By studying the immune system through the application of reductionist principles, its mediators have been thoroughly analyzed over recent decades, which has yielded tremendous scientific advances. However, studying the properties of its isolated components is limited in terms of elucidating how system properties emerge, because they may strongly rely on and arise from interactions between its numerous constituents. In this study we present a substantial amount of data on immunologically relevant proteins, for which often only scant or no information within the context with SS has been published. Multiplexing of analytes thus significantly diminished the sample volume required and enabled us to investigate the analytes' connectivity through correlation networks. Such a study cannot be as conclusive in defining the role of a single protein as a component-focused experimental study design. However, it represents a novel way to analyze the implications of multiple molecules in a specific condition, by providing insight into the inter-relationships that define a specific system state.
Analyses of protein levels instead of mRNA expression data can allow exclusion of certain factors that cause uncertainty, such as RNA stability and correlations between mRNA levels and corresponding protein levels. A recent study combined global gene expression analyses with quantitative proteomics based on two-dimensional gel electrophoresis and mass spectrometry in saliva obtained from patients with SS [25
]. Indeed, the correlation between mRNA levels and proteins was proven to be poor. Using mass spectrometry, 42 proteins were identified that were significantly altered when comparing pooled saliva from SS patients with healthy control individuals [25
]. None of the proteins identified by Hu and coworkers [25
] was included in our MAP. Two-dimensional gel electrophoresis is limited in terms of its sensitivity in identifying proteins with concentrations in the pg/ml range, especially if they may be masked by abundant proteins present in the biofluid of interest. However, methods such as stable-isotope protein tagging or subtractive proteomics may improve the number of immune system related proteins identified by mass spectrometry. Nevertheless, the two approaches of antibody-based biomarker identification and mass spectrometry-based global proteome profiling may well complement one another in delineating SS-specific disease signatures. Nevertheless, further technological advances are required in both fields to achieve more complete coverage of crucial immune mediators. Unfortunately, molecules of proven importance in SS, such as B-cell activating factor [26
] and type I interferons [27
] were not part of our MAP. Including such crucial molecules in future studies and the present correlation network would indeed greatly increase completeness and enahce the plausibility of an integrated model of SS.
By accounting for SS-related extraglandular autoimmune manifestations and insulitis, we are confident that analyses in serum reflect to a large extent systemic aspects of SS. Nevertheless, although we could not associate the presence of extraglandular disease manifestations with the situation in the salivary glands, it would be of interest to investigate further the involvement of kidneys and lungs in both experimental and human SS [28
]. The potential presence of SS-unrelated histopathology or of other subclinical autoimmune diseases in mouse models for SS represents a common problem of all mouse models used in SS research [13
]. This divergence of these models from clinical SS may lead one to draw erroneous conclusions from measurements in serum. Indeed, biomarker profiling solely focusing on serum or plasma may require considerable validation efforts to prove their specificity for a specific autoimmune disease such as SS.
In contrast, because it is directly collected from the site of inflammation, saliva may largely reflect the immunological situation in the salivary glands. The presence and local production of some inflammatory mediators and autoantibodies in the salivary glands were previously described (Additional file 1
[Supplementary table 1]); these findings were largely confirmed by our study. Importantly, we did not observe a concentration effect related to lesser fluid secretion. In a noninflammatory situation (represented by Balb/c mice), the average correlation coefficient of analytes included in the model did not show a negative association with salivary flow (r = -0.076). The noninvasive collection method and the lack of extraction procedures predispose biomarkers, measured in saliva, as potential surrogate markers of disease and disease activity [25
]. In addition, they may reflect the disease independent from other inflammatory conditions in patients. The identification of a set of biomarkers in human saliva, similar to the three chemokines we identified in mice, that predicts the presence of glandular inflammation with high accuracy would represent a major advance in the field.
Traditionally, loss of secretory capacity, degree of lymphoid infiltration and production of specific autoantibodies have been anticipated to correlate with each other and to indicate disease state and severity [5
]. However, the correctness of this assumption was difficult to prove. Our findings strongly argue against such close interrelationships and suggest that there is much independence of the various hallmarks of SS. Only RI and SSB correlated directly with each other, and the separation between proteins in terms of whether they were associated with hyposalivation or with glandular inflammation was absolute.
