G.J.M. Pruijn

Anti-Ro52 antibodies frequently co-occur with anti-Jo-1 antibodies in sera from patients with idiopathic inflammatory myopathy

We analysed 112 idiopathic inflammatory myopathy (IIM) sera for the presence of anti‐Ro, anti‐La and anti‐histidyl‐tRNA synthetase (Jo‐1) autoantibodies, and subsequently mapped B cell epitopes on the Ro52 protein recognized by anti‐Ro52+ IIM sera. Sera were characterized by immunoblotting, ELISA and RNA precipitation. Both anti‐Ro60 and anti‐La activity was found in 4% of IIM sera. Anti‐Ro52 antibodies were present in 20% of IIM sera. However, in anti‐Jo‐1+ IIM sera (21%), the frequency of the anti‐Ro52 antibodies was found to be much higher (58%). No cross‐reactivity between anti‐Ro52 and anti‐Jo‐1 antibodies could be detected in these sera. To learn more about the nature of anti‐Ro52 antibodies occurring in IIM sera, we analysed the major epitopes of the Ro52 protein targeted by anti‐Ro52+ IIM sera by immunoprecipitation of in vitro translated Ro52 deletion mutants. The major epitope was mapped in the region bordered by amino acids 126 and 252. This part of the protein includes a long α‐helical region which contains two potential coiled‐coil domains as well as a leucine zipper motif. Although no difference in Ro52 epitope recognition between anti‐Jo‐1+ and anti‐Jo‐1+IIM sera could be observed, our results suggest that the autoimmune response against Ro52 and Jo‐1 in IIM patients is coupled.

The La (SS-B) autoantigen, a key protein in RNA biogenesis, is dephosphorylated and cleaved early during apoptosis.

In the past few years, a role for apoptotic processes in the development of autoimmune diseases has been suggested. An increasing number of cellular proteins, which are modified during apoptosis, has been described, and many of these proteins have been identified as autoantigens. We have studied the effects of apoptosis on the La protein in more detail and for the first time demonstrate that this autoantigen is rapidly dephosphorylated after the induction of apoptosis. Dephosphorylation of the La protein was observed after induction of apoptosis by several initiators and in various cell types. Furthermore, we demonstrate that at least a subset of the La protein is proteolytically cleaved in vivo, generating a 45 kDa fragment. Dephosphorylation as well as cleavage of La is inhibited by ZnSO4 as well as by several tetrapeptide caspase inhibitors, indicating that these processes require the activation of caspases. Dephosphorylation of La is inhibited by low concentrations of okadaic acid, suggesting that a PP2A-like phosphatase is involved. Generation of the 45 kDa fragment is consistent with proteolytic cleavage at amino acids 371 and/or 374. The possible significance of the apoptotic changes in the La protein for autoantibody production is discussed.

Detection and occurrence of the 60- and 52-kD Ro (SS-A) antigens and of autoantibodies against these proteins.

The simultaneous detection of anti‐La. anti‐60‐kD Ro and anti‐52‐kD Ro antibodies by immunoblotting is greatly improved by changing the erosslinking level in the gel to an acrylamide/bisacrylamide ratio of 19:1. Using this method for the analysis of a number of systemic lupus erythematosus (SLE) and Sjögrens syndrome patient sera it was observed that antibody to the 52‐kD Ro protein without anti‐60‐kD Ro antibody was restricted to Sjogrens syndrome patients (9/26), whereas antibody to the 60‐kD Ro protein without contaminating anti‐52‐kD Ro antibody was only found in SLE patients (8/38). Moreover, in Sjogrens sydrome patient sera anti‐Ro antibody was found only in combination with anti‐La antibody (20/26). whereas in SLE patient sera anti‐Ro antibody could be found without detectable anti‐La specificity (4/38). Double immunofluorescence microscopy revealed that the 52‐kD Ro and the 60‐kD Ro proteins co‐localize in the cytoplasm as well as in the nucleus, whereas immunoprecipitation of [32P]‐labelled HeLa cell extract with monospecific anti‐52‐kD Ro and anti‐60‐kD Ro sera showed that both proteins are associated with the Ro RNAs. These data suggest the presence of both the 52‐kD and the 60‐kD Ro proteins in the same ribonucleoprolein complexes. To study the evolutionary conservation of the 52‐kD Ro, the 60‐kD Ro and the La proteins, extracts of cell lines derived from various mammalian species were analysed on Western blols using monospecific human antibodies. In contrast to the 60‐kD Ro and the La antigens which are well conserved in evolution, the 52‐kD Ro antigen could be detected in primate cells only by this immunological approach.

Ro ribonucleoprotein assembly in vitro: Identification of RNA-protein and protein-protein interactions☆

The human Y RNAs, small RNAs with an unknown function, are complexed with at least three proteins: the 60,000 M(r) Ro protein (Ro60), the 52,000 M(r) Ro protein (Ro52) and the La protein (La). In this study we examined the intermolecular interactions between the components of these so-called Ro ribonucleoprotein (Ro RNP) complexes. Incubation of 32P-labelled hY1 RNA in HeLa S100 extract allows the reconstitution of Ro RNP complexes, which were analysed by immunoprecipitation with monospecific antisera. By immunodepletion of HeLa S100 extracts for either Ro60, Ro52 or La, followed by supplementation with recombinant Ro60 or La, it was demonstrated that both Ro60 and La bind to hY1 RNA directly without being influenced by one of the other proteins. However, binding of Ro52 to hY1 RNA required the presence of Ro60, which strongly suggests that the association of Ro52 with Ro RNPs is mediated by protein-protein interactions between Ro60 and Ro52.

