Group Title: Arthritis Research & Therapy
Title: Biomarker profiles in serum and saliva of experimental Sjögren's syndrome: associations with specific autoimmune manifestations
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Title: Biomarker profiles in serum and saliva of experimental Sjögren's syndrome: associations with specific autoimmune manifestations
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Language: English
Creator: Delaleu, Nicolas
Immervoll, Heike
Cornelius, Janet
Jonsson, Roland
Publisher: Arthritis Research and Therapy
Publication Date: 2008
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Abstract: INTRODUCTION:Sjögren's syndrome (SS) is a systemic autoimmune disease that mainly targets the exocrine glands. The aim of this study was to investigate the involvement of 87 proteins measured in serum and 75 proteins analyzed in saliva in spontaneous experimental SS. In addition, we intended to compute a model of the immunological situation representing the overt disease stage of SS.METHODS:Nondiabetic, nonobese diabetic (NOD) mice aged 21 weeks were evaluated for salivary gland function, salivary gland inflammation and extraglandular disease manifestations. The analytes, comprising chemokines, cytokines, growth factors, autoantibodies and other biomarkers, were quantified using multi-analyte profile technology and fluorescence-activated cell sorting. Age-matched and sex-matched Balb/c mice served as a reference.RESULTS:We found NOD mice to exhibit impaired salivary flow, glandular inflammation and increased secretory SSB (anti-La) levels. Thirty-eight biomarkers in serum and 34 in saliva obtained from NOD mice were significantly different from those in Balb/c mice. Eighteen biomarkers in serum and three chemokines measured in saliva could predict strain membership with 80% to 100% accuracy. Factor analyses identified principal components mostly correlating with one clinical aspect of SS and having distinct associations with components extracted from other families of proteins.CONCLUSION:Autoimmune manifestations of SS are greatly independent and associated with various immunological processes. However, CD40, CD40 ligand, IL-18, granulocyte chemotactic protein-2 and anti-muscarinic M3 receptor IgG3 may connect the different aspects of SS. Processes related to the adaptive immune system appear to promote SS with a strong involvement of T-helper-2 related proteins in hyposalivation. This approach further established saliva as an attractive biofluid for biomarker analyses in SS and provides a basis for the comparison and selection of potential drug targets and diagnostic markers.
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Research article

Biomarker profiles in serum and saliva of experimental Sj6gren's

syndrome: associations with specific autoimmune manifestations
Nicolas Delaleu1, Heike Immervoll2,3, Janet Cornelius4 and Roland Jonsson1,5,6


1Broegelmann Research Laboratory, The Gade Institute, University of Bergen, Haukelandsveien, Bergen 5021, Norway
2Section of Pathology, The Gade Institute, University of Bergen, Jonas Liesvei, Bergen 5021, Norway
3Department of Pathology, Haukeland University Hospital, Jonas Liesvei, Bergen 5021, Norway
4Department of Pathology, Immunology and Laboratory Medicine, University of Florida, SW Archer Road, Gainesville, FL 32610, USA
5Department of Rheumatology, Haukeland University Hospital, Bergen, Jonas Liesvei, Bergen 5021, Norway
6Department of Otolaryngology, Head and Neck Surgery, Haukeland University Hospital, Bergen, Jonas Liesvei, Bergen 5021, Norway

Corresponding author: Nicolas Delaleu, nicolas.delaleu@gades.uib.no

Received: 28 Nov 2007 Revisions requested: 8 Jan 2008 Revisions received: 5 Feb 2008 Accepted: 20 Feb 2008 Published: 20 Feb 2008

Arthritis Research & Therapy 2008, 10:R22 (doi:1 0.11 86/ar2375)
This article is online at: http://arthritis-research.com/content/1 0/1/R22
C 2008 Delaleu et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract


Introduction Sjogren's syndrome (SS) is a systemic
autoimmune disease that mainly targets the exocrine glands.
The aim of this study was to investigate the involvement of 87
proteins measured in serum and 75 proteins analyzed in saliva
in spontaneous experimental SS. In addition, we intended to
compute a model of the immunological situation representing
the overt disease stage of SS.

Methods Nondiabetic, nonobese diabetic (NOD) mice aged 21
weeks were evaluated for salivary gland function, salivary gland
inflammation and extraglandular disease manifestations. The
analytes, comprising chemokines, cytokines, growth factors,
autoantibodies and other biomarkers, were quantified using
multi-analyte profile technology and fluorescence-activated cell
sorting. Age-matched and sex-matched Balb/c mice served as a
reference.

