- Email: [email protected]
Fibrillarin Autoantibodies
CHAPTER
Fibrillarin Autoantibodies
38 K. Michael Pollard1 and Per Hultman2
1Department
of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, 2Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology, Linköping University, Linköping, Sweden
Historical notes The sera of patients with rheumatic diseases were reported to contain antibodies to nucleolar structures by Beck in 1961, and further studies in the 1970s showed that such antinucleolar antibodies (ANoA) were more frequent in patients with scleroderma. Immunofluorescence (IF) microscopy was used to describe different nucleolar staining patterns using sera from patients with scleroderma. The pattern described as “clumpy” was later shown to be due to antibodies to a nucleolar protein [1] named fibrillarin because of its localization to the dense fibrillar and fibrillar center regions of the nucleolus. In 1983, Reddy and coworkers reported that some scleroderma sera precipitated particles containing U3 ribonucleic acid (RNA). U3 RNA is a member of a family of small nucleolar RNAs (snoRNAs) called box C/D snoRNA because of the presence of conserved sequences close to the 5’ and 3’ ends of the RNA. Fibrillarin, together with a number of other proteins, is a constituent of box C/D snoRNA-protein complexes called small nucleolar ribonucleoprotein particles, or snoRNP particles [2].
Autoantigen Definition Mammalian fibrillarin (pronounced fi-brill-a-rin) was detected using serum from a patient with scleroderma, which contained autoantibodies monospecific for a protein with an apparent molecular weight of 34 kDa. Fibrillarin shows a high degree of evolutionary conservation as an analogous protein, called B-36, which occurs in the slime mold Physarum polycephalum.
Biological function Fibrillarin is involved in pre-ribosomal ribonucleic acid (pre-rRNA) processing. Specifically, fibrillarin utilizes the methyl donor S-adenosyl-L-methionine to catalyze the methylation of 2’-hydroxyl ribose moieties in pre-rRNA. It has been determined that box C/D snoRNAs have a methylation guide function that consists of antisense sequence elements that are complementary to a site of rRNA 2’-O-ribose Autoantibodies. http://dx.doi.org/10.1016/B978-0-444-56378-1.00038-1 Copyright © 2014 Elsevier B.V. All rights reserved.
319
320
CHAPTER 38 Fibrillarin Autoantibodies
methylation [2]. It is believed that highly conserved, basic residues on the surface of fibrillarin promote rRNA interaction and aid in accurate transfer of the methyl group.
Origin/sources Fibrillarin is present in all nucleated cell types. Purified recombinant human fibrillarin is commercially available from Creative Biomart, New York, US and Prospec, New Jersey, US. Antifibrillarin antibodies are commercially available from numerous sources including Abcam, Massachusetts, US (rabbit and mouse serum), Cytoskeleton, Colorado, US (mouse monoclonal 72B9), EnCor Biotechnology, Florida, US (mouse monoclonal 38F3), and Everest Biotech, Upper Heyford, UK (goat serum).
Methods of purification Fibrillarin was initially purified from nucleoli using LiCl and urea followed by phosphocellulose column chromatography. More recent methods include purification of recombinant protein from bacterial and insect cell lines transfected with expression vectors containing fibrillarin complementary deoxyribonucleic acid (cDNA). Fibrillarin and/or fibrillarin-containing ribonucleoprotein (RNP) particles have been purified by affinity chromatography using reagents specific for either fibrillarin or its associated RNA components.