Cell death: a trigger of autoimmunity?

Systemic autoimmune diseases are characterized by the production of antibodies against a broad range of self‐antigens. Recent evidence indicates that the majority of these autoantigens are modified in various ways during cell death. This has led to the hypothesis that the primary immune response in the development of autoimmunity is directed to components of the dying cell. In this article, we summarize data on the modification of autoantigens during cell death and the possible consequences of this for autoimmunity. BioEssays 22:627–636, 2000.

Ribonucleoprotein complexes as autoantigens

Many intracellular proteins and nucleic acids, that are involved in important biosynthetic pathways, are targeted by autoantibodies occurring spontaneously in the sera of patients with systemic autoimmune diseases. Frequently, the autoantigens are assembled into multicomponent complexes containing both nucleic acid(s) and proteins. Recently, progress has been made in the study of autoantigenic ribonucleoprotein complexes, the most important of which are spliceosomal ribonucleoproteins, nucleolar ribonucleoproteins, Ro/La ribonucleoproteins and complexes of aminoacyl-tRNA synthetase and tRNA. In addition to new structural and functional information, important results have been obtained on epitope spreading, as well as on a potential role for apoptosis during the development of an autoimmune response against these complexes.

Autoantibodies against small nucleolar ribonucleoprotein complexes and their clinical associations

Sera from patients suffering from systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) have been shown to contain reactivities to nuclear components. Autoantibodies specifically targeting nucleolar antigens are found most frequently in patients suffering from SSc or SSc overlap syndromes. We determined the prevalence and clinical significance of autoantibodies directed to nucleolar RNA‐protein complexes, the so‐called small nucleolar ribonucleoprotein complexes (snoRNPs). A total of 172 patient sera with antinucleolar antibodies were analysed by immunoprecipitation. From 100 of these patients clinical information was obtained by chart review. Autoantibodies directed to snoRNPs were detected not only in patients suffering from SSc and primary Raynauds phenomenon (RP), but also in patients suffering from SLE, rheumatoid arthritis (RA) and myositis (PM/DM). Antibodies against box C/D small snoRNPs can be subdivided in antifibrillarin positive and antifibrillarin negative reactivity. Antifibrillarin‐positive patient sera were associated with a poor prognosis in comparison with antifibrillarin negative (reactivity with U3 or U8 snoRNP only) patient sera. Anti‐Th/To autoantibodies were associated with SSc, primary RP and SLE and were found predominantly in patients suffering from decreased co‐diffusion and oesophagus motility and xerophthalmia. For the first time autoantibodies that recognize box H/ACA snoRNPs are described, identifying this class of snoRNPs as a novel autoantigenic activity. Taken together, our data show that antinucleolar patient sera directed to small nucleolar ribonucleoprotein complexes are found frequently in other diseases than SSc and that categorization of diagnoses and clinical manifestations based on autoantibody profiles seems particularly informative in patient sera recognizing box C/D snoRNPs.

Sera from patients with rheumatic diseases recognize different epitope regions on the 52-kD Ro/SS-A protein.

Patients suffering from systemic lupus erythematosus (SLE) or Sjögrens syndrome (SS) often contain autoantibodies directed to the Ro(SS‐A) complex. In this study the antigcnic determinants on two of the components of the Ro complex, i.e. the Ro60 and the Ro52 polypeptides, were investigated. Anti‐Ro+ sera were selected by counter‐immunoelectrophoresis. Depending on the detection method. 59–68% of the SLE patients produced anti‐Ro but not anti‐La antibody, while 72–81 % of the SS patients produced both anti‐Ro and anti‐La antibody. Immunoprecipitation of recombinant Ro‐proteins showed that 61 sera (87%) were reactive with both Ro proteins, seven sera with Ro60 only, one serum with Ro52 only, and one serum did not precipitate the proteins at all. The anti‐Ro60 reactivity of human sera is strongly associated with the native form of Ro60. suggesting that conformational autoepitopes are an important feature of Ro60. In the case of Ro52, frequently the residues located between amino acids 216 and 292 were essential for reactivity with the antibodies. With 70% of the lupus sera tested this appeared to be the only region important for reactivity. The antibodies of SS patients generally recognized multiple B cell epitopes located between amino acids 55 and 292. The results of this study indicate that the antigenic determinants on Ro52 are different for autoantibodies produced by lupus patients compared with those of SS patients.

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule

During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B′. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5–6 nucleotides including the 2,2,7-trimethylguanosine (TMG) cap. The observations that U1 snRNA cleavage is very specific (no such modifications were detected for the other U snRNAs tested) and that U1 snRNA cleavage is markedly inhibited in the presence of caspase inhibitors, indicate that an apoptotically activated ribonuclease is responsible for the specific modification of U1 snRNA during apoptosis.

Caspase-dependent cleavage of nucleic acids

Autoimmune diseases are frequently characterized by the presence of autoantibodies directed against nucleic acid-protein complexes present in the nucleus of the cell. The mechanisms by which these autoantigenic molecules escape immunological tolerance are largely unknown, although a number of recent observations suggest that modified self-proteins generated during apoptosis may play an important role in the development of autoimmunity. It has been hypothesized that the recognition of these modified self-proteins by the immune system may promote autoantibody production. While apoptosis is specifically characterized by posttranslational modification of proteins, recent findings also show that nucleic acids are modified. This review summarizes the specific cleavages of some of these key nucleic acids, i.e. chromosomal DNA, ribosomal RNA and small structural RNAs (U1 snRNA, Y RNA), in apoptotic cells. Cell Death and Differentiation (2000) 7, 616–627