Sjögren's syndrome and the danger model
A. Bredberg,
G. Henriksson,
Å. Larsson,
R. Manthorpe and
A. Sallmyr
Sjögren's Syndrome Research Centre, Department of Medical Microbiology, University Hospital UMAS, Lund University, Malmo, Sweden.
Correspondence to: A. Bredberg, Department of Medical Microbiology, University Hospital, S-20502 Malmo, Sweden. E-mail: anders.bredberg{at}mikrobiol.mas.lu.se
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Introduction
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This review seeks to cover the data on primary Sjögren's syndrome (SS) that has accumulated especially during recent years, and that apparently fits into a common theme of cellular stress abnormalities. This includes demonstrations of hyperactivity of danger-sensing antigen-presenting cells (APC), and of a strong response to DNA damage in many different cell types. The relevance of these findings for the pathogenesis of SS will be addressed by applying them to the danger model of immune activation [1, 2].
This model has been put forward by Matzinger, stating that immune activation, including the innate system as well as the adaptive lymphocyte response, is initiated by danger in a broad sense rather than solely by the presence of infectious non-self material. By means of danger signals, tissue stress is postulated to activate the APC of the innate immune system, in turn influencing also the adaptive immune cells, i.e. T and B lymphocytes. Thus, a consequence of the danger model of immune activation is that danger signals can be assumed to be a primary determinant of autoimmunity.
A number of factors that threaten the integrity of a tissue or the host may act as danger signals. These are produced by, for example, necrotic cells, hypoxia, low pH and mechanical damage to blood vessels. Danger signals from necrotic cells that strongly stimulate APC to up-regulate HLA and costimulatory molecule expression include heat shock proteins transferred upon cell death to the cell surface [3], and the release from dying cells of high concentrations of crystalline uric acid [4]. CD40L on activated extravasating platelets, hyaluron modified in traumatized tissue, reactive oxygen species (ROS) and non-self molecules such as bacterial cell wall lipopolysaccharides can also act as potent danger signals, by the mechanism of binding to Toll-like receptors and other surface markers on APC [1, 2, 58]. Interestingly, physiological cell death manifested as apoptosis, posing no threat to the tissues, has been found to have a relatively minor influence on APC, in accordance with the lack of inflammation being typical of apoptosis [9].
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DNA damage: a high level in salivary glands, and an exaggerated defence reaction in SS cells
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Perhaps the observation by Fox et al. in 1988 [10] of failure to undergo immunoglobulin (Ig) gene somatic hypermutation in SS can be taken as a first report on the stress response in SS. This concept of aberrant Ig gene diversification in SS has now gained support from a number of later observations. Some of the molecular components of V(D)J recombinase, Ku protein and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [11] are not only targets of autoantibody formation in SS [12, 13] but also display increased activity in SS cells [14, 15]. These components constitute the first line of defence against DNA double-strand breaks, which are generated continuously in human cells by, for example, ROS and ionizing radiation [16]. A wider impact of a V(D)J recombinase abnormality on SS is therefore indicated by findings of strong cell cycle arrest, facilitating cellular recovery, and apoptosis in gamma-irradiated SS cells [14, 15], both of which are known to be p53-dependent downstream signalling consequences of DNA-PKcs-mediated protein phosphorylation [11]. Since DNA-PKcs is part of the V(D)J recombinase complex, involved in both V(D)J Ig gene variable sequence recombination and class-switch recombination, it can be speculated that its high activity in SS cells contributes to the autoimmune features seen in these patients.