Autoantibody Definition Historically antifibrillarin antibodies have been called anti-U3-RNP or anti-(U3) small nuclear ribonucleic particle (snRNP) antibodies. However, fibrillarin is not a U3 snoRNP-specific protein. Patient sera that immunoprecipitate individual snoRNPs (e.g., U3 or U8) do not contain antifibrillarin antibodies, as determined by immunoblot of recombinant fibrillarin [3]. U3 snoRNP-specific antigenic proteins include Mpp10 and hU3-55K. In a study examining immunoprecipitation (IP) of individual snoRNP proteins, patient sera positive for anti-hU3-55K were found to contain antifibrillarin, but several sera were identified that immunoprecipitated Mpp10 but not fibrillarin [4]. Together with a number of other proteins, such as Nop56, Nop58/Nop5, and Snu13, fibrillarin is a component of the snoRNPs that contain box C/D RNAs (e.g., U3, U8, U13, U22). IP of U3, U8, and U13 snoRNAs (i.e., box C/D) has been used to identify antifibrillarin antibodies, as all positive sera also immunoblot recombinant fibrillarin [3]. However, care must be taken in assuming that IP of box C/D snoRNPs is a specific feature of antifibrillarin antibodies. IP of box C/D components (Nop58, Nop56) is difficult, as they interact with endogenous fibrillarin in the reticulocyte lysate used in transcription/translation assays and are coprecipitated by antifibrillarin antibodies [4]. It has proven difficult to exclude the possibility that box C/D snoRNPs contain antigenic components other than fibrillarin. Given the potential antigenic complexity of the snoRNPs we propose the abbreviation AFA (antifibrillarin antibody) be used when denoting use of detection assays that use fibrillarin as a pure antigen (see “Methods of detection” below). Nomenclature that uses snoRNP/RNA terminology (anti-U3, anti-box C/D) should not be used as surrogates for AFA.
Autoantibody
321
Pathogenic role Similar to other autoantibodies reacting with intracellular antigens, AFA have not been shown to interact directly with intracellular fibrillarin in vivo. Microinjection of antifibrillarin antibodies modifies the ultrastructural organization of the nucleoli and prenucleolar bodies (PNBs). This antibody-mediated blocking of fibrillarin translocation reduces or inhibits RNA pol I transcription.
Genetics Studies show that AFA appear more frequently in patients of African ancestry with scleroderma than their Caucasian counterparts. In addition, patients with AFA show significantly greater frequency of the HLA-DQ6 alleles DQB1*0602 and/or *0604 (carried on HLA-DR2 and DR13 haplotypes) [5].
Methods of detection The most convenient screening method for detection of AFA is the indirect IF (IIF) test using cultured cells as the substrate in order to detect nucleolar staining. Various ANoA patterns were described in the early 1980s, and it was subsequently shown that the pattern described as “clumpy” with bright granules decorating the nucleoli corresponds to AFA (Fig. 38.1). Another characteristic that aids in identification is a distinct staining of the condensed chromosomes in metaphase cells, whereas interphase cells show no staining of the nucleoplasm. This differentiates AFA staining from that caused by PM/Scl and RNApol I, which give other patterns in metaphase cells and a weak staining of the nucleoplasm in interphase cells. Recognition of the different nucleolar patterns requires experience and is facilitated by viewing
FIGURE 38.1 Indirect immunofluorescence (IIF) pattern of antifibrillarin autoantibodies on HEp-2 cells. The nucleolar fluorescence reveals a characteristic “clumpy” pattern in interphase cells.
322
CHAPTER 38 Fibrillarin Autoantibodies
the nucleolar staining in various focal planes. Diluting the serum may also be helpful. The clumpy nucleolar staining is often combined with two to six brightly staining dots in the nucleoplasm identified as Cajal or coiled bodies that contain fibrillarin. Using monospecific antiserum as a reference, AFA can be readily identified by immunoblotting (IB) using purified nuclei or nucleoli as an antigenic source. IP of radiolabeled extracts from cell culture lines has been used to show that fibrillarin is an antigenic component of macromolecular complexes containing protein and RNA. Radiolabeling of protein with [35S]-methionine reveals the presence of at least six proteins of 12.5, 13, 30, 36 (fibrillarin), 59, and 74 kDa, the latter two being phosphoproteins. Use of 32PO4 to label nucleic acids showed the association of fibrillarin with a number of snRNAs, the most predominant of which is the 217 nucleotide long U3 snoRNA. It must be noted that the above techniques (IIF, IB, and IP (of radiolabelled cell extracts)) do not unequivocally identify antifibrillarin autoantibodies because a positive reaction could be due to interaction with an snoRNP component