Circulating MIP-1α and MCP-5, which we found to be associated with higher salivary flow, are Th1-related chemokines that are negatively regulated by STAT6 [33
]. In opposition, eotaxin and MDC, correlating with decreased salivary flow, are dependent on STAT6 [33
] and are considered to be Th2-related chemokines [34
]. In accordance with these findings, STAT6-deficient NOD mice did not develop hyposalivation [20
]. Both eotaxin and MDC are produced by Th2-promoting dendritic cell types upon engagement of CD40/CD40L [35
]; in our study, these two proteins were associated with low salivary secretion capacity as well. CD40 and CD40L are expressed on salivary gland epithelial cells and infiltrating lymphocytes in biopsies obtained from SS patients [36
]. The observed correlation between CD40/CD40L and anti-M3R IgG3
levels may therefore be related to the primary role played by CD40 and CD40L in B-cell survival, B-cell proliferation, antibody production and antibody isotype switching [37
]. We found all of the antibody isotypes binding M3R that we measured to be upregulated, with (perhaps most importantly) positive associations between M3R IgG3
and secretory CD40, CD40L, IL-18, GCP-2 and MMP-9. Previous studies identified IgG1
as the crucial isotype in anti-M3R antibody mediated inhibition of salivary flow in STAT6-/-
] and IL4-/-
] deficient NOD mice. Nevertheless, IgG2
subclasses and IgG3
are generally considered to be significantly more potent in mediating pathogenic effects [38
]. In contrast to other isotypes, the effect of IgG3
is FcR independent and strictly related to complement activation; the latter component of the immune system was recently related to SS pathology [39
]. In addition, IgG3
can form complexes through self-association and generate cryoglobulins [38
], a feature observed in SS [6
]. With regard to the quality of the antibody response, we found total circulating and secretory IgA to be related to lower disease activity.
Apart from B-cell fate, CD40/CD40L ligation plays a central role in converting a tolerogenic antigen presentation into pathogenic immune activation [40
]. In conditions with chronic inflammation, CD40 and other inflammatory mediators can have adverse effects on tissue renewal and repair processes [41
]. Indeed, we found specific growth factors to be negatively correlated with CD40 and CD40L and secreted autoantibodies. Furthermore, we found circulating IL-10 to correlate negatively with CD40/CD40L; the anti-inflammatory properties of IL-10 may apply to our model, because increased circulating IL-10 did correlate with higher salivary flow and lower autoantibody concentrations in saliva. IL-10 gene transfer in NOD mice partially suppressed the appearance of SS-like features [42
]. However, we found IL-10 in saliva to be associated with glandular inflammation, which corroborates reports indicating that IL-10 transgenic mice develop progressive histopathology and hyposalivation, evocative of SS [43
With the exception of vWF, increased concentrations of proteins in saliva related to acute tissue injury were all associated with a lower degree of glandular inflammation. These events may be followed later by inflammatory cell invasion into the glandular tissue and/or mirror an imbalance between processes that restore homeostasis and factors that promote chronic inflammation. Higher levels of IL-1 family members did correlate with both worsening of hyposalivation and increased glandular inflammation. IL-1β is also a major inducer of GCP-2 [44
] and was, similar to IL-1β, associated with glandular inflammation. Leukaemia inhibitory factor, inducible through IL-1β [45
] and loaded on the same component together with IL-1β and IL-10, has been shown to have parallels with IL-1, tumour necrosis factor-α and IL-6 within the context of promoting inflammation in rheumatoid arthritis [45
Among the biomarkers analyzed in saliva, GCP-2, IP-10 and RANTES exhibited the greatest potential in predicting strain membership. GCP-2 is expressed at neutrophil and macrophage dominated inflammatory sites, IP-10 is related to Th1 immune responses, and RANTES can be involved in Th1 and Th2 immune responses [46
]. GCP-2 and IP-10 have opposite roles in angiogenesis [47
]; this issue has not yet been addressed in SS, despite the recognized importance of neovascularization in promoting the influx of inflammatory cells [48
]. Proteins such as GCP-2, vascular endothelial growth factor, epidermal growth factor, IL-1, VCAM-1 and MMP-9, which we found to be significantly increased in NOD mice, are involved in angiogenesis [48
]. In addition, angiostatic molecules such as IP-10, tissue inhibitor of metalloproteinase-1 and endostatin-1 were also altered [48
]. Based on our findings, pathogenic neovascularization deserves to receive more attention in SS research.