Results We found NOD mice to exhibit impaired salivary flow,
glandular inflammation and increased secretary SSB (anti-La)
levels. Thirty-eight biomarkers in serum and 34 in saliva obtained


Introduction
Over recent decades the immune system has been subject to
much investigation. Growing complexity has often been a
major byproduct of the discoveries reported, and subse-


from NOD mice were significantly different from those in Balb/c
mice. Eighteen biomarkers in serum and three chemokines
measured in saliva could predict strain membership with 800/0 to
1000/0 accuracy. Factor analyses identified principal
components mostly correlating with one clinical aspect of SS
and having distinct associations with components extracted
from other families of proteins.


Conclusion Autoimmune manifestations of SS are greatly
independent and associated with various immunological
processes. However, CD40, CD40 ligand, IL-18, granulocyte
chemotactic protein-2 and anti-muscarinic M3 receptor IgG3
may connect the different aspects of SS. Processes related to
the adaptive immune system appear to promote SS with a
strong involvement of T-helper-2 related proteins in
hyposalivation. This approach further established saliva as an
attractive biofluid for biomarker analyses in SS and provides a
basis for the comparison and selection of potential drug targets
and diagnostic markers.


quently models were established to cope with such complex-
ity. Regarding autoimmune diseases in general, and Sjogren's
syndrome (SS) in particular, verifying and expanding such
models is desirable, because it has proved difficult to extrapo-


CD40L = CD40 ligand; CXCL = C-X-C chemokine ligand; DA = discriminant analyses; FITC = fluorescein isothiocyanate; FS = focus score; GCP
= granulocyte chemotactic protein; IL = interleukin; IP = inducible protein; IS = insulitis score; MAP = multi-analyte profile; MCP = monocyte chem-
oattractant protein; MDC= macrophage-derived chemokine; MIP= macrophage-inflammatory protein; MMP = matrix metalloproteinase; M3R= M3
receptor; NOD = nonobese diabetic; PANTHER = Protein ANalysis THrough Evolutionary Relationships; PCA = principal component analysis;
RANTES = regulated upon activation, normal T-cell expressed and secreted; RI = ratio index; SGOT = serum glutamic-oxaloacetic transaminase; SS
= Sj6gren's syndrome; STAT = signal transducer and activator of transcription; Th= T-helper; VCAM = vascular cell adhesion molecule; vWF= von
Willebrand factor.


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Arthritis Research & Therapy Vol 10 No 1 Delaleu et al.



late findings to existing models that were often developed in
different contexts [1-3]. Recent technological advances have
greatly increased the amount of information and the number of
proteins that can be investigated in any given system and put
into a scientific context simultaneously. These technologies,
termed transcriptomics, proteomics, metabolomics and other
'-omics', were followed by the an increase in systems-based
thinking across different scientific disciplines [4]. This trend
has promoted systems biology from a technology-driven enter-
prise to an innovative tool in drug discovery, and it may lead to
a more complete perspective on how specific components
contribute at different system levels to the immune response.
Contextualization and integration have been key drivers of
such approaches.

SS (for review [5,6]), a systemic autoimmune disease, is man-
ifested by severe impairment of exocrine gland function and
focal mononuclear cell infiltrates within the salivary and lac-
rimal glands. The identification of anti-M3 receptor (M3R)
autoantibodies for the first time attributed a defined patho-
genic role to an autoantibody in SS [7-9]. The roles of other
autoantibodies in the pathogenesis of SS (especially SSA
[anti-Ro] and SSB [anti-La], which are frequently present in
patients with SS) remain to be determined. The disease can
involve organs other than the exocrine glands, and the worst
disease outcome lymphoid malignancy develops in up to
5% of patients with SS. Currently, applied treatments provide
merely marginal symptomatic relief [10,11].