other than fibrillarin, or a nuclear component with the same cellular localization or molecular weight as fibrillarin (see “Autoantibody: Definition” above). Molecular cloning of cDNA encoding fibrillarin from a variety of species has allowed in vitro synthesis of the protein, which can then be used for unambiguous detection of antifibrillarin autoantibodies. Two methods can be used. Radiolabeled and nonradiolabeled protein produced by in vitro transcription and translation using rabbit reticulocyte lysate [4] produces protein most closely resembling that produced in vivo. Synthesis of protein using such a cell-free eukaryotic system can include posttranslational modifications and disulfide bond formation, thereby aiding in protein folding and attainment of correct three-dimensional structure. Such material can then be used in IP under fluid phase conditions to maintain structural integrity. Fibrillarin has also been expressed from cDNA in bacteria as a fusion protein and used in immunoblot to detect antifibrillarin antibodies [6]. Bacterially expressed recombinant proteins must be purified to reduce nonspecific reaction with bacterial antigens, and their use in immunoblot means denaturation of the protein and possible loss of reactivity with autoantibodies directed against conformational determinants. This is of importance in detecting antifibrillarin autoantibodies, as a common reactivity appears to be against a highly conserved conformational epitope [7]. The recommended methodology for detection of antifibrillarin autoantibody (AFA) would include IF detection of the “clumpy” nucleolar staining pattern, and if possible colocalization of staining to nuclear Cajal bodies. To confirm the reactivity, IP should be performed with radiolabeled and nonradiolabeled protein from transcribed/translated fibrillarin cDNA or IB using purified recombinant protein. IB of nuclei or nucleoli should be considered an alternative only if IP techniques are unavailable. In all assays, standardized positive and negative control serum must be used. Commercial assays for antifibrillarin include the Systemic Sclerosis (Nucleoli) Profile Euroline (IgG) lineblot assay (LB) (Euroimmun, Germany) and the Phadia EliA connective tissue disease (CTD) screen test (Phadia, Sweden).
Clinical utility Disease association Although antifibrillarin antibodies occur in less than 15% of patients with systemic sclerosis (scleroderma, SSc) (Table 38.1), they are highly associated with diffuse cutaneous scleroderma. AFA have
Clinical utility
323
Table 38.1 Antinucleolar and Antifibrillarin Antibodies in Selected Studies Study Group
ANoA (Total)
ANoA SSc
AFA
Reference SSc#
4500 sera
172*
25%
7% (1/14) 28% (3/11) SLE# 20% (2/10) pRP# 8% (8/100)ANoA+#
[3]
220 SSc
59*
27%
14% (31/220) SSc## 52% (31/59) ANoA+##
[4]
646 SSc
53**
8%
48% (22/46) ANoA+$
[1]
335 SSc
69*
21%
8% (27/335) SSc## 39% (27/69) ANoA+##
[5]
ANA substrate: ANoA: antinucleolar antibody; AFA: antifibrillarin antibody, SSc: systemic sclerosis (scleroderma); SjS: Sjögren syndrome; SLE: systemic lupus erythematosus; pRP: primary Raynaud phenomenon. *HEp-2 cells **rat liver. AFA detection #box C/D snoRNA immunoprecipitation, recombinant fibrillarin immunoblot ##in vitro transcribed and translated fibrillarin immunoprecipitation $HeLa nucleoli immunoblot.
been found more frequently in SSc patients of African descent than Caucasian and in patients with internal organ involvement including pulmonary hypertension, myositis, and renal disease [5,8]. AFA have been found associated with severe scleroderma when an antigen-specific assay has been used (i.e., recombinant or in vitro transcribed/translated protein) [3,5]. A recent study has questioned the restriction of AFA to SSc [3]. This study selected ANoA-positive sera from over 4500 serum samples tested for autoantibodies by IIF on HEp-2 cells. ANoA were found to occur more frequently in scleroderma (25%) than other autoimmune diseases (Sjögren syndrome 11%, systemic lupus erythematosus (SLE) < 7%). However, AFA were found more frequently in ANoA-positive SLE (28%) and primary Raynaud phenomenon (20%) than SSc (8%) (Table 38.1). These results are somewhat different when compared to other studies where the numbers of AFA-positive sera in ANoA-positive scleroderma patients lie between 40% and 50% (Table 38.1). Confirmation of the presence of AFA in diseases other than scleroderma and their clinical relevance requires further investigation.
Disease prevalence In the US, the estimated incidence of scleroderma is 19 cases per million population, and the prevalence is 240 cases per million population. However, it must be noted that very limited data exists to estimate the incidence of autoimmune diseases, as many published studies estimate incidence in relatively small or geographically limited populations.
