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1 Departments of Medicine, 2 Cell Biology, 3 Pathology, and 4 Dermatology, Johns Hopkins University School of Medicine, 5200 Eastern Avenue, Mason F. Lord Building, Center Tower, Suite 4100, Baltimore, MD 21221;
* corresponding author, arosen{at}jhmi.edu
Abstract Introduction Autoantibodies: Probes of the Perturbed State Several Intracellular Molecules are the Targets of a High-titer Autoantibody Response in SS. Sjögren’s Syndrome Autoantigens are Redistributed in Apoptotic Cells. Susceptibility to Efficient Cleavage by Caspases Unifies a Subgroup of SS Autoantigens. Novel Autoantigen Fragments are Produced during Cytotoxic Lymphocyte-induced Target Cell Apoptosis. Autoantigen Cleavage in SS: Insights from the NFS/sld Mouse Model Unique Protease Expression in the SS Salivary Gland: Potential for Generation of Altered Autoantigen Structure in the Unique Disease Microenvironment Model of SS Acknowledgments REFERENCES
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Key words. Apoptosis, autoimmunity, protease, autoantigen, autoantibody, structure, caspase, granzyme B
| Introduction |
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| Autoantibodies: Probes of the Perturbed State |
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There is universal acknowledgment of the critical role of proteolysis within the endosome-lysosome pathway during the processing of antigens for presentation on MHC class II (Tortorella et al., 2000). These proteolytic events are responsible for the stereotyped and predictable outcome of the antigen-processing pathways, followed by whole protein antigens. For many antigens, antigen structure and initial antigen-processing events play a large role in the selection of determinants that predominate when loaded onto specific MHC class II molecules (dominant determinants). Since, for self-antigens, T-cell tolerance is predominantly focused on such dominant determinants (which are generated and presented at suprathreshold concentrations during natural processing of whole protein antigens), a potential exists for T-cell autoreactivity directed against ‘cryptic’ determinants (which are generated at subthreshold concentrations during normal antigen processing (reviewed in Gammon et al., 1991; Sercarz et al., 1993). Such T-cells recognizing the cryptic self never encounter their antigen during natural antigen presentation, and are therefore not tolerized. Indeed, several experimental systems have now clearly demonstrated the existence of such T-cells in vivo (reviewed in Lehmann et al., 1992; Lanzavecchia, 1995). When the structure of antigens is changed (for example, by novel autoantigen cleavage [Mamula, 1995]) or altered autoantigen processing is induced by high-affinity ligand binding (e.g., to an antibody or receptor molecule [Watts and Lanzavecchia, 1993; Salemi et al., 1995; Simitsek et al., 1995]), the hierarchy of epitopes which are efficiently loaded onto MHC class II molecules is altered, sometimes resulting in presentation of previously cryptic epitopes. We have proposed that autoantigens in systemic autoimmune diseases are structurally altered during disease initiation, thus allowing for the efficient loading of previously cryptic epitopes onto class II. This in turn results in the activation of autoreactive T-cells, which provide effective T-cell help to autoantibody-producing B-cells and probably cytotoxic lymphocytes (Casciola-Rosen et al., 1994a, 1996, 1999). High-titer autoantibodies therefore provide excellent probes of the cell biology and biochemistry of autoantigens during different clinically relevant perturbed states, to search for those circumstances in which autoantigens become clustered, concentrated, and structurally modified (Casciola-Rosen et al., 1994a, b, 1995, 1996, 1999; Casiano et al., 1996, 1998; Utz et al., 1997, 1998; Andrade et al., 1998; Odin et al., 2001). The association of particular autoantibodies with specific disease phenotypes may allow for the identification of unique modifications of autoantigen structure that occur during a pro-immune initiating event in the relevant target tissue (Andrade et al., 1998, 2001; Casciola-Rosen et al., 1999; Rosen and Casciola-Rosen, 1999).