The lymphoma-associated chromosome translocation t(14;18), shown to be produced by illegitimate (i.e. not restricted to Ig genes) V(D)J recombination, can be detected at a low level in normal lymphocytes of healthy control subjects. The observation of a strongly reduced frequency in SS peripheral blood mononuclear cells of this bcl2-activating translocation [17] may seem to be in conflict with the finding of heightened DNA-PKcs activity in SS, since DNA-PK is a component of the V(D)J complex. However, efficient recognition and further processing of DNA base damage, together with a prolonged G1 cell cycle phase arrest, will most probably lower the presence of single-strand lesions, such as ROS-dependent DNA strand breaks, in cells entering the DNA synthesis phase. In this way, SS cells will eliminate an important source of DNA double-strand breaks, preventing the recombination of two broken chromosomes generating a translocation. Likewise, closely spaced multiple DNA base substitutions in a UV-damaged shuttle plasmid vector, sharing many characteristics with Ig somatic hypermutation [18] contributing to the varied antibody repertoire, were lacking in SS B-cell lines [19]. Thus, it is possible that such second-line error-prone DNA repair defence mechanisms are not called into action in SS cells proficient in first-line DNA repair systems.
In addition to V(D)J recombinase, abnormalities have been found in other proteins capable of modifying nucleic acids. An abnormal pattern of DNA-binding proteins in SS lymphoblastoid cell lines exposed to different DNA-damaging agents has been demonstrated with gel shift methods [20]. The level of the DNA repair factor O6-methyl-guanine methyltransferase (MGMT) is diminished in SS peripheral blood lymphocytes [21]. Although this observation is of interest in the light of induction of autoimmunity and lymphoma by methylating agents in susceptible mouse strains [22], MGMT reduction has been reported only for lymphocytes, and in a number of autoimmune conditions [22]. It may therefore well be secondary to lymphocyte activation rather than reflecting some basic aspect of SS pathogenesis. This potential source of false conclusions was pointed out by Zeher et al., by providing evidence that the predisposition of SS blood lymphocytes to apoptosis induction is, in fact, coupled to increased lymphocyte activation [23]. At this point, it should be noted that much of the stress response literature on SS uses salivary gland biopsies or cell lines established from SS patients and cultured in vitro for more than 10 generations, demonstrating that stress abnormalities do not occur only in activated lymphocytes. High levels of Ku protein and DNA strand breaks have been found in salivary gland epithelial cells [24, 25].
The diversified cellular defence reaction to DNA damage is orchestrated not only by DNA-binding repair enzymes; after this initial phase comes complex cascades including the p53 transcription factor, engaged in a wide range of stress situations, and then mediating end-points such as apoptosis and cell cycle arrest [26]. A number of signalling network components have been found to be up-regulated in SS. Protein kinase C (PKC), activated by G-coupled receptors and tyrosine kinases, and influencing a number of target genes, is present at an increased level in SS gland cells [27, 28]. A high level of PKC activity may also be an important factor for the B-cell influence on SS pathology, since PKCß has been shown to enhance B-cell proliferation and survival [29]. High levels of both p21 and p53 have been demonstrated in glandular tissue, p21 being the proximal signalling element known to activate p53 into mediating G1 cell cycle arrest [30], which may be linked to the previously related observation on Ku and DNA strand breaks in biopsies [24]. In accordance with these findings is the DNA-PKcs hyperactivity in SS B- and T-cell lines [14, 15], which was noted to be preformed, i.e. it was not only induced by radiation but also displayed by protein extracted from unirradiated cells [15]. Much attention has been given to apoptosis, a predominance of results indicating up-regulation in SS acinar and ductal cells [31, 32], including demonstration of activated caspase 3 and caspase-degraded forms of poly(ADP)ribose polymerase (PARP) [33], as well as in peripheral blood lymphocytes [23]. However, a shift in the consensus of a high apoptosis level in SS may be under way, since apoptosis was found to be a rare event in a recent in situ study, determining both Fas ligand expression and DNA fragmentation [34]. Such minor apoptosis might well be a physiological event present in any healthy tissue, which is predicted by the danger model to convey only minor danger signals and cause no significant up-regulation of costimulatory molecules on APC.
To summarize the data on DNA damage and the cellular defence reaction evoked by this type of danger signal: (i) a high frequency of DNA strand breaks in the salivary glands has been reported [24], suggesting a constantly elevated danger level in a main target organ of SS; and (ii) a number of cell types in SS patients have been demonstrated to display an exaggerated DNA damage response, including the PKC- and p53-dependent signalling networks. The role of this exaggerated danger response for the immune activation seen in SS patients will be outlined in the following section.