324
CHAPTER 38 Fibrillarin Autoantibodies
Diagnostic value Specificity Initial studies on the presence of antifibrillarin antibodies suggested a restriction to scleroderma. This was based on the observation that numerous sera selected from other autoimmune diseases were negative for AFA. However examination of the presence of AFA in ANoA-positive sera has argued that AFA can occur in several systemic autoimmune diseases (e.g., SLE, Raynaud phenomenon, and SSc) [3]. Thus, the presence of AFA in a serum sample should not be considered to have diagnostic significance.
Sensitivity A number of studies have examined the frequency of AFA in scleroderma and found that 7–14% of patients can be positive. Several examples are given in Table 38.1. However, it must be noted that the presence of AFA can be influenced by both gender and ethnic background [1,5].
Prognostic value Disease activity In studies with scleroderma patients, antifibrillarin antibodies have been found to occur most often in patients with the diffuse cutaneous form. It has been suggested that the antibody identifies a subset of SSc patients who are often of African descent and with serious cutaneous and visceral disease [5,9]. Although there have been no published studies on the prognostic value of AFA, it has been argued that clinical features common in AFA-positive patients identify a group with a poorer prognosis [3,9,10].
Organ involvement/damage In scleroderma patients, cardiac, renal, and gastrointestinal involvement correlate with the presence of AFA [4,5,10]. However, as noted above, care must be taken when interpreting the clinical features of scleroderma patients with antifibrillarin antibodies, as one study has suggested that the presence or absence of other U3 snoRNP specific antibodies (e.g., anti-Mpp10, anti-hU3-55K) may influence clinical associations [4].
Clinical utility of the different methods of detection As noted above (see “Autoantibody: Definition”), fibrillarin is a component of box C/D snoRNP particles. Studies have suggested that autoantibodies to nonfibrillarin components can precipitate the U3 snoRNP [3,4], and there is potential for recognition of nonfibrillarin components of box C/D snoRNP [4]. These observations stress the importance of using AFA assays that are specific for fibrillarin. At present only two assays, IP of in vitro transcribed and translated fibrillarin [4,5] and IB of recombinant protein [3], provide the necessary specificity. Neither assay is in widespread clinical use.
Take-home messages • A ntifibrillarin antibodies recognize a highly conserved 34-kDa nucleolar protein that is a major component of box C/D small nucleolar ribonucleoprotein (snoRNP) particles. • Fibrillarin is an S-adenosylmethionine-dependent methyltransferase of rRNA.
References
325
• D ue to the antigenic complexity of snoRNPs the term antifibrillarin antibody (or AFA) should only be used when denoting use of detection assays that use fibrillarin as a pure antigen. Nomenclature that uses snoRNP/RNA terminology (anti-U3, anti-box C/D) should not be used as surrogates for AFA (antifibrillarin antibody). • Antifibrillarin antibodies are more frequent in scleroderma patients but are not diagnostic, as they have been found patients with SLE and primary Raynaud phenomenon. • In scleroderma, antifibrillarin antibodies occur in less than 15% of patients and identify a subset of SSc patients who are often of African descent and with serious cutaneous and visceral disease.
Acknowledgments The authors’ studies cited in this article were supported by National Institutes of Health grants ES014847, and ES020388 to KMP, and a grant from the Swedish Medical Research Council (project no. 9453) to PH.