This review highlights the susceptibility of SS autoantigens to undergoing such structural changes during immune effector pathways, and synthesizes a model of SS incorporating these concepts. Of necessity, elements of this model are speculative, but the different components are eminently testable in vitro and in vivo.
| Several Intracellular Molecules are the Targets of a High-titer Autoantibody Response in SS. |
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The ubiquitously expressed autoantigen targets in SS include the ribonucleoprotein autoantigens Ro/SS-A and La/SS-B (Harley et al., 1986), as well as several coiled-coil-containing molecules (including nuclear mitotic apparatus protein [NuMA] and members of the golgin family) (Price et al., 1984; Chan and Fritzler, 1998; Mancini et al., 2000). There have also been reports of autoantibodies recognizing 
- and ß-fodrin in Japanese cohorts of patients with SS, but not other autoimmune rheumatic diseases (Haneji et al., 1997; Kuwana et al., 2001). While the prevalence of such antibodies is apparently very high in Japan, it appears to be lower in cohorts in the US and Europe, and less specific for primary SS (Witte et al., 2000, and unpublished data). Antibodies to -fodrin, however, are found with high prevalence in the NFS/sld mouse model of SS, and might provide important pathogenic insights in that model (see below). Other molecules described as autoantigens in SS include poly(ADP)ribose polymerase (PARP) (Yamanaka et al., 1987), NuMA (Price et al., 1984), Golgins (Fritzler et al., 1993, 1995; Chan and Fritzler, 1998), Ku (Yaneva and Arnett, 1989), 90-kDa nucleolar organizer region protein (NOR-90) and p80 coilin (von Muhlen and Tan, 1995), and CENP-C (Gelber et al., 2002). It is important to note that these molecules have little in common in terms of structure, subcellular distribution, and function. Furthermore, there are few data about whether antibodies directed against these targets have any direct functional effects on the salivary gland epithelium.
Several recent studies have defined autoantigens in SS that have a much more restricted expression pattern than the molecules described above. These antigens include an islet cell autoantigen (ICA), ICA-69, which is the target of autoantibodies in patients with SS, as well as in the NOD mouse model of SS (Winer et al., 2002). ICA-69 is a 69-kDa molecule expressed in pancreatic islets, brain, and salivary and lacrimal glands. Patients with SS have B-cell and T-cell reactivity directed against ICA-69, as do NOD mice, which exhibit an autoimmune sialadenitis and dacryoadenitis. Interestingly, disruption of the ICA-69 locus in NOD mice prevented lacrimal disease and greatly reduced salivary gland disease, suggesting that immunoreactivity against ICA-69 might play a role in ongoing gland damage in this model (Winer et al., 2002).
Another specificity that may be of pathogenic relevance is that directed against the type III muscarinic receptor (M3R) (Bacman et al., 1996, 1998, 2001; Robinson et al., 1998; Waterman et al., 2000). Since transduction of the autonomic signal in the salivary gland is predominantly through M3R, antibodies to this molecule would be of particular consequence through functional inhibition of salivary epithelial signaling. While most of the publications referenced above demonstrated that sera from patients with SS or NOD mice do inhibit signaling though M3R, it remains unclear whether this inhibitory activity is mediated by autoantibodies, and the precise specificity of the activity remains to be established. Defining additional salivary-specific targets of the autoantibody response in SS remains a high priority.
| Sjögren’s Syndrome Autoantigens are Redistributed in Apoptotic Cells. |
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). Thus, small surface blebs (which contain fragmented rough endoplasmic reticulum [ER]) are highly enriched in 52-kDa Ro, as well as those autoantigens found within the ER lumen (e.g., calreticulin) (Rosen et al., 1995). This marked enrichment of autoantigens in small surface blebs is accompanied by their concomitant depletion from the cytosol (Casciola-Rosen et al., 1994a). Nuclear autoantigens also undergo a striking redistribution and concentration during apoptosis (Fig.). Thus, 60-kDa Ro/SS-A, La/SS-B, the snRNPs, Ku, and PARP, which normally have a diffuse nuclear distribution, initially become concentrated as a rim around the condensing chromatin in early apoptosis (Casciola-Rosen et al., 1994a, 1996). As apoptosis progresses and the nucleus becomes fragmented into multiple membrane-bound apoptotic bodies, nuclear autoantigens remain rimmed around the condensed chromatin. Such clustering of autoantigens during apoptosis has been observed in response to several additional stimuli (Rosen et al., 1995; Casciola-Rosen et al., 1999), and in numerous different cell types, including salivary epithelial cells (Ohlsson et al., 2002a). The phenomenon is not observed in cells dying by necrosis.
