In (NZB/NZW)F₁ mice, histopathological manifestations of SS coincide with features reminiscent of SLE 18 19 . In NZW mice, exocrine gland inflammation is more pronounced in females compared with males, whereas this phenomenon is generally less apparent in NZB mice 20 21 .

Although other SS-related disease manifestations are not very pronounced in (NZB/NZW)F₁ mice, a more recent study demonstrated that an unspecific inflammatory stimulus, evoked by Freund's incomplete adjuvant, can trigger a significant drop in salivary gland function already in an early phase of the disease, while this intervention protocol affected anti-Ro levels at a latter disease stage 22 . Thought to alter the sizes of T-cell subsets, administration of anti-CD25 monoclonal antibodies shortly after birth also exacerbates sialoadenitis and auto-antibody production in this strain 23 . Lastly, Toll-like receptor 3 engagement through polyinosinic:polycytidylic acid has been studied in an attempt to recapitulate the effect of a dsRNA virus infection on the SS-like disease manifested in these mice 24 . As a result, inflammatory mediators downstream of Toll-like receptor 3, such as type-1 interferon, were transcribed and a concomitant transient loss in salivary gland secretory function was observed 24 .

In 1982 the MRL strain, at the time already established as a model of SLE, was reported to develop periductal lymphoid infiltrates in the salivary glands 25 . MRL/lpr mice differ from MRL mice with respect to a mutation involving the Fas gene 26 ; however, negative selection in the thymus does not seem to be impaired in either strain 27 . In addition, irrespective of the lpr mutation in the Fas gene, MRL/lpr mice express a detectable amount of apoptosis-related FAS protein on lymphoid cells 28 . Nevertheless, defective apoptosis associated with the lpr mutation results in increased susceptibility and severity of the disease, most probably through acceleration of the disease course 26 28 .

Immunohistochemical analyses of the organs targeted by the inflammation show the presence of activated T cells 29 30 , whose importance was further confirmed in T-cell transfer experiments 31 . Inflammatory lesions in the salivary glands of MRL/lpr mice contain B cells producing IgA and IgM rheumatoid factor 32 and were, in addition, identified to be sites of IFNγ production 30 . Of potential concern, despite female predominance and the rare occurrence of anti-Ro autoantibodies, the clinical hallmarks of SS - hyposalivation and keratoconjunctivitis sicca - are absent in this model.

The NFS/sld mouse provides a model in which aberrant immune responses against α-fodrin are elicited 33 . A defect in salivary gland development leads to aberrant enzymatic proteolysis of the structural protein fodrin by caspase 33 . Indeed, some patients with SS produce antibodies specific to the 125 kDa subunit of α-fodrin 34 . However, the association between antibodies to α-fodrin and SS does not seem to be as strong as originally thought 35 . Thymectomy performed in NFS/sld mice 3 days after birth results in development of T-cell dominated infiltrates in the salivary and lacrimal glands, and - secondary to the SS-like disease - the NFS/sld mice undergoing thymectomy 3 days after birth also tend to develop inflammatory lesions in other organs 36 .

The IQI/Jic strain was developed from the same stock that gave rise to the NOD mouse. Selection, however, was for mice that exhibited a SS-like disease comparable with NOD mice but in the absence of T1D. IQI/Jic mice develop focal inflammation in the salivary and lacrimal glands, accompanied by parenchymal destruction 37 . Sialoadenitis progresses over time and becomes more prominent in females compared with males. IQI/Jic mice also develop inflammatory lesions in several other organs, including the lung, pancreas and kidneys 38 .

Interestingly, kallikrein-13 has recently been suggested to play a role in the etiology of the SS-like disease manifested in IQI/Jic mice 39 . Kallikreins, together with other proteases, were found to be part of the salivary proteome characteristic for patients with SS 40 .

The NOD strain descends from a cataract-prone strain of outbred Jcl/ICR mice and is today the most extensively characterized model of SS and T1D. Although some genetic loci related to diabetes (idd^s loci) contribute to the inflammatory changes in the exocrine glands, it seems that diabetes and SS develop independently of each other 41 42 43 . T1D in NOD mice is restricted to the expression of the class II major histocompatibility complex (MHC) haplotype H2 ^g7 44 . Whereas NOD.B10-H2^b mice are resistant to the onset of overt T1D, they still exhibit the main disease manifestations of SS 42 . The exact extent and cellular composition of the glandular inflammation in NOD.B10-H2^b mice, however, remains to be defined.