References [1] Reimer G, Steen VD, Penning CA, Medsger Jr TA, Tan EM. Correlates between autoantibodies to nucleolar antigens and clinical features in patients with systemic sclerosis (scleroderma). Arthritis Rheum 1988;31:525–32. [2] Champion EA, Baserga SJ. Autoantibody recognition of macromolecular structures and their subunits. In: Pollard KM, editor. Autoantibodies and Autoimmunity: Molecular Mechanisms in Health and Disease. Weinheim: Wiley-VCH; 2006. p. 379–417. [3] Van Eenennaam H, Vogelzangs JH, Bisschops L, Te Boome LC, Seelig HP, Renz M, et al. Autoantibodies against small nucleolar ribonucleoprotein complexes and their clinical associations. Clin Exp Immunol 2002;130:532–40. [4] Yang JM, Hildebrandt B, Luderschmidt C, Pollard KM. Human scleroderma sera contain autoantibodies to protein components specific to the U3 small nucleolar RNP complex. Arthritis Rheum 2003;48:210–7. [5] Arnett FC, Reveille JD, Goldstein R, Pollard KM, Leaird K, Smith EA, et al. Autoantibodies to fibrillarin in systemic sclerosis (scleroderma). An immunogenetic, serologic, and clinical analysis. Arthritis Rheum 1996;39:1151–60. [6] Pearson DL, Reimonenq RD, Pollard KM. Expression and purification of recombinant mouse fibrillarin. Protein Expr Purif 1999;17:49–56. [7] Mahler M, Bluthner M, Pollard KM. Advances in B-cell epitope analysis of autoantigens in connective tissue diseases. Clin Immunol 2003;107:65–79. [8] Cepeda EJ, Reveille JD. Autoantibodies in systemic sclerosis and fibrosing syndromes: clinical indications and relevance. Curr Opin Rheumatol 2004;16:723–32. [9] Reveille JD, Fischbach M, McNearney T, Friedman AW, Aguilar MB, Lisse J, et al. Systemic sclerosis in 3 US ethnic groups: a comparison of clinical, sociodemographic, serologic, and immunogenetic determinants. Semin Arthritis Rheum 2001;30:332–46. [10] Steen VD. Autoantibodies in systemic sclerosis. Semin Arthritis Rheum 2005;35:35–42.
Fibrillarin Autoantibodies
38 K. Michael Pollard1 and Per Hultman2
1Department
of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, 2Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology, Linköping University, Linköping, Sweden
Historical notes The sera of patients with rheumatic diseases were reported to contain antibodies to nucleolar structures by Beck in 1961, and further studies in the 1970s showed that such antinucleolar antibodies (ANoA) were more frequent in patients with scleroderma. Immunofluorescence (IF) microscopy was used to describe different nucleolar staining patterns using sera from patients with scleroderma. The pattern described as “clumpy” was later shown to be due to antibodies to a nucleolar protein [1] named fibrillarin because of its localization to the dense fibrillar and fibrillar center regions of the nucleolus. In 1983, Reddy and coworkers reported that some scleroderma sera precipitated particles containing U3 ribonucleic acid (RNA). U3 RNA is a member of a family of small nucleolar RNAs (snoRNAs) called box C/D snoRNA because of the presence of conserved sequences close to the 5’ and 3’ ends of the RNA. Fibrillarin, together with a number of other proteins, is a constituent of box C/D snoRNA-protein complexes called small nucleolar ribonucleoprotein particles, or snoRNP particles [2].
Autoantigen Definition Mammalian fibrillarin (pronounced fi-brill-a-rin) was detected using serum from a patient with scleroderma, which contained autoantibodies monospecific for a protein with an apparent molecular weight of 34 kDa. Fibrillarin shows a high degree of evolutionary conservation as an analogous protein, called B-36, which occurs in the slime mold Physarum polycephalum.
Biological function Fibrillarin is involved in pre-ribosomal ribonucleic acid (pre-rRNA) processing. Specifically, fibrillarin utilizes the methyl donor S-adenosyl-L-methionine to catalyze the methylation of 2’-hydroxyl ribose moieties in pre-rRNA. It has been determined that box C/D snoRNAs have a methylation guide function that consists of antisense sequence elements that are complementary to a site of rRNA 2’-O-ribose Autoantibodies. http://dx.doi.org/10.1016/B978-0-444-56378-1.00038-1 Copyright © 2014 Elsevier B.V. All rights reserved.
319
320
CHAPTER 38 Fibrillarin Autoantibodies
methylation [2]. It is believed that highly conserved, basic residues on the surface of fibrillarin promote rRNA interaction and aid in accurate transfer of the methyl group.
Origin/sources Fibrillarin is present in all nucleated cell types. Purified recombinant human fibrillarin is commercially available from Creative Biomart, New York, US and Prospec, New Jersey, US. Antifibrillarin antibodies are commercially available from numerous sources including Abcam, Massachusetts, US (rabbit and mouse serum), Cytoskeleton, Colorado, US (mouse monoclonal 72B9), EnCor Biotechnology, Florida, US (mouse monoclonal 38F3), and Everest Biotech, Upper Heyford, UK (goat serum).