The nonobese diabetic (NOD) mouse, which spontaneously
develops both SS-like histopathology and hyposalivation, is
the most widely accepted model for SS [12,13]. Based on the
findings of studies conducted in these mice [14], it is thought
that the various SS-related manifestations develop according
to a specific time course. However, similar to human SS, the
immunological relationship between the two hallmarks of SS,
namely salivary gland inflammation and hyposalivation, is far
from being understood in NOD mice. Although some diabe-
tes-related genetic loci might contribute to the SS-like disease
in NOD mice, both autoimmune diseases can develop inde-
pendently from each other [15]. Onset of SS in NOD mice is
not critically dependent on the diabetes-related H2g7 haplo-
type. NOD.B10.H2b congenic mice also exhibit an SS-like
disease in the absence of overt diabetes [16], which prevents
exclusion of diabetic animals from studies conducted in SS.
However, similar to nondiabetic parental NOD mice, they
exhibit lymphoid infiltration in the pancreas and, rarely, insulitis
[17]. Another model of SS, the C57BL/6.NOD-AeclAec2
strain, has not been screened for SS-unrelated autoimmune
manifestations other than insulitis [15]. However, the back-
ground strain C57BL/6 develops spontaneous organ-specific
autoimmune lesions in salivary glands, pancreas, kidneys,
lungs and liver, and produces a variety of autoantibodies [18].


The analytes investigated in this study, which were previously
studied in humans or NOD mice within the context of SS, are
listed in Additional file 1 (Supplementary table 1). Few studies
have assessed interactions between several immune mole-
cules and their association with disease parameters.

Summarizing findings regarding immune mediators such as
cytokines in SS, it was concluded that T-helper (Th)2
cytokines are predominant in an early phase of SS, whereas
Thl cytokines are associated with a later stage of the disease
[19]. In opposition stands the proposed principle that
decreased salivary flow, potentially associated with Th2
cytokines, follows the emergence of glandular inflammation,
which was linked to a Thl response [14,20]. In addition, the
transition between the preclinical and the overt disease state
has been associated with shifts in cytokine profiles in NOD
mice [14] and the IL-4/signal transducer and activator of tran-
scription (STAT)6 pathway [20]. Chemokines, small secreted
proteins, have been implicated in leucocyte chemoattraction,
angiogenesis, fibrosis and malignancy [21]. Despite their
uncontested potential as targets for therapeutic intervention,
few studies have examined chemokines within the context of
SS (Additional file 1 [Supplementary table 1]).

The purpose of the present study was to expand knowledge
regarding 87 analytes in serum and 75 proteins in saliva. Thirty
and 62 of these molecules have not yet been investigated in
SS patients and SS-like disease in NOD mice, respectively.
Thirty-six and 54 of the biomarkers have not yet been analyzed
in serum and saliva obtained from patients with SS, and nei-
ther have 70 of the analytes in serum and 62 biomarkers in
saliva from NOD mice been evaluated in a SS-specific context.
Based on direct comparison with Balb/c mice, we intended to
identify differentially expressed proteins and investigate their
potential to discriminate between the disease model and the
control strain. This pool of data should also allow computation
of a correlation network, representing associations of biomar-
kers with relevant clinical features of SS in nondiabetic NOD
mice, both systemically and locally.

Materials and methods
Animals and assessment of diabetes
Twenty-two female NOD/LtJ (stock #001976) and 19 female
Balb/cJ (stock #000651) mice (The Jackson Laboratory, Bar
Harbor, ME, USA) were housed in individually ventilated cages
at the animal facility of the Department of Physiology, Univer-
sity of Bergen, Bergen, Norway. The study was approved by
the Committee for Research on Animals/Forsoksdyrutvalget
(project #12-05/BBB).

To serve as controls for subsequent immunostimulatory inter-
vention studies, all mice were injected subcutaneously at 7
weeks of age with 25 ipl incomplete Freund's adjuvant emulsi-
fied in phosphate-buffered saline. From 10 weeks onward
NOD mice were screened weekly for diabetes. Two repeated


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measurements of glucosuria (>50 mg/dl; Keto-Diabur-Test
strips, Roche, Mannheim, Germany) were considered to rep-
resent onset of diabetes. At weeks 20 and 21, all mice were
screened for hyperglycaemia (>300 mg/dl; Ascensia-microfill,
Bayer Healthcare, Mishawaka, IN, USA). At 21 weeks, 10 out
of 22 mice (45.5%) were considered to be diabetic and were
excluded from all subsequent analyses. We excluded these
animals in order to eliminate from our findings any SS-unre-
lated impact of hyperglycaemia on the physiological process
of saliva secretion and the anticipated distorting effect of
hyperglycaemia on biomarker profiles.