NOD mice in which the original MHC H2 ^g7 haplotype was replaced with an H2 ^q or H2 ^p haplotype were also investigated. In summary, while the difference in H2 haplotype did not seem to affect the frequency of sialoadenitis, the disease severity varied among these strains 43 . Interestingly, introduction of the H2 ^q haplotype directed the autoimmune response towards the production of SLE-associated autoantibodies and a higher incidence of kidney pathology 43 .

Autoimmune manifestations in NOD mice represent a complex disease involving genetics, sensitivity to exogenous factors and defects in central and peripheral tolerance 44 . These factors have also been reported to contribute to the susceptibility of the strain to develop autoimmune thyroiditis 45 , SLE 46 , myasthenia gravis 47 and autoimmune encephalomyelitis 44 subsequent to specific intervention.

In NOD mice, focal inflammation in the submandibular salivary glands and the lacrimal glands develops from approximately 8 weeks of age onwards. The foci appear comparable in structure and cellular composition with infiltrates found in human salivary glands (Figure 1) 48 49 , and gender-related differences in the degree of exocrine gland inflammation have also been reported in this strain 50 . As in patients with SS, in NOD mice the relationship between histopathological changes and hyposalivation is not always obvious - which indicates a certain autonomy of the autoimmune manifestations of SS (Figure 2) 51 . Exocrine gland inflammation in NOD mice appears to precede the onset of hyposalivation by a considerable amount of time 52 . Interestingly, transition to an overt disease does not necessarily need to be associated with a significantly higher degree of glandular inflammation 52 , but hyposalivation and reduction in lacrimation were rather correlated with the occurrence of B-cell response-related gene transcripts in the exocrine glands 53 54 .

Supporting the notion of a certain independence between degree of inflammation and glandular hypofunction, introduction of an NZW-derived interval of chromosome 7 (annotated Ssial3) into NOD mice moderated sialoadenitis without ameliorating salivary gland function 55 . Analyses of dozens of inflammatory mediators in serum and saliva obtained from NOD mice, furthermore, only revealed a minimal number of biomarkers correlating with several SS-related disease manifestations in an association network 56 . In addition, successful prevention of hyposalivation - through administration of 60 kDa heat-shock protein and of 60 kDA heat-shock protein-derived peptide amino acids 437 to 460 - did not coincide with a corresponding decrease in salivary gland inflammation 57 . In contrast, biomarker signatures generated from saliva, indicating qualitative changes in salivary gland inflammation, predicted treatment outcome and salivary gland function with high accuracy 57 . Several lines of evidence indicate that as T1D progresses from early insulitis to overt diabetes there is a loss of immune cell subsets, such as regulatory T cells (T_regs) and invariant natural killer T cells within the islets 17 . Unfortunately, little is still known about the role of these cell subsets in the progression of SS. Nevertheless, NOD mice deficient for E2F transcription factor 1 - a regulator of T-cell proliferation, differentiation, and apoptosis - have a pronounced decrease in CD4⁺CD25⁺ T_regs and seem to be highly predisposed not only to T1D but also to SS 58 . In order to investigate the effects of E2F transcription factor 1 deficiency prior to the involvement of the adaptive immune system, the SS disease profile was later assessed in NOD-E2f1^-/- mice, which, in addition, carried the severe combined immunodeficiency (scid) mutation. Interestingly, this strain's saliva secretion capacity was found impaired 59 irrespective of the severe deficiencies in adaptive immunity and the absence of exocrine gland inflammation reminiscent of SS mediated by the scid mutation 60 .

Another possible connection between SS and T1D in NOD mice might involve common autoantigens. Disruption of the islet-cell autoantigen 69 kDa gene in NOD mice, a self-antigen associated with diabetes that is expressed not only in the pancreas but also in the exocrine glands, reduced SS-related histopathology and glandular hypofunction 61 . A study investigating a large cohort of patients with SS could not, however, confirm a role or true frequency of islet-cell autoantigen 69 kDa autoimmunity in patients with SS 62 . Studying the role of autoimmune regulator deficiency and central tolerance in the context of SS in NOD and Balb/c mice identified odorant binding protein 1a as a potential autoantigen involved in the etiology of autoimmune-mediated lacrimal gland pathogenesis 63 .