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While exposure of autoantigens at the surfaces of apoptotic cells may provide a mechanism for activating and amplifying immune effector mechanisms that propagate tissue damage, a critical question remains as to how the autoantibody response recognizing such clustered antigens is primarily initiated. Several mechanisms which alter the structure and expression of autoantigens during apoptosis have recently been described, and include novel proteolysis, phosphorylation, glutathiolation, transglutamination, citrullination, or formation of novel protein-protein or protein-nucleic acid complexes (Utz and Anderson, 1998; Rosen and Casciola-Rosen, 1999; Doyle and Mamula, 2002). We will use the cleavage of SS autoantigens during different forms of cell death as the paradigmatic modification with which to view the role of altered autoantigen structure in the pathogenesis of autoimmunity.
| Susceptibility to Efficient Cleavage by Caspases Unifies a Subgroup of SS Autoantigens. |
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- and ß-fodrin, Ku/DNA-PKcs, NuMA, and CENP-C (Casciola-Rosen et al., 1994b, 1995, 1996, 2001; Martin et al., 1995; Casiano et al., 1996; Waterhouse et al., 1996; Haneji et al., 1997; Utz et al., 1998; Ayukawa et al., 2000; Schachna et al., 2002). Since the total number of proteins efficiently cleaved by the caspases during apoptosis appears to be restricted to several hundred molecules at most, the striking enrichment of SS autoantigens among this group of substrates is of great interest. In each case, cleavage appears to separate important functional and regulatory domains, with functional consequences. For example, cleavage of PARP removes the regulatory domain and initiates unregulated ADP-ribosylation activity (Scovassi and Poirier, 1999). Similarly, caspase-mediated cleavage of La/SS-B removes the C-terminal domain containing the nuclear localization sequence, which results in redistribution of La/SS-B from the nucleus to the cytoplasm (Ayukawa et al., 2000). It is important to note that while caspase-mediated cleavage of purified substrates in vitro is frequently highly efficient, some molecules (e.g., La/SS-B) are inefficiently cleaved in intact cells during apoptosis. Defining the immunological relevance of caspase-mediated cleavage remains an important priority (see below). While susceptibility to efficient cleavage by a caspase is a frequent feature of several SS autoantigens, it is not a universal feature of all the autoantigens targeted in this disease. For example, caspase-mediated proteolytic cleavage of components of the frequently targeted Ro/SS-A particle has not been observed (Casciola-Rosen et al., 1996; Casiano et al., 1996). Furthermore, susceptibility to caspase-mediated cleavage does not appear to be specific to autoantigens. This imperfect, albeit striking, correlation of susceptibility to caspase cleavage and status as an autoantigen suggests that additional (and potentially partially overlapping) properties might be relevant. For example, (i) caspases may have evolved to cleave a specific regulatory motif in proteins—the association with autoantigen status may be with the presence of this protein structure rather than with cleavage during apoptosis; (ii) such structure may also be the target of additional proteases during specific forms of apoptosis or inflammation in unique tissues/microenvironments (e.g., granzyme B [GrB], matrix metalloproteases; see below); and (iii) such a regulatory motif may be subject to additional post-translational modifications either during apoptosis or during other physiologic states relevant to disease propagation (e.g., phosphorylation, glutathiolation, transglutamination, citrullination, or formation of novel protein-protein or protein-nucleic acid complexes). In this regard, it is of interest that numerous autoantigens are phosphorylated during a variety of physiologic perturbations (including apoptosis) (Utz et al., 1997). Furthermore, in several cases, recognition by autoantibodies is dependent on the phosphorylation state of the antigens (Satoh et al., 1994; Neugebauer et al., 2000).