Measurement of stimulated salivary flow
Mice were fasted but given water ad libitum and anaesthetized
with an intramuscular injection of ketamine and medetomidine.
Salivary secretion was induced by intraperitoneal injection of
0.5 uig pilocarpine/g body weight (#P6503; Sigma, St. Louis,
MO, USA) and collected during 10 minutes. Pre-weighed
tubes were weighed again after collection to determine the
amount of saliva (1 uig = 1 uil). Protease inhibitor cocktail
(#P8340; Sigma) was added at a concentration of 1:500 and
samples were kept at -800C until analysis.

Blood sampling and organ collection
Blood was collected from the saphenous vein from nonanaes-
thetized mice and by heart puncture on the day of euthanasia.
The blood was allowed to clot and centrifuged for 10 minutes
at 800 g to obtain serum. The organs were fixed in 4% formalin
before embedding in paraffin, sectioning, and staining with
haematoxylin and eosin. Sections obtained from the kidneys
were also stained using the periodic acid-Schiff staining
technique.

Evaluation of salivary gland inflammation and insulitis in
the pancreas
After qualitative evaluation of three independent sections, the
section with the highest degree of inflammation was recorded
as a whole, creating a multiple image-composite picture. A
graph tablet was used to select and morphometrically meas-
ure the total glandular area and the individual size of each
focus. Subsequently, focus score (FS; number of foci of 50 or
more mononuclear cells/mm2 glandular tissue) and ratio index
(RI; area of inflammation/area of glandular tissue) were
determined.

To determine the insulitis score (IS), at least five haematoxylin
and eosin stained tissue sections of the pancreas were ana-
lyzed in a blinded manner. On average, 32 islets per mouse
were scored, as described by Leiter [22].

Multi-analyte profiles from serum and saliva
A bead-based multiplex sandwich immunofluorescence assay
was used to generate multi-analyte profiles (MAPs) from
serum and saliva from the 12 nondiabetic NOD and 12 Balb/
c mice, comprising 82 analytes for serum and 75 for saliva


Available online http://arthritis-research.com/content/10/1/R22



(Additional file 1 [Supplementary table 2]). Analyses were con-
ducted at Rules Based Medicine Inc. (Austin, TX, USA) using
a fully automated system. For each multiplex, eight-point cali-
brators and three-level controls were included on each micro-
titre plate. Antibodies used in the MAP to recognize and
quantify the specific autoantibodies were directed against all
isotypes.

Quantification of anti-M3R antibodies
Levels of anti-M3R autoantibodies were measured as
described previously [23]. In brief, aliquots of 2 x 105 Chinese
hamster ovary cells, transfected with pcDNA5/FRT/V5-His
MsM3R-Flp-In cells, were incubated for 1.5 hours at 40C with
10 p of serum before incubation with one of the following flu-
orescein isothiocyanate (FITC)-conjugated goat anti-mouse
detection antibodies (purchased from Southern Biotech)
diluted 1:50: isotype control, goat IgG (#0110-02); IgG (H+L;
#1031-02); IgG, F(ab')2 (#1072-02); IgG2b F(ab')2 (#1092-
02); IgG20 F(ab')2 (#1079-02); and IgG3 F(ab')2 (#1102-02).
The cells were analyzed using a FACSCalibur flow cytometer
using Cell Quest software (BD Biosciences, San Jose, CA,
USA) and FlowJo (Tree Star Inc., Ashland, OR, USA). The
quantities of anti-M3R autoantibodies were analyzed by gating
on the FITC-positive population situated above the threshold,
set by the sample stained with the secondary antibody alone.
The percentage of positive cells was calculated to represent
the quantity of anti-M3R autoantibodies.

Statistical analyses
Means were compared using independent Student's t-test
(two-tailed). Bivariate linear associations, used to generate the
correlation matrixes, were computed using two-tailed Pearson
correlation (r). Strain membership prediction was assessed by
discriminant analyses (DA) and subsequent cross-validated
(leave one out) group prediction. The quality of the DA function
is expressed by its canonical correlation (R*).