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High level of danger signals in SS causes exaggerated APC activity
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Irrespective of whether apoptosis has an aetiological role in SS, it is often seen as a principal mediator of ANA formation [12, 35]. Nuclear protein is expressed on surface blebs of apoptotic cells, being engulfed by APC, and is assumed to lead to lymphocyte stimulation by these autoantigens [35]. Many targets of ANA, e.g. DNA-PKcs and SS-A, are cleavage products of caspase 3, strongly implying apoptosis as a partner in ANA formation. Many ANA targets are also cleaved by the granzyme B protease, used by cytotoxic T cells to induce apoptosis in target cells [36]. However, all ANA antigens are not known to act as substrates for apoptotic enzymes (e.g. Ku86 and SS-B) [36], and virus infection, signalling danger to neighbouring cells, may provide an additional route for the production of ANA [37]. EpsteinBarr virus (EBV), capable of breaking host cell DNA, resulting in chromosomal integration, has long held a prominent position in the SS field [3739]. Cell surface expression of SS-B, used by EBV for RNA multiplication, in non-apoptotic infected cells, with SS-B in physical association with viral-encoded non-self protein, may well be a source of anti-SS-B production [40]. ZEBRA mRNA (a product of the EBV gene BZLF1 and coding for a transcriptional activation that mediates a genetic switch between the latent and lytic states of the virus) has been found in SS salivary gland tissue [39], which is known to be accompanied by host cell DNA strand breaks [41]. However, cell surface expression of viral factors is not limited to EBV; furthermore, the presence in salivary glands of the coxsackievirus P2A gene and protein were quite recently reported to be highly significantly associated with SS [42]. Thus, it is conceivable that a number of viruses, and possibly other infectious agents, contribute to the development of SS by increasing the tissue stress level. Using differential display technology, Moutsopoulos' group has identified a number of genes expressed preferentially in SS salivary glands, which may be causally related to an increased level of virus-induced danger signals. These include, as anticipated, some genes expressed in lymphocytes, such as CD4, but also CRISP-3 (cysteine-rich secretory protein) belonging to a gene family active preferentially in exocrine glands and mucosal surfaces, suggested to play a role in non-specific defence reactions [43].
Both SS-A and SS-B peptides bind to small RNA molecules; the cellular function of SS-A is unknown, whereas SS-B is an RNA polymerase cofactor and has been documented to bind viral RNA [12, 44]. It can be speculated that a possible mechanism whereby anti-SS-A/anti-SS-B acts as a danger signal is the stimulation of a Toll-like receptor with this type of antibody in complex with double-stranded RNA [44]. Increased or aberrant (aberrant is used by these authors to mean abnormal intracellular distribution) expression of the RNA-modifying SS-A/SS-B factors have been shown in SS salivary gland epithelial cells [45], but with undefined relation to EBV infection status. SS-A and SS-B were quite recently shown to be present at a high concentration in normal salivary gland epithelium, and to be up-regulated in the salivary gland ductal cells of SS patients and in many stress situations in normal individuals, such as acute appendicitis and non-autoimmune sialoadenitis [46].
These data provide links between tissue alarm and anti-SS-A/SS-B that might possibly even form a circulus vitiosus; danger may induce the development of anti-SS-A/SS-B, with capacity to further drive immune activation by acting as a danger signal. Thus, a picture emerges in which DNA/RNA, originating from either a non-self microbe or from cellular self-metabolism, can act as chronic danger signals that stimulate APC.