To determine whether B cells contribute to the SS-like disease, experiments were carried out in NOD-Igμ^null mice, which lack mature B cells 64 . The results indicate that in SS, in contrast to T1D, B cells do not significantly participate in the initiation phase of the disease 44 64 . However, B-cell activity appears to be critical in the transition to an overt disease stage in these mice, since, despite the presence of T cells in the salivary glands, NOD-Igμ^null mice fail to develop hyposalivation 64 . Subsequent studies also documented the concomitant lack of hyposalivation and anti-muscarinic acetylcholine type-3 receptor (M3R) autoantibodies of the IgG₁ isotype in IL-4-deficient and signal transducer and activator of transcription 6-deficient NOD strains 65 66 . In connection with possible non-inflammatory mechanisms underlying the onset of hyposalivation, an altered aquaporin 5 distribution - similar to the patterns observed in human specimens - has also been described in exocrine glands obtained from NOD mice 67 68 .

Protection from T1D in NOD mice has been associated with a shift from a Th1 to a Th2 cytokine expression profile in autoreactive T cells 17 . Results obtained in subsequent studies, however, indicated that compartmentalization into disease-promoting Th1 and protective Th2 cytokines cannot be applied to the overall pathogenesis manifested in NOD mice 69 . The emergence of novel immune-cell subsets such as T_regs and Th17 cells further questions the validity of such models 70 71 .

Cytokine expression in the exocrine glands obtained from NOD mice has been analyzed 72 73 . In a later study, taking advantage of recent technological developments, more comprehensive sets of inflammatory mediators were analyzed in serum and saliva obtained from NOD mice 56 . Furthermore, blocking of either lymphotoxin βR or TNFR1 signaling has given insight into the implication of these two TNF family members in the development of the SS-like disease in NOD mice 48 74 . Whereas lymphotoxin βR signaling appears to affect the degree and cellular composition of salivary gland inflammation 48 , inhibition of TNFR1 engagement has been suggested to exacerbate the manifestation of hyposalivation 74 . In an earlier study, however, transgenic overexpression of TNFR1 inhibited exocrine gland inflammation 75 . Investigation of antibody-mediated inhibition of lymphocyte migration as a potential treatment strategy demonstrated that α₄β₁-integrin, leukocyte selectin and leukocyte function-associated antigen 1 expression on lymphocytes and that vascular cell adhesion molecule 1 expression and peripheral node addressin on endothelial cells are required for lymphocyte homing to the lacrimal gland of NOD mice 76 .

The functional roles of Th1 and Th2 cytokines in the pathogenesis of SS have been assessed in some detail by comparing a set of gene knockout mice: NOD-Il4^-/- 77 , NOD.B10-H2^b -Il4^-/- 65 , NOD.B10-H2^b -C.Stat6^-/- 66 , NOD-Ifnγ^-/- and NOD-Ifnγr^-/- 78 mice. Il4^-/- NOD mice and Stat6^-/- NOD mice retain salivary secretion rates similar to Balb/c mice despite the fact that they continued to present with exocrine gland inflammation 65 66 . NOD-Ifnγ^-/- mice and NOD-Ifnγr^-/- mice were found to develop neither sialoadenitis or hyposalivation nor to present the signs of delayed salivary gland organogenesis present in the salivary gland of the parental NOD strain 60 78 . Of note, the mononuclear cell infiltrates within the lacrimal glands persisted in these two latter strains 78 . Results regarding the more recently described Th17-cell subset suggest that the Th17/IL-23 system is activated in a NOD-derived strain during the overt state of the disease 79 . Interestingly, local IL-17A expression as a result of adenovirus vector-associated Il17a delivery to the salivary gland of SS nonsusceptible C57BL/6 mice recapitulated to a large extent the SS-like disease phenotype described in the NOD strain 80 . Subsequent investigation of IL-17 as a therapeutic target at different disease stages showed that gene therapy-induced inhibition of IL-17, through expression of its receptor in the salivary gland, had the capacity to significantly reduce several important features of the SS-like disease, including salivary gland inflammation and severity of hyposalivation 81 .

To investigate the importance of specific gene regions with regard to SS-like disease manifestations, NOD-specific genetic loci were introduced into either a C57BL/6 background 41 or a C57BL/10 background 82 . For both strains, gene expression of the salivary gland tissues was compared with their respective parental strain 54 82 83 . Unfortunately, the C57BL/10-based model termed B10.Q-Nss1/Idd5 has not been assessed for salivary gland hypofunction 82 .