Principal component analyses (PCAs) were computed from
MAP obtained from NOD mice with the purpose being to
uncover the latent structure within protein families. Protein
family membership was defined based on the Protein ANalysis
THrough Evolutionary Relationships (PANTHER) classification
system [24]. PCA seeks a linear combination of variables so
that the maximum variance is extracted from the variables. It
then removes this variance and seeks a second linear combi-
nation, and so forth. Loadings greater than 0.6 were consid-
ered defining parts of the component. For proper model
specification, variables being either differentially expressed
between the two strains (P < 0.05) and/or significantly corre-
lated (r > 0.6; P < 0.05) with one of the disease parameters
were included. As a rotation method, Varimax was chosen. The
number of components was determined using the Kaiser crite-
rion (Eigenvalue > 1.0). An explanatory criterion (>80/o) was
applied, in addition, for growth factors and cytokines in serum.
Variables being defining parts of a component were also com-


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Arthritis Research & Therapy Vol 10 No 1 Delaleu et al.



bined for DA and entered simultaneously. In serum no data
were missing and in saliva missing values were excluded pair-
wise from all analyses except PCA and DA. All analyses were
computed using SPSS 13 (SPSS Inc., Chicago, IL, USA).

Results
Autoimmune disease manifestations
At 21 weeks of age salivary secretion (expressed as uil/minute
per g bodyweight) in NOD mice (n = 12; 0.367 0.026) was
decreased by 42% compared with Balb/c mice (n = 12;
0.637 0.024; P < 0.001; Additional file 1 [Supplementary
table 2]). Salivary secretion rate classified 100% of the mice
according to their strain membership, confirming the onset of
overt SS in all NOD mice.

FS averaged 1.007 0.087 foci/mm2 and RI 0.045 0.007
mm2/mm2 in NOD, whereas Balb/c mice were free from glan-
dular inflammation. IS in nondiabetic NOD mice averaged
0.454 0.052 (Additional file 1 [Supplementary table 2]), rep-
resenting mild to intermediate insulitis expressed on a scale
from 0 (no inflammation) to 1 (all islets are to a large extent
invaded by lymphocytes). Importantly, among all variables only
serum glutamic-oxaloacetic transaminase (SGOT; r = -0.615,
P = 0.033), serum IgA (r = -0.581, P = 0.048) and anti-M3R
IgG1 (r = 0.702, P= 0.011) correlated with IS.

Histopathological evaluation of the kidneys, thyroid gland, thy-
mus, heart, lungs, liver, stomach, small and large intestines,
appendix and skin revealed a subset of NOD mice exhibiting
mononuclear cell infiltration in the kidneys (n = 5; Additional
file 1 [Supplementary figure 1A]), accompanied by hyaline
casts in two cases (Additional file 1 [Supplementary figure
1 B]). In one case, hyaline casts were found in the absence of
lymphoid infiltration. In addition, some kidneys exhibited
glomeruli with increased numbers of mesangial cells. In one
kidney hyaline material was found in glomerular capillaries
(Additional file 1 [Supplementary figure 1C]). Necrosis or
crescent formation in the glomeruli, however, was not
observed. NOD mice exhibiting signs of kidney pathology (n =
6) had significantly lower P3-microglobulin and lower anti-pro-
teinase 3 antibody levels compared with mice free from such
alterations (n = 6). Sections of lungs from 11 NOD mice pre-
sented foamy cells in the alveoli (Additioinal file 1 [Supplemen-
tary figure 1 D, E]), which in three cases were accompanied by
focal lymphoid infiltrates in the lungs (Additional file 1 [Supple-
mentary figure 1D, F]). However, convincing histological pat-
terns of interstitial lung disease were absent. Using light
microscopic screening, no signs of other extraglandular dis-
ease manifestations or other independent autoimmune dis-
eases were found.

Univariate analyses
In serum the levels of 87 biomarkers, including 14 autoanti-
bodies and four anti-M3R antibody subclasses, were meas-
ured (Additional file 1 [Supplementary table 2]). Ten proteins,


mostly cytokines, were undetectable in serum of NOD and
Balb/c. Interferon-y was detectable in one NOD mouse and
circulating fibroblast growth factor-9 in one Balb/c mouse
only. In saliva 75 biomarkers were analyzed, of which IL-3 and
leptin were undetectable in Balb/c mice; IL-3, leptin and glu-
tathione S-transferase-ac were detectable in only one, four and
four NOD mice, respectively (Additional file 1 [Supplementary
table 2]). Thirty-eight of the analytes assessed in serum were
found at significantly different concentrations in NOD mice
compared with Balb/c mice, whereas in saliva 34 analytes
were significantly different (Table 1).