It was recently discovered that the innate immune system, represented by B-cell-activating factor (BAFF), secreted by APC such as monocytes, macrophages and dendritic cells, strongly influences the development of SS in both animal models and patients [47, 48]. This important finding was followed up by Jonsson's group in a report on attenuated apoptosis in BAFF-expressing cells in SS salivary gland biopsies [49]. There are numerous observations of B-cell subpopulation disturbances, which may be secondary to the strong BAFF activity in SS, including accumulation of CD27+ memory B cells in the salivary glands, which has been suggested to possibly reflect an influence on lymphocyte homing pathways [5055]. APC are known to secret cytokines such as IL-1, IL-6, TNF-
and BAFF after recognition of danger signals [2, 58, 56], and to up-regulate cell surface expression of HLA class I or II, and of costimulatory molecules such as CD40L and B-7 [57]. Interestingly, many of these cytokines also become expressed in stress situations in epithelial cells, including the salivary glands [2, 10, 37, 58, 59]. Most recently, much attention has become focused on plasmacytoid dendritic cells (PDC), reported to be present at an increased level in the affected organs of SS (and SLE) patients, releasing IFN-
and carrying a molecular signature characterized by expression of IFN genes and IFN-dependent transcripts [60, 61]. As for all IFNs, this cytokine is produced during virus infection and is known to significantly up-regulate HLA expression on APC and also on most other cell types, thereby acting to strongly enhance HLA-dependent lymphocyte antiviral defence [57]. Accordingly, IFN-
has been shown to act as a potent danger signal, by activating dendritic cells [62]. Although at present the role of danger signals in the activation of PDC has not been clearly defined, these findings on IFN-
provide additional support for a role of innate immunity in SS. Obviously, it also adds weight to the hypothesis of a chronic virus infection in SS as a source of danger signals.
The collected data on APC and glandular cells lend support to the mucosal epithelitis model formulated by the group of Moutsopoulos, implying a principal pathogenetic role of the salivary gland epithelium [6365]. This fits with the statement of the danger model that the immune system is activated by danger signals. Furthermore, a critical role of exocrine glands in SS is in line with the most recent update of this model, implying that the cells that control immunity are not immune cells; rather it is the tissue cell that emits alarm signals [66]. A central role in SS for the perception of danger stimuli, possibly with an exaggerated response by both APC and epithelium, seems to emerge. An attempt is made in Table 1 to provide an overview of the diverse kinds of danger signals, including DNA damage, that evoke enhanced responses in various SS cell types.
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Acetylcholine deficiency may contribute to the exaggerated APC activity
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It is tempting to link the observation of a receptor for acetylcholine (Ach) on macrophages to the pathogenesis of SS [67, 68]. Gram-negative bacteraemic shock (known to be mediated by APC cytokine release) has been shown in mice to be alleviated by stimulating neural cells to release Ach, illustrating the physiological relevance of the receptor [69, 70]. Engagement of this nicotinic-like type Ach receptor down-regulates macrophage secretion of cytokines; thus, low receptor activity is a possible factor in the high BAFF level in SS. In addition to BAFF, APC is a major source of circulating TNF-
; however, results from clinical trials of TNF blockade and from mouse models have shown clearly that this cytokine does not significantly influence the pathology of SS [71, 72]. It may well be that a cytokine profile dominated by TNF-
is coupled to a more acute inflammation type, with a rise in CRP and neutrophil count, as can be seen in rheumatoid arthritis patients. Nevertheless, a caveat has been provided by a report of renal manifestations in a subgroup of SS patients carrying a TNF-
polymorphism [73]. The relatively sparse density of macrophages in SS salivary glands probably should not be assumed to exclude influence from the regulatory APC, which may be compared with the relatively low number of dendritic cells in lymph-node germinal centres.
Support for a significant influence on SS by this APC receptor is given by the strong association between cigarette smoking and reduced lymphocyte infiltration in SS salivary gland biopsies [74], which may be mediated by the Ach-mimetic effect of nicotine on this receptor. Intriguingly, this forms an analogy to the case of the M3 muscarinic Ach receptor on ductal and acinar cells, widely considered to provide a plausible explanation for glandular dysfunction in SS, with autoantibodies against the receptor as a suggested mechanism [7577]. Although the implications for SS of this knowledge about Ach cannot be fully established at present, it seems indisputable that reduced Ach receptor activity forms a possible basis for both decreased saliva production (via the M3 receptor type) and increased APC activity (via the nicotinic-type receptor), as well as for the diverse autonomic neurological manifestations reported for SS patients [7880].