Autoantigen clustering and cleavage occur in almost all forms of apoptosis described to date, which often occur in actively anti-inflammatory and non-immune contexts (Voll et al., 1997; Fadok et al., 1998; Hoffmann et al., 2001; Huynh et al., 2002). The marked frequency of apoptosis in normal development and homeostasis, coupled with the infrequency of systemic autoimmunity in the population, strongly suggests that only a very restricted subset of apoptotic events (e.g., those occurring in a pro-immune setting; see below), in individuals who are genetically predisposed to the generation of novel autoantigen structure (e.g., from abnormalities in the clearance and degradation of apoptotic material in tissues; see below), will initiate a self-sustaining autoimmune response.
| Novel Autoantigen Fragments are Produced during Cytotoxic Lymphocyte-induced Target Cell Apoptosis. |
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. GrB also initiates caspase-independent pathways which contribute to target cell death through direct targeting of several downstream caspase substrates that mediate critical components of the apoptotic phenotype. Thus, GrB directly cleaves Bid at a site close to that utilized by caspase-8, and recruits the downstream mitochondrial pathway (Li et al., 1998; Heibein et al., 2000; Sutton et al., 2000; Alimonti et al., 2001; Pinkoski et al., 2001). The release of pro-apoptotic mediators (e.g., SMAC/Diablo) from the mitochondrial matrix into the cytoplasm also plays a critical role in deregulating the restraint imposed by ‘inhibitors of apoptosis’ proteins (IAPs) on caspase activity (Goping et al., 2003; Sutton et al., 2003). Similarly, GrB cleaves inhibitors of the caspase-activated DNAs (ICAD), allowing for activation of the caspase-activated DNAase, thereby generating the inter-nucleosomal DNA degradation that is characteristic of caspase-mediated apoptotic death (Thomas et al., 2000). We have demonstrated that the majority of autoantigens targeted across the spectrum of systemic autoimmunity are directly and efficiently cleaved by GrB, both in vitro and in cells undergoing granule-induced cytotoxicity (Andrade et al., 1998; Casciola-Rosen et al., 1999, 2001; Nagaraju et al., 2001). GrB-mediated cleavage of these substrates generates unique fragments not generated during any other form of apoptosis studied to date. Interestingly, efficient cleavage by GrB (with the generation of distinct fragments) has also been observed for several of those systemic disease autoantigens that are not cleaved by caspases. These molecules include the scleroderma autoantigens CENP-B, fibrillarin, and B23, PMS1 targeted in myositis, and the M3R targeted in Sjögren’s syndrome (Casciola-Rosen et al., 1999, 2001; Nagaraju et al., 2001). The recent demonstration that GrB cleaves subunit III of the glutamate receptor (GluR3) (an autoantigen in Rasmussen’s encephalitis) is also of significance (Gahring et al., 2001). Interestingly, the GrB cleavage site is located within the epitope targeted by antibodies in this disease. In GluR3, the GrB cleavage site faces the outside of the plasma membrane, and cleavage efficiency is markedly influenced by the glycosylation state of the receptor (Gahring et al., 2001). Thus, when GluR3 is fully glycosylated, it is relatively resistant to cleavage by GrB; the deglycosylated or non-glycosylated form of GluR3 is efficiently cleaved. The authors have proposed that generation of an ‘under’-glycosylated form of GluR3 during inflammatory states may allow for GluR3 cleavage by GrB, and the generation of previously cryptic peptide fragments (Gahring et al., 2001). Demonstrating that such circumstances occur in vivo will provide important evidence to support such a mechanism.
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The cytotoxic lymphocyte granule pathway has been demonstrated to be active in several circumstances in vivo. These include lymphocyte-mediated killing of virally infected target cells or cells that have undergone malignant transformation (Russell and Ley, 2002; Trapani and Smyth, 2002). A critical role for the granule pathway in down-modulation of the antigen-specific immune response has also been demonstrated (Badovinac et al., 2000). Such circumstances may have particular relevance for the initiation of systemic autoimmune diseases like Sjögren’s syndrome, and provide a paradigm within which we can understand disease initiation and propagation. For example, viral infection or transformation of salivary epithelial cells may play important roles in making these cells the targets for initial immune attack by the cytotoxic lymphocyte granule pathway. During this initial event, the granule pathway may generate altered structure of autoantigens (preferentially expressed at this site or specifically increased during this perturbation) and initiate an autoimmune response (see below).