DA were subsequently computed to investigate each analyte's
individual potential and relative importance in accurately pre-
dicting mouse strain. Cross-validated classification revealed
18 biomarkers in serum that predicted strain membership with
80% to 100% accuracy (hit rate) and with specificity and sen-
sitivity up to 100%, whereas such capacity was identified for
three chemokines measured in saliva (Table 2). Compared
with nonvalidated group prediction, cross-validated prediction
is based on all cases except the case being classified and it is
thought to give a better estimate of the hit rate in the popula-
tion. For each analyte shown in Table 2 the specificity (per-
centage of correct predictions in the NOD group) and
sensitivity (percentage of correct predictions in the Balb/c
group) were calculated.

Multivariate analyses
An immune response is an orchestrated process that involves
several protein families and several molecules with similar
molecular function. The PANTHER classification system was
used to classify the analytes into families of proteins with
shared function based on scientific experimental evidence and
evolutionary relationships. A correlation matrix comprising the
variables identified to be suitable for modelling purposes
exhibited profound differences in protein associations within
and among protein families (Figure 1). To gain further under-
standing of these interrelationships in NOD mice, PCA was
computed to uncover the underlying dimensions of the
immune response (Additional file 1 [Supplementary table 3]).
PCA identifies patterns in data and uses a correlation matrix to
find the linear combination of original variables, which
accounts for most of the variance. It then represents those
objects in terms of these new linear combinations, which are
called principal components. The first component will be
defined to account for the maximum of variation possible
(Additional file 1 [Supplementary figure 3]). The second com-
ponent will subsequently account for the maximum of the
remaining variation, and so forth, until a certain criterion set by
the researcher is met. In our dataset such patterns were clearly
recognizable (Figure 1). Consequently, a large number of the
original variables, entered according to protein family member-
ship, could be combined into 27 components that still
accounted for at least 80% of the original variance (Additional
file 1 [Supplementary table 3]).


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Table 1

Significantly different biomarker concentrations in NOD and Balb/c mice

Serum Fold change t-test P value Saliva Fold change t-test P value
MIP-2 (CXCL-2) 0.74 0.0130 GCP-2 (CXCL-5) 1.81 < 0.0001
IP-10 (CXCL-10) 1.70 0.0145 MIP-2 (CXCL-2) 1.83 0.0236
LTN (XCL-1) 1.40 0.0037 IP-10 (CXCL-10) 4.58 0.0001
MCP-1 (CCL-2) 1.73 0.0007 LTN (XCL-1) 2.21 0.0204
MCP-3 (CCL-7) 1.99 0.0006 MCP-1 (CCL-2) 3.95 0.0304
MIP- 1 u(CCL-3) 1.37 0.0059 MCP-3 (CCL-7) 2.97 0.0024
MIP-1y (CCL-9) 1.43 < 0.0001 MIP-1 p (CCL-4) 3.14 0.0155
MDC (CCL-22) 1.28 0.0002 RANTES (CCL-5) 4.32 < 0.0001
MIP-3p (CCL-19) 1.57 0.0004 Eotaxin (CCL-11) 2.80 0.0179
OPN (SPP-1) 2.32 < 0.0001 MDC (CCL-22) 2.65 0.0102
IL-10 1.17 0.0201 IL-17 2.27 0.0294
CD40L 0.61 0.0136 GM-CSF (CSF-2) 2.06 0.0377
CD40 1.59 0.0007 IL-7 2.52 0.0244
IL-ua 0.67 0.0003 IL-10 1.36 0.0351
IL-18 1.58 0.0005 CD40 6.31 0.0277
EGF 1.44 0.0111 IL-11 12.18 0.0018
Growth hormone 3.41 0.0001 LIF 2.99 0.0116
SCF (Kitl) 1.29 0.0092 OSM 3.99 0.0102
VEGF-A 0.64 0.0051 IL-18 2.62 0.0193
Endothelin-1 1.64 0.0184 FGF-9 (ng/ml) 2.82 0.0021
Insulin 1.48 < 0.0001 M-CSF (CSF-1) 1.68 0.0289
CRP 1.85 < 0.0001 SCF (Kitl) 2.27 0.0349
Haptoglobin 1.14 0.0359 TPO 5.40 0.0003
SAP 1.43 < 0.0001 Leptin 0.0374
SGOT 1.33 0.0062 VCAM-1 1.49 0.0424
Factor III 1.45 0.0173 MMP-9 1.72 0.0191
Factor VII 1.54 0.0092 TIMP-1 1.58 0.0444
Fibrinogen 11.88 < 0.0001 MPO 1.97 0.0100
VCAM-1 1.39 < 0.0001 Anti-Insulin 1.32 0.0415
MMP-9 0.60 0.0170 SSB (anti-La) 1.20 0.0155
Cystatin-C 1.56 < 0.0001 Anti-RNP 1.46 0.0174
MPO 1.67 0.0001 Anti-beta 2GPI 1.56 0.0117
Apo Al 1.20 0.0024 Anti-mitochondrial 1.72 0.0392
IgA 0.47 < 0.0001 Anti-SCL70 1.40 0.0021
Anti-M3R IgG, 18.56 0.0029
Anti-M3R IgG2b 11.23 0.0019
Anti-M3R IgG20 77.28 < 0.0001
Anti-M3R IgG3 2.14 0.0219
Fold change in biomarker concentrations between nonobese diabetic (NOD) and Balb/c mice. Proteins significantly different between the two
strains (P < 0.05) are listed. For the remaining proteins, please refer to Additional file 1 (Supplementary table 2). Abbreviations not defined in the
text: Apo, apolipoprotein; beta 2GPI, p2-glycoprotein; EGF, epidermal growth factor; FGF, fibroblast growth factor; GM-CSF, granulocyte
macrophage colony-stimulating factor; Kitl, Kit ligand; LIF, leukaemia inhibitory factor; LTN, lymphotactin; M-CSF, macrophage colony-stimulating
factor; MPO, myeloperoxidase; OPN, osteopontin; OSM, oncostatin M; SAP, serum amyloid P; SCF, stem cell factor; SCL, scleroderoderma;
TPO, thrombopoietin; VEGF, vascular endothelial cell growth factor.