These new findings about neuroimmune interaction provide one example of a factor contributing to the enhanced stress response in SS, namely Ach deficiency contributing to APC activation. A schematic overview of possible influences on APC in SS acting to increase the level of BAFF is shown in Fig. 1.

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FIG. 1. Schematic view of the APC, illustrating the role of Ach and danger signals for BAFF production.
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Can the danger model explain the preferential and chronic engagement of the salivary glands?
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Konttinen and Käsnä-Ronkainen [81] speculate that the salivary glands are targeted in SS due to a combination of factors including constant exposure to microorganisms ascending from the oral cavity, and a need for androgen stimulation [82]. Contributing to a locus minoris resistentiae might also be a peculiar anatomical structure, presumably with release of apoptotic acinar cells into secretory ducts hindering them from physiological uptake by interstitial macrophages. Hereby, alveolar self-components will be exposed to APC in periductal immunological active foci, presumed to occur at low frequency in healthy persons [81]. As outlined in this review, this speculation is supported by several lines of experimental data. An elevated local danger level is indicated by signs of persistent virus infection [3742] and by the high frequency of DNA strand breaks [24]. These DNA breaks were determined by an in situ Klenow polymerase method usually employed for apoptosis analysis, labelling free DNA ends. However, numerous ductal cells and some acinar cells showed a positive pattern, with no morphological signs of apoptosis [24]. The origin of this DNA breakage is therefore unlikely to be apoptotic DNA degradation; two possible sources are ROS production and virus infection [16, 41]. Interestingly, this DNA damage was accompanied by staining for Ku protein, i.e. a major DNA strand break repair factor, adding force to the view of an elevated danger level in this target organ. In mice lacking the ataxia-telangiectasia mutated (ATM) gene, which codes for a regulatory protein essential for cellular recovery from DNA strand breakage [26], a major survival-limiting factor after exposure to low doses of ionizing radiation is salivary gland dysfunction [83, C. Barlow, personal communication]. Perhaps the damage caused by the radiation became added to an organ-specific high chronic baseline level of DNA strand breaks [24], making residual repair in the absence of ATM insufficient in the salivary glands of these knock-out mice.
In addition to the observation of Ku in SS glands, there are numerous other reports of an exaggerated cellular response to danger signals in SS salivary glands, including the PKC and p53 pathways, high concentrations of SS-A/SS-B and CRISP, which are suggested to play a role in cellular defence reactions [43, 45, 46], and PDC release of IFN-
[60, 61]. To summarize, there is ample experimental data in support of chronic danger signals in SS salivary glands and of a persistent exaggerated local cellular response to this alarm.
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Conclusions
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In vitro experiments clearly show enhanced reactivity to DNA damage in SS compared with cells from healthy persons exposed to the same genotoxic treatment. When these findings are put together with the salivary gland data, it becomes obvious that a dangerous liaison may be at hand: overreacting danger-sensing cells are meeting a heavy load of danger signals in a target organ. According to the danger model, this exaggerated cellular response to danger signals may explain the immune activation in SS, and may therefore constitute a primary and major aetiological determinant in this syndrome. The complex and multiorgan clinical picture of SS patients can therefore be traced back to a general disposition of cells in many tissues to overreact when confronted with diverse danger signals, ranging from DNA damage to virus infection. Such a disposition will influence multiple, potentially pathogenetic mechanisms, such as apoptosis and lymphocyte activation.
This increased understanding of the underlying causes of a systemic autoimmune disease will hopefully stimulate future research on APC using both immunohistochemistry of affected organs and in vitro Toll-like receptor stimulation experiments. The promise of improved therapy may seem weak at present, but experiences from animal transplantation models with blockers of costimulatory molecules [84] provide some clues.
The authors have declared no conflicts of interest.
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Submitted 30 December 2004;
revised version accepted 10 March 2005.