Methods of purification Fibrillarin was initially purified from nucleoli using LiCl and urea followed by phosphocellulose column chromatography. More recent methods include purification of recombinant protein from bacterial and insect cell lines transfected with expression vectors containing fibrillarin complementary deoxyribonucleic acid (cDNA). Fibrillarin and/or fibrillarin-containing ribonucleoprotein (RNP) particles have been purified by affinity chromatography using reagents specific for either fibrillarin or its associated RNA components.
Autoantibody Definition Historically antifibrillarin antibodies have been called anti-U3-RNP or anti-(U3) small nuclear ribonucleic particle (snRNP) antibodies. However, fibrillarin is not a U3 snoRNP-specific protein. Patient sera that immunoprecipitate individual snoRNPs (e.g., U3 or U8) do not contain antifibrillarin antibodies, as determined by immunoblot of recombinant fibrillarin [3]. U3 snoRNP-specific antigenic proteins include Mpp10 and hU3-55K. In a study examining immunoprecipitation (IP) of individual snoRNP proteins, patient sera positive for anti-hU3-55K were found to contain antifibrillarin, but several sera were identified that immunoprecipitated Mpp10 but not fibrillarin [4]. Together with a number of other proteins, such as Nop56, Nop58/Nop5, and Snu13, fibrillarin is a component of the snoRNPs that contain box C/D RNAs (e.g., U3, U8, U13, U22). IP of U3, U8, and U13 snoRNAs (i.e., box C/D) has been used to identify antifibrillarin antibodies, as all positive sera also immunoblot recombinant fibrillarin [3]. However, care must be taken in assuming that IP of box C/D snoRNPs is a specific feature of antifibrillarin antibodies. IP of box C/D components (Nop58, Nop56) is difficult, as they interact with endogenous fibrillarin in the reticulocyte lysate used in transcription/translation assays and are coprecipitated by antifibrillarin antibodies [4]. It has proven difficult to exclude the possibility that box C/D snoRNPs contain antigenic components other than fibrillarin. Given the potential antigenic complexity of the snoRNPs we propose the abbreviation AFA (antifibrillarin antibody) be used when denoting use of detection assays that use fibrillarin as a pure antigen (see “Methods of detection” below). Nomenclature that uses snoRNP/RNA terminology (anti-U3, anti-box C/D) should not be used as surrogates for AFA.
Autoantibody
321
Pathogenic role Similar to other autoantibodies reacting with intracellular antigens, AFA have not been shown to interact directly with intracellular fibrillarin in vivo. Microinjection of antifibrillarin antibodies modifies the ultrastructural organization of the nucleoli and prenucleolar bodies (PNBs). This antibody-mediated blocking of fibrillarin translocation reduces or inhibits RNA pol I transcription.
Genetics Studies show that AFA appear more frequently in patients of African ancestry with scleroderma than their Caucasian counterparts. In addition, patients with AFA show significantly greater frequency of the HLA-DQ6 alleles DQB1*0602 and/or *0604 (carried on HLA-DR2 and DR13 haplotypes) [5].
Methods of detection The most convenient screening method for detection of AFA is the indirect IF (IIF) test using cultured cells as the substrate in order to detect nucleolar staining. Various ANoA patterns were described in the early 1980s, and it was subsequently shown that the pattern described as “clumpy” with bright granules decorating the nucleoli corresponds to AFA (Fig. 38.1). Another characteristic that aids in identification is a distinct staining of the condensed chromosomes in metaphase cells, whereas interphase cells show no staining of the nucleoplasm. This differentiates AFA staining from that caused by PM/Scl and RNApol I, which give other patterns in metaphase cells and a weak staining of the nucleoplasm in interphase cells. Recognition of the different nucleolar patterns requires experience and is facilitated by viewing
FIGURE 38.1 Indirect immunofluorescence (IIF) pattern of antifibrillarin autoantibodies on HEp-2 cells. The nucleolar fluorescence reveals a characteristic “clumpy” pattern in interphase cells.