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Arthritis Research & Therapy Vol 10 No 1 Delaleu et al.


Table 2

Strain membership-prediction potential of individual biomarkers

R* Specificity

Salivary flow 0.849

Serum

IgA 0.921

Anti-M3R IgG20 0.902

Cystatin-C 0.856

Insulin 0.837

CRP 0.812

MIP-1y (CCL-9) 0.784

VCAM-1 0.783

OPN (SSP-1) 0.778

SAP 0.757

Fibrinogen 0.747

MPO 0.708

Growth hormone 0.703

MDC (CCL-22) 0.693

IL-l a 0.678

MIP-3p (CCL-19) 0.665

IL-18 0.653

MCP-3 (CCL-7) 0.648

CD40 0.645

MCP-1 (CCL-2) 0.643

Anti-M3R IgG2b 0.600


Sensitivity


100%


100%

92%

83%

92%

92%

100%/

92%

83%

92%

83%

75%

67%

75%

83%

92%

75%

67%

67%

67%

67%0/


Hit rate


100%


100%/

100%/

92%

83%

92%

92%

92%

92%

83%

100%

92%

100%

92%

83%

92%

100%/

92%

92%

100%/

100O/o


100%


100%/o

96%

88%

88%

92%

96%

92%

88%

88%

92%

83%

83%

83%

83%

92%

88%

79%

79%

83%

83%


Saliva

RANTES (CCL-5) 0.799 75%/o 92%/o 83%/o

GCP-2 (CXCL-5) 0.745 75%/o 92%/o 83%/o

IP-10 (CXCL-10) 0.731 75%/o 100%/o 88%/o

TPO 0.678 67%/o 83%/o 75%/o

Anti-SCL70 0.645 50% 100% 80%

IL-11 0.603 58%/o 92%/o 75%/o

Results from uni-variate DA sorted according to their canonical correlation. Only predictors yielding a canonical correlation >0.6 were included.
Specificity (percentage of correct predictions in the NOD group), sensitivity (percentage of correct predictions in the Balb/c group) and hit ratio
(%/o of correctly classified cases) represent results obtained from cross-validated (leave-one-out) group prediction analyses. CRP, C-reactive
protein; MPO, myeloperoxidase; OPN, osteopontin; SAP, serum amyloid P; TPO, thrombopoietin.


Subsequently, linear interrelationships were analyzed using
Pearson correlation. The extraction of principal components
reduced the correlation matrix considerably, from 9'604 (Fig-
ure 1) to 1'994 coefficients (Figure 2). Components associ-
ated with disease manifestations are presented in Figures 2
and 3, whereas correlations between components are shown
in Figures 2 and 4. Variables for which no reasonable
component structure could be computed were included as


original variables, if a significant correlation with an autoim-
mune manifestation was detected (Figures 2 and 3). Results
form multivariate DA, based on the simultaneous entry of the
variables combined in the different components, are listed in
Additional file 1 (Supplementary table 4). They represent the
relative importance of the collinear variables, combined in an
individual component, in predicting strain membership.