The principal aim for the development of the C57BL/6-based model named C57BL/6.NOD-Aec1Aec2 was primarily to circumnavigate three problems associated with its parental NOD strain: the known impact of overt T1D on the physiological process of saliva and tear secretion as well as the possible interference of T1D, overt or asymptomatic, with biological readouts obtained from the NOD strain; the fact that there is no appropriate comparative nondiseased control strain for NOD mice; and the presence of a multitude of immune system-associated defects in the NOD strain 44 .

The genes within the genetic regions designated Aec1 (Idd5 on chromosome 1) and Aec2 (Idd3 on chromosome 3) appear sufficient for the manifestation of a SS-like disease phenotype comparable with the one manifested in NOD mice 41 . First steps towards fine-mapping of Aec2 were undertaken with the purpose of identifying candidate genes potentially regulating SS-associated autoimmunity 84 . Nevertheless, although considered nonsusceptible to the development of a SS-like disease, the genomes of C57BL/6J or C57BL/10 might still contribute to the congenic strain's disease phenotype by enhancing the primary effects introduced by the congenic regions 85 . Such phenomena render it more difficult to discriminate between disease-causing and disease-promoting gene segments. In addition, the two recipient strains may develop spontaneous sialoadenitis as they age 82 86 . The improved applicability of the C57BL/6.NOD-Aec1Aec2 strain compared with the original NOD mice, however, facilitated the study of proteases in the initiation phase of the disease 87 , a more distinct delineation of the salivary and lacrimal gland transcriptome prior to and during the onset of the SS-like disease 53 54 , as well as assessment of a potential role of complement 3 in SS 88 .

Repeated intraperitoneal injection of the Ro peptides - Ro amino acids 480 to 494 or Ro amino acids 274 to 290 - emulsified in complete Freund's adjuvant and later in Freund's incomplete adjuvant has been shown to recapitulate some manifestations of SS in Balb/c mice 136 . These mice present with hyposalivation, SS-like histopathology and production of anti-Ro antibodies and anti-La antibodies by 38 weeks of age 136 . Unfortunately, the actual penetrance rate of the SS-like disease proved to be low, thereby limiting the potential value of the model 136 . Oral feeding of Ro or Ro peptides abolished the susceptibility of Balb/c mice to SS-like disease induction through the experimental procedure described above 137 . While these studies were designed to determine whether Ro, as an autoantigen, is important in the etiology of SS, there is still a question as to how Ro might actually be presented to the immune system 138 . In light of a recent study indicating that Ro52 is a negative regulator of proinflammatory cytokine production 139 , if and how these newly described properties of Ro52 contribute to SS remain to be investigated.

As alluded to above, antibodies targeting the M3R may directly mediate the inhibition of exocrine gland secretion by inhibiting neuronal innervation of acinar cells. A recent study assessed the question further by vaccinating C57BL/6-M3r^-/- mice with a six-valent mixture of free-form extracellular peptides of M3R 140 . Indeed, the inoculation of splenocytes or CD3⁺ T cells into immunodeficient C57BL/6-Rag1^-/- mice triggered the development of marked mononuclear cell inflammation in the exocrine glands accompanied by salivary gland hypo-function 140 . This study further supports the notion of a direct pathogenic role of anti-M3R immunity in SS 10 .

A subset of patients with autoimmune diseases, including patients with SS, produces autoantibodies against carbonic anhydrase II 141 . Studies carried out in mice revealed that experimental sialoadenitis can be induced through carbonic anhydrase II immunization of PL/J mice 142 as well as congenic strains of PL/J mice carrying the a H2 ^s or a H2 ^u haplotype 142 . Additional studies are required, however, to be able to estimate in more detail the resemblance of the disease manifested in this model with SS in humans.

Intraperitoneal injection of murine cytomegalovirus has been documented to lead to sialoadenitis and production of anti-Ro autoantibodies and anti-La autoantibodies in genetically modified C57BL/6 mice 143 . The modifications, affecting either FAS-mediated or TNFR1-mediated apoptosis, resulted in an incomplete clearance of murine cytomegalovirus, suggesting that any defect in this response may evoke chronic inflammation that resembles the histopathologlogical changes characteristic for SS 143 . In a subsequent study, C57BL/6-gld/gld mice, which are Fas ligand deficient, were treated with an adenoviral viral vector inducing the overexpression of Fas ligand 144 . In light of high levels of Fas ligand expression following injection of the vector, fewer than 5% of ductal and acinar cells proved to be apoptotic. Nevertheless, the intervention caused significant reductions in the number of inflammatory foci and the degree of tissue destruction in the salivary glands 144 .