modiÞed self-proteins could act as means to induce autoimmunity [166]. After the modiÞed self-proteins are taken up, digested, and processed in antigen-presenting cells (APCs), potentially self-reactive T and B cells can recognize them, resulting in the breaking of tolerance. One explanation for the mechanisms of loss of tolerance to unmodiÞed proteins following immune response to cryptic epitope is through a process called epitope spreading in part contributed by intermolecularÐintrastructural help [80]. The immune response may spread to unmodiÞed self-proteins and thus generating an autoimmune reaction [166]. The most frequently observed posttranslational modiÞcations include glycosylation, phosphorylation, acetylation, citrullination, and cleavage by apoptosis [5, 149, 167Ð169]. Several studies have shown that posttranslational modiÞcations can affect autoantibodies binding onto different autoantigens in various systemic autoimmune diseases [5, 149, 167Ð169]. Interests in Ro60 epitope (aa 169Ð 190) have been renewed recently, as McClain et al. demonstrated that this sequence could be involved, through molecular mimicry mechanisms with viral proteins, in the initiation of the autoimmune response [81]. They also showed that puriÞed antibodies against the epitope crossreacted with the peptide aa 58Ð72 sequence of the latent viral protein EpsteinÐBarr virus nuclear antigen-1 (EBNA-1). Phosphorylation of the linear B-cell epitope of SS-B/La (aa 349Ð368) enhances autoantibody binding and its relative avidity [59]. Although susceptibility to efÞcient cleavage by a caspase is a frequent feature of several SS autoantigens, it is not a universal feature of all the autoantigens. For example, caspasemediated proteolytic cleavage SS-A/Ro has not been observed [170]. Thus Rosen and co-workers have speculated alternative mechanisms such as cleavage by granzyme B, which is another effector protease especially in cell killing mediated by CD8+ cytotoxic T lymphocytes and natural killer cells [5]. Furthermore, it is known that apoptosis also can mediate not only proteolytic cleavage but also citrullination or oxidation [171]. For example, oxidized low-density lipoproteins have been found on apoptotic cells, and mice immunized

with apoptotic cells develop high titers of autoantibodies to various oxidized epitopes, whereas mice immunized with viable or necrotic cell do not [172]. These data indicate that the posttranslationally modiÞed self-antigens found on apoptotic cell are ÒneoantigensÓ capable of eliciting an immune response.

9.3.3Establishment Phase: Autoreactive T and B Lymphocytes Dysregulation and Aberrant Cytokines Production

The production and persistence of autoantibodies in autoimmune conditions may be caused by immune dysregulation with autoreactive T- lymphocyte and B-lymphocyte hyperactivation and aberrant cytokines production. InÞltrating lymphocytes and dendritic cells in affected tissues interact with salivary gland epithelial cells and contribute to the perpetuation and progression of the disease. In this context, dysregulation of cytokines production (e.g., Th1/Th2 imbalance, elevated BAFF production) and chronic B-cell hyperactivity are consistent and prominent immunoregulatory abnormalities in SS [2, 173Ð176]. Recent studies have demonstrated the B-cell hyperactivity in the inßamed tissues, especially in SS patients with detectable ectopic GClike structures [177]. Similar ectopic lymphoid structures have also been described in the target tissues of several other autoimmune conditions that are accompanied by B-cell disturbance such as RA [178, 179], SLE [178, 180], and chronic autoimmune thyroiditis [181, 182]. Delineation of common and diverse mechanisms of SS may underlie the B-cell disturbances, and the development of ectopic GC-like structures in SS entities should be important for our understanding of their immunopathogenesis.

Chronic focal periductal lymphocytic sialadenitis is a hallmark of SS and it is generally thought to be a stepwise process [2, 173, 174]. This process may include (1) a sequence of scattered tiny perivascular lymphoid inÞltrates,

(2) subsequent development of the typical focal periductal lymphoid sialadenitis/formation of

ectopic GC-like structures, and (3) eventually the destruction and replacement of the affected glandular tissue [177]. In this process, cytokine and/or autoantibody-mediated endocrine glandular tissue dysfunction may occur [106, 183]. These ectopic GC-like structures of the inßamed tissues bear a histological resemblance to the native GCs that are physiologically generated from primary B-cell follicles of secondary lymphoid organs during T-cell-dependent immune response. The ectopic GC-like structures contain T and B cells aggregate with proliferating lymphocytes, a network of follicular dendritic cells, and activated endothelial cells with the morphology of high endothelial venules [163, 179, 180, 184, 185]. Hanson et al. have explained abnormal B-cell differentiation pathways resulting in autoantibody production from the ectopic lymphoid tissues by ÒnicheÓ theory [177]. The ectopic GCs represent a niche where B cells, which are recruited by chemokines into the microenvironment of chronically inßamed tissues, may escape from peripheral check points against autoreactivity and are abnormally stimulated. B cells may proliferate and incompletely differentiate via T-cell-dependent or independent pathway into memory B cells and plasma cells, resulting in autoantibody production. Furthermore, the autoreactive B cells may be additionally stimulated by cytokines such as BAFF.

9.3.4Establishment Phase: Intermolecular–Intrastructural Help

Autoantigens are usually macromolecular complexes such as SS-A/Ro and SS-B/La, and linkages of autoimmune response among the components of the complex are often observed in patients with SS and other systemic autoimmune diseases [186, 187]. For instance, anti-Sm antibodies always occur with anti-U1RNP, and anti-SS-B/La is also associated with antibodies against SS-A/Ro and the Y5 small RNA molecule [188Ð190]. This phenomenon was also observed in autoantibodies to PCNA [191]. Such

linked sets of autoantibodies, reacting with multiple components of the same macromolecular complex, may be the products of intermolecularÐ intrastructural help, which plays a key role in the immune system [192]. The importance of intermolecularÐintrastructural help in the progression of autoimmunity is also supported by peptide immunization studies in SS-A/Ro and SS-B/La antigenÐantibody systems in which mice immunized with recombinant murine SS- B/La develop not only anti-SS-B/La antibodies but also anti-SS-A/Ro antibodies [74, 76]; conversely, immunization with SS-A/Ro causes the production of both anti-SS-B/La and anti-SS- A/Ro antibodies [74, 76]. Gordon et al. have also reported similar results in which sera from mice immunized by Ro52 and Ro60 showed reactivity against the fragment of SS-B/La protein [73]. The explanation for these observations is that T cells speciÞc for the immunized antigen can provide help to B cells carrying receptor for other components such as in the case of SS-A/Ro-SS- B/La complex. Alternatively, or in addition, the spread of autoimmunity to other proteins within a macromolecular complex may come from distinct products of the antigen processing by APC [193, 194]. This suggests that activated autoreactive B cells (or professional APC) can prime autoreactive T cells.

9.3.5 Effector Phase

Although tissue destruction mediated by autoreactive cytotoxic T cells and aberrant cytokines such as TNF-α is considered as major factor of the tissue damage of SS, antibody-mediated tissue destruction is also believed to play an important role in the process. Clancy et al. recently reported novel effector mechanisms of tissue destruction mediated by autoantibodies [67]. They demonstrated that anti-SS-A/Ro and anti-SS-B/La antibodies bound to apoptotic cardiomyocytes caused an impaired clearance of the apoptotic cells. Blocking physiologic apoptotic cell clearance by autoantibody binding of apoptotic cells would be expected to skew the pool of IgG autoantibodyÐapoptotic