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Figure 1
Sahvarv fl


Correlation matrix from original variables measured in NOD and Balb/c mice. Correlation matrix of proteins differently expressed (P< 0.05) between
nonobese diabetic (NOD) and Balb/c mice or significantly associated with autoimmune manifestations in NOD (r > 0.6, P < 0.05) sorted according
to protein family membership and principal component structure. The lower left triangle displays the coefficients obtained from NOD, and the upper
right triangle the values obtained from Balb/c mice. Red indicates positive correlation, and green negative correlation. Colour saturation indicates the
strength of the association. Protein names printed in black are variables that could not be fitted in principal component analyses. Red lettering iden-
tifies the variable as being a significant part of component 1, blue of component 2, green of component 3, and so on, of the corresponding of protein
family. *Variables representing measurements in saliva. The figure was drawn using iVici 0.91. Abbreviations not defined in the text: Apo, apolipopro-
tein; beta 2GPI, 32-glycoprotein; CRP, C-reactive protein; EGF, epidermal growth factor; FGF, fibroblast growth factor; GM-CSF, granulocyte mac-
rophage colony-stimulating factor; GRO, melanoma growth stimulatory activity protein; LIF, leukaemia inhibitory factor; LTN, lymphotactin; M-CSF,
macrophage-colony stimulating factor; MPO, myeloperoxidase; OPN, osteopontin; OSM, oncostatin M; SAP, serum amyloid P; SCF, stem cell fac-
tor; SCL, scleroderoderma; TIMP, tissue inhibitor of metalloproteinase; TPO, thrombopoietin; VEGF, vascular endothelial cell growth factor.


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Arthritis Research & Therapy Vol 10 No 1 Delaleu et al.


Figure 2


Chemokine Se-C-2
S Cytotokine Se-C-4
o Growth factor Se-C-2
CRP serum
SSGOT serum
.? VCAM-1 serum
Z= IgA serum
U) CCL; XCL Sa-C-2
Cytokine Sa-C-3
c Cytokine Se-C-2
o .0 Acute-phase reac. Sa-C-1
B3 Coagulation factor Sa-C-2
0 E Cytokine Sa-C-2
. IgA saliva
,0 CXCL Sa-C-2
E- Cytokine Sa-C-1
Cytokine Se-C-5
SAP serum
Growth factor Se-C-1
_ MMP-9 saliva
Vo Chemokine Se-C-3
0 Growth factor Se-C-3
Coagulation factor Se-C-1
Endothelin-1 serum
CCL; XCL Sa-C-1
CXCL Sa-C-1
Growth factor Sa-C-1
Chemokine Se-C-1
Insulin serum


El N





U,-q


o S a 2 0v 000 f2l 2 200 p0 . .000 0 0 00o 2os 0000.

-c.ct... ooO o- OiOc o ,- o ooo E O-OO O.o-c
n- -a cc U s Oca)o Ut UUn < ) i
) c oo.oD: CDn CD U g S*
00 0 0 0a 2 o 0
<0 0 0 0 0

Autoimmune Salivary flow Glandular Autoantibodies
maifestations inflammation
Correlation matrix of principal components and original variables associated with autoimmune manifestations. Correlation matrix of principal compo-
nents and original variables sorted according to their associations with Sjogren's syndrome (SS) disease manifestations. The upper right triangle
indicates significant P values with yellow fill (P < 0.05). The lower left triangle displays the corresponding r. Red indicates positive correlation and
green negative correlation, and colour saturation indicates the strength of the association. Abbreviations not defined in the text: CRP, C-reactive pro-
tein; SAP, serum amyloid P.


Salivary flow flow (Figure 3). The defining variables of the components are
Correlation analyses indicated no linear association between specified in parentheses. Chemokine Se-C-2 (macrophage-
salivary flow and the parameters of glandular inflammation inflammatory protein [MIP]-1la, MIP-1y and monocyte chem-
(Figure 2 and 3). PCA identified three components in serum oattractant protein [MCP]-5), cytokine Se-C-4 (IL-10) and
(Se-C) and two in saliva (Sa-C) that correlated with salivary growth factor Se-C-2 (macrophage-colony stimulating factor


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Figure 3


Serum
Chemokine