322
CHAPTER 38 Fibrillarin Autoantibodies
the nucleolar staining in various focal planes. Diluting the serum may also be helpful. The clumpy nucleolar staining is often combined with two to six brightly staining dots in the nucleoplasm identified as Cajal or coiled bodies that contain fibrillarin. Using monospecific antiserum as a reference, AFA can be readily identified by immunoblotting (IB) using purified nuclei or nucleoli as an antigenic source. IP of radiolabeled extracts from cell culture lines has been used to show that fibrillarin is an antigenic component of macromolecular complexes containing protein and RNA. Radiolabeling of protein with [35S]-methionine reveals the presence of at least six proteins of 12.5, 13, 30, 36 (fibrillarin), 59, and 74 kDa, the latter two being phosphoproteins. Use of 32PO4 to label nucleic acids showed the association of fibrillarin with a number of snRNAs, the most predominant of which is the 217 nucleotide long U3 snoRNA. It must be noted that the above techniques (IIF, IB, and IP (of radiolabelled cell extracts)) do not unequivocally identify antifibrillarin autoantibodies because a positive reaction could be due to interaction with an snoRNP component other than fibrillarin, or a nuclear component with the same cellular localization or molecular weight as fibrillarin (see “Autoantibody: Definition” above). Molecular cloning of cDNA encoding fibrillarin from a variety of species has allowed in vitro synthesis of the protein, which can then be used for unambiguous detection of antifibrillarin autoantibodies. Two methods can be used. Radiolabeled and nonradiolabeled protein produced by in vitro transcription and translation using rabbit reticulocyte lysate [4] produces protein most closely resembling that produced in vivo. Synthesis of protein using such a cell-free eukaryotic system can include posttranslational modifications and disulfide bond formation, thereby aiding in protein folding and attainment of correct three-dimensional structure. Such material can then be used in IP under fluid phase conditions to maintain structural integrity. Fibrillarin has also been expressed from cDNA in bacteria as a fusion protein and used in immunoblot to detect antifibrillarin antibodies [6]. Bacterially expressed recombinant proteins must be purified to reduce nonspecific reaction with bacterial antigens, and their use in immunoblot means denaturation of the protein and possible loss of reactivity with autoantibodies directed against conformational determinants. This is of importance in detecting antifibrillarin autoantibodies, as a common reactivity appears to be against a highly conserved conformational epitope [7]. The recommended methodology for detection of antifibrillarin autoantibody (AFA) would include IF detection of the “clumpy” nucleolar staining pattern, and if possible colocalization of staining to nuclear Cajal bodies. To confirm the reactivity, IP should be performed with radiolabeled and nonradiolabeled protein from transcribed/translated fibrillarin cDNA or IB using purified recombinant protein. IB of nuclei or nucleoli should be considered an alternative only if IP techniques are unavailable. In all assays, standardized positive and negative control serum must be used. Commercial assays for antifibrillarin include the Systemic Sclerosis (Nucleoli) Profile Euroline (IgG) lineblot assay (LB) (Euroimmun, Germany) and the Phadia EliA connective tissue disease (CTD) screen test (Phadia, Sweden).
Clinical utility Disease association Although antifibrillarin antibodies occur in less than 15% of patients with systemic sclerosis (scleroderma, SSc) (Table 38.1), they are highly associated with diffuse cutaneous scleroderma. AFA have
Clinical utility
323
Table 38.1 Antinucleolar and Antifibrillarin Antibodies in Selected Studies Study Group
ANoA (Total)
ANoA SSc
AFA
Reference SSc#
4500 sera
172*
25%
7% (1/14) 28% (3/11) SLE# 20% (2/10) pRP# 8% (8/100)ANoA+#
[3]
220 SSc
59*
27%
14% (31/220) SSc## 52% (31/59) ANoA+##
[4]
646 SSc
53**
8%
48% (22/46) ANoA+$
[1]
335 SSc
69*
21%
8% (27/335) SSc## 39% (27/69) ANoA+##
[5]
ANA substrate: ANoA: antinucleolar antibody; AFA: antifibrillarin antibody, SSc: systemic sclerosis (scleroderma); SjS: Sjögren syndrome; SLE: systemic lupus erythematosus; pRP: primary Raynaud phenomenon. *HEp-2 cells **rat liver. AFA detection #box C/D snoRNA immunoprecipitation, recombinant fibrillarin immunoblot ##in vitro transcribed and translated fibrillarin immunoprecipitation $HeLa nucleoli immunoblot.