Another important issue to consider in evaluation of the potential role of the cytotoxic lymphocyte granule pathway in the initiation of autoimmune diseases is that viral infection and transformation are encountered very frequently throughout the life of the individual, while autoimmunity is decidedly more uncommon. Numerous studies have now demonstrated that the CTL granule pathway efficiently activates downstream caspases, and directly recruits the mitochondrial pathway through cleavage of Bid (Li et al., 1998; Heibein et al., 2000; Sutton et al., 2000; Alimonti et al., 2001; Pinkoski et al., 2001). This recruitment of downstream caspase pathways is a highly amplified event, ensuring that cells die with the predominant biochemical features of the default caspase pathway under most circumstances. For example, La/SS-B, golgins, PARP, NuMA, and fodrin are all cleaved by both caspase-3 and GrB, at distinct sites. During CTL granule-induced death in normal target cells, almost all of the fragment generated is from caspase-3 activity (Andrade et al., 1998; Casciola-Rosen et al., 1999). In contrast, when endogenous caspases in the target cells are specifically inhibited, generation of the caspase-mediated fragments is abolished, and fragments directly generated by GrB are formed. This is of particular interest in SS salivary gland epithelial cells, which express high concentrations of anti-apoptotic Bcl-2 family members (Kong et al., 1998). In contrast to Bcl-2 family members, knowledge about the expression of other anti-apoptotic proteins in SS is still rudimentary. Of particular interest is the expression of the inhibitors of apoptosis (IAP) proteins, including X-IAP. It is likely that the initial generation of unique autoantigen structure occurs under such circumstances.
It is also possible that other unique structural autoantigen modifications occur during caspase-independent death pathways, and that GrB-induced fragments are only one relevant example of a more general phenomenon. For example, the lack of glutathiolation of PDC-E2 observed in cholangiocytes (in which high-level expression of Bcl-2 abrogates this modification normally observed in other cells) may generate a unique form of this primary biliary cirrhosis autoantigen that is not generated at sites of tolerance development (Odin et al., 2001).
| Autoantigen Cleavage in SS: Insights from the NFS/sld Mouse Model |
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-fodrin as a prominent antigen in the majority of affected animals. Interestingly, this fragment co-migrated with that generated by caspase-mediated cleavage of fodrin, generated during apoptotic cell death. The specific fodrin fragment could be demonstrated in salivary glands from affected animals, but not in those from controls. When the fragment was used to tolerize animals early in life, development of sialadenitis was prevented, demonstrating a role for an immune response to cleaved fodrin in the initiation and propagation of ongoing salivary gland pathology in these animals (Saegusa et al., 2002). Similarly, prevention of caspase activity in vivo partially ameliorates development of salivary gland dysfunction in affected animals (Saegusa et al., 2002). Although a similar -fodrin fragment is generated by caspases, there is no direct evidence that caspases are responsible for generating this fragment in vivo. Indeed, additional proteases active in the salivary gland may play important roles (see below). Although the prevalence of anti-fodrin autoantibodies in human SS patients and the presence of fodrin cleavage fragments in SS salivary glands remain controversial, the Hayashi studies have important implications. First, they suggest that autoantigen cleavage occurs in affected tissues in the mouse model. Second, they demonstrate that such cleavage may play an important role in the selection of autoantigens for an autoimmune response, and in generating ongoing tissue damage. It is of interest that, at the hinge region between the N- and C-terminal domains, fodrin is highly susceptible to cleavage by several other proteases, including calpains (Siman et al., 1984) and GrB (Nagaraju et al., 2001). Defining the physiologically critical protease responsible for the initial cleavage of fodrin in this model is important.
| Unique Protease Expression in the SS Salivary Gland: Potential for Generation of Altered Autoantigen Structure in the Unique Disease Microenvironment |
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| Model of SS |
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). Such forces generate suprathreshold concentrations of antigens with structure not previously tolerized by the host, and facilitate effective capture of such antigens by antigen-presenting cells, and presentation to T-cells. The molecules targeted are unified by their susceptibility to structural modification during the perturbing process, likely revealing previously cryptic structure. Once primary immunization has occurred, the effector pathways activated by the primed immune system include several which themselves may generate an ongoing supply of modified antigen (e.g., cytokine effects, novel protease expression), driving further immune response and effector pathways. Our understanding of the pathways relevant to disease initiation and propagation will provide a rational basis for therapy for a disease which places an extremely high burden on those who suffer from it, and in which targeted and effective therapies remain elusive.
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| Acknowledgments |
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