid, spleen, and calf thymus (Clark et al. 1969, Alspaugh and Tan 1975). The method is easy and quick; however, the substrates are rather unspe- cific and thus may lead to misleading results, especially when appropriate control sera are not available. In terms of specificity, immunoblotting and immunprecipitation are the methods of choice. Extracts of MOLT-4 and HeLa cell lines have been used suc- cessfully for the detection of Ro/SSA antigens by immunoblotting (Ben-Chetrit et al.
1988, Rader et al. 1989). Immunoblotting enables the exact identification of the target antigens by determination of their molecular weight. With the cloning of Ro/SSA52 and Ro/SSA60 proteins, purified recombinant proteins have become the preferred substrates in ELISAs (Chan et al. 1991). However, ELISAs are expensive and prone to false-positive results, since the technique does not allow definition of molecular and cell biological properties of the substrate.
In contrast, a combination of immunofluorescence and immunoblotting provides
information on the subcellular distribution and molecular weight (= integrity) of tar-
get antigens, and thus allows exact identification of ANA specificities. Moreover, the
use of confocal microscopes delivers subnuclear immunofluorescent staining pat- terns in such detail that nuclear autoantigens can be distinguished by exclusive employment of this imaging technique (see previously). Since the new instruments are easy to use and become less and less expensive, it is safe to assume that confocal laser scanning microscopy will find its way from the research laboratories to the clini- cal routine laboratories.
B-Cell Epitopes of Ro/SSA
A major goal of immunochemistry is to define the molecular basis of receptor recog- nition, which requires the analysis of structure and function of (1) the receptor, (2) the ligand, and (3) the interface between the two. The portion of the antigen that is recognized by an antibody during the binding reaction is called “antigenic determi- nant” or “epitope”. Structurally defined antibody-protein complexes share common features: 15–22 amino acid residues contact each other on both antibody and antigen molecules (Laver et al. 1990, Smith-Gill 1994). Epitopes are usually classified as either continuous or discontinuous, consisting of linear peptides or amino acid residues brought together by folding of the polypeptide chain, respectively.
Patients with autoimmune connective tissue diseases such as SLE characteristically produce autoantibodies that recognize continuous or discontinuous B-cell epitopes.A considerable heterogeneity of epitopes has been targeted by patient sera (Chou et al.
1992, van Venrooij et al. 1994). Properties of autoantigenic epitopes seem to be similar to those of foreign protein antigens in that they reside in hydrophilic and highly con- served domains (Elkon et al. 1988). However, ANAs are often directed against func- tional regions of the conserved antigenic molecules (Chan and Tan 1989), and auto- antigens of systemic rheumatic diseases are generally either basic or contain extended, multivalent, charge-rich sequence motifs such as coiled-coils (Dohlman et al. 1993).
Numerous studies described the definition of important epitopes on the RoRNP complex. Autoepitopes of Ro/SSA60 have been reported by several authors using dif- ferent biochemical methods. The most commonly used methods are to test autoim- mune sera (a) with successively truncated recombinant fragments of the autoantigen by immunoblot or immunoprecipitation or (b) with overlapping synthetic peptides in ELISA. Although detection techniques differed between the studies, positions of major epitopes within Ro/SSA60 and Ro/SSA52 were found to be consistent. The major antigenic region of Ro/SSA60 resides in the middle of the protein, between amino acid residues 150 and 320 (Saitta et al. 1994, Scofield and Harley 1991, Wahren et al. 1996). Accordingly, sera from most patients with NLE, SCLE, and Sjögren’s syn- drome recognized an epitope within residues 139 and 326 (McCauliffe et al. 1994).
The major epitope of Ro/SSA52 has been mapped to the center of the protein within residues 136–292 (Buyon et al. 1994, Frank et al. 1994, Kato et al. 1995, Dorner et al.
1996). It turned out that the major antigenic determinant of the Ro/SSA60 antigen is the most hydrophilic and presumably the most exposed part of the protein. The most frequently identified antigenic domains of Ro/SSA52 contain a putative coiled-coil domain (Kastner et al. 1992) and a leucine zipper motif (Chan et al. 1991, Itoh et al.
1991). Taken together, the data suggest that the similarities in the autoimmune
response against Ro/SSA as observed with different (a) patient sera and (b) detection
methods results from an immune response that is stimulated by endogenous autoantigens. Such immune responses are also referred to as antigen driven.
The idea that Ro/SSA autoantibodies generate during antigen-driven immune responses is corroborated by results from animal experiments. In contrast to the epi- tope mapping with patient sera, monoclonal antibodies raised in mice recognized various antigenic regions on Ro/SSA60 and Ro/SSA52 that were not confined to the middle part but that are distributed all over the proteins (Schmitz et al. 1997, Veld- hoven et al. 1995). Immunization of mice (Topfer et al. 1995) and rabbits (Scofield et al. 1996) with recombinant Ro/SSA antigens or synthetic Ro/SSA peptides leads to intramolecular and intermolecular spreading of the immune reactivity, referred to as epitope spreading. It is thought that once an autoimmune response to one antigenic determinant has developed it might spread to associated proteins. However, to date, it is far from clear whether such an epitope spreading (a) occurs in patients during the onset of systemic autoimmune responses or (b) merely represents an artifact resulting from immunization of animals with exogenous proteins.
To address the issue of discontinuous epitopes on the RoRNP particle, Saitta et al.
(Saitta et al. 1994) generated 10 overlapping recombinant polypeptides of the human 60-kDa Ro/SSA protein and compared their reactivities to immunoprecipitation of in vitro translated, native Ro60. Patient sera that immunoprecipitated full-length Ro/SSA60 had low or undetectable anti-Ro/SSA60 titers by ELISA and immunoblot- ting using recombinant antigens. These results suggest that discontinuous epitopes are the predominant targets of anti-Ro/SSA60 autoantibodies. It is important to note that during immunoprecipitation and immunofluorescence, the autoantigens retain their native structure, whereas in ELISA and immunoblotting, the substrate proteins are more or less denatured, which favors the exclusive detection of linear epitopes.
Since ANAs in general represent very useful tools for immunoprecipitation, it was concluded that they preferentially recognize discontinuous epitopes on RNP com- plexes. Using ribonuclease protection assays, Wolin and Steitz (Wolin and Steitz 1984) discovered the major epitope of anti-Ro/SSA antibodies on the RoRNP particle. For each of the three human Ro RNAs whose sequence was known at that time, the most highly protected portion was found in immunoprecipitates corresponding to the lower section of the stem formed by base pairing the 5' and 3' ends of the RNA (see Fig. 24.2). These results confirmed that the complex of Ro/SSA60 and the lower stem of Ro RNA forms a major, discontinuous epitope for anti-Ro/SSA autoantibodies.
Outlook/Perspectives
Although the structure of Ro/SSA autoantigens and the antigenic targets (epitopes) for anti-Ro/SSA autoantibodies have been characterized in detail, two important challenges should be addressed in future research: (a) the function of the RoRNP par- ticle is still unknown and (b) the molecular causes and mechanisms leading to sys- temic autoimmune responses against RoRNPs remain to be identified.
Since autoantibodies cannot penetrate into the cells, the central question is: How
is an immune response against intracellular, nuclear proteins generated? Processing
and presentation of antigens represents the prerequisite for every antigen-driven
(auto)immune response. To date, little information exists on how nuclear autoanti-
gens are processed and where this processing takes place in the cell, in situ. Recent evidence suggests that nuclear autoantigens are subjected to proteasomal proteolysis (Desai et al. 1997, Everett et al. 1999) within the nucleus (Chen et al. 2002, Rockel and von Mikecz 2002). Since proteasomes constitute a major proteolytic system that delivers peptides to antigen presentation, these new findings are the basis for the fol- lowing hypothesis on the generation of systemic autoimmune responses: alteration of nuclear structure and function may result in recruitment of autoantigens to antigen- processing proteases, for example, the proteasome system. Previously cryptic nuclear proteins may be efficiently processed into peptides and subjected to antigen presen- tation. Thus, new antigenic peptides might appear on the surface of antigen-present- ing cells, which are mistakenly interpreted as “non self ” or foreign by T lymphocytes, and an autoimmune response is initiated against components of the body’s own cells.
Sensitivity to sun is a cardinal feature of SLE, and UV light may be involved in its pathogenesis (Chen et al. 2000). In this respect, it is intriguing that the presence of anti-Ro/SSA antibodies in patients suffering from rheumatic disease is highly corre- lated with photosensitive dermatitis (von Muhlen and Tan 1995). According to the above-mentioned hypothesis, UV irradiation might induce recruitment of Ro/SSA to proteasomal proteolysis. Thus, future investigations on processing and presentation of RoRNPs and their intracellular trafficking may permit more insight into the molecular mechanisms that cause the generation of autoimmune responses and the production of anti-Ro/SSA antibodies.
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