been found more frequently in SSc patients of African descent than Caucasian and in patients with internal organ involvement including pulmonary hypertension, myositis, and renal disease [5,8]. AFA have been found associated with severe scleroderma when an antigen-specific assay has been used (i.e., recombinant or in vitro transcribed/translated protein) [3,5]. A recent study has questioned the restriction of AFA to SSc [3]. This study selected ANoA-positive sera from over 4500 serum samples tested for autoantibodies by IIF on HEp-2 cells. ANoA were found to occur more frequently in scleroderma (25%) than other autoimmune diseases (Sjögren syndrome 11%, systemic lupus erythematosus (SLE) < 7%). However, AFA were found more frequently in ANoA-positive SLE (28%) and primary Raynaud phenomenon (20%) than SSc (8%) (Table 38.1). These results are somewhat different when compared to other studies where the numbers of AFA-positive sera in ANoA-positive scleroderma patients lie between 40% and 50% (Table 38.1). Confirmation of the presence of AFA in diseases other than scleroderma and their clinical relevance requires further investigation.
Disease prevalence In the US, the estimated incidence of scleroderma is 19 cases per million population, and the prevalence is 240 cases per million population. However, it must be noted that very limited data exists to estimate the incidence of autoimmune diseases, as many published studies estimate incidence in relatively small or geographically limited populations.
324
CHAPTER 38 Fibrillarin Autoantibodies
Diagnostic value Specificity Initial studies on the presence of antifibrillarin antibodies suggested a restriction to scleroderma. This was based on the observation that numerous sera selected from other autoimmune diseases were negative for AFA. However examination of the presence of AFA in ANoA-positive sera has argued that AFA can occur in several systemic autoimmune diseases (e.g., SLE, Raynaud phenomenon, and SSc) [3]. Thus, the presence of AFA in a serum sample should not be considered to have diagnostic significance.
Sensitivity A number of studies have examined the frequency of AFA in scleroderma and found that 7–14% of patients can be positive. Several examples are given in Table 38.1. However, it must be noted that the presence of AFA can be influenced by both gender and ethnic background [1,5].
Prognostic value Disease activity In studies with scleroderma patients, antifibrillarin antibodies have been found to occur most often in patients with the diffuse cutaneous form. It has been suggested that the antibody identifies a subset of SSc patients who are often of African descent and with serious cutaneous and visceral disease [5,9]. Although there have been no published studies on the prognostic value of AFA, it has been argued that clinical features common in AFA-positive patients identify a group with a poorer prognosis [3,9,10].
Organ involvement/damage In scleroderma patients, cardiac, renal, and gastrointestinal involvement correlate with the presence of AFA [4,5,10]. However, as noted above, care must be taken when interpreting the clinical features of scleroderma patients with antifibrillarin antibodies, as one study has suggested that the presence or absence of other U3 snoRNP specific antibodies (e.g., anti-Mpp10, anti-hU3-55K) may influence clinical associations [4].
Clinical utility of the different methods of detection As noted above (see “Autoantibody: Definition”), fibrillarin is a component of box C/D snoRNP particles. Studies have suggested that autoantibodies to nonfibrillarin components can precipitate the U3 snoRNP [3,4], and there is potential for recognition of nonfibrillarin components of box C/D snoRNP [4]. These observations stress the importance of using AFA assays that are specific for fibrillarin. At present only two assays, IP of in vitro transcribed and translated fibrillarin [4,5] and IB of recombinant protein [3], provide the necessary specificity. Neither assay is in widespread clinical use.
Take-home messages • A ntifibrillarin antibodies recognize a highly conserved 34-kDa nucleolar protein that is a major component of box C/D small nucleolar ribonucleoprotein (snoRNP) particles. • Fibrillarin is an S-adenosylmethionine-dependent methyltransferase of rRNA.
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• D ue to the antigenic complexity of snoRNPs the term antifibrillarin antibody (or AFA) should only be used when denoting use of detection assays that use fibrillarin as a pure antigen. Nomenclature that uses snoRNP/RNA terminology (anti-U3, anti-box C/D) should not be used as surrogates for AFA (antifibrillarin antibody). • Antifibrillarin antibodies are more frequent in scleroderma patients but are not diagnostic, as they have been found patients with SLE and primary Raynaud phenomenon. • In scleroderma, antifibrillarin antibodies occur in less than 15% of patients and identify a subset of SSc patients who are often of African descent and with serious cutaneous and visceral disease.
Acknowledgments The authors’ studies cited in this article were supported by National Institutes of Health grants ES014847, and ES020388 to KMP, and a grant from the Swedish Medical Research Council (project no. 9453) to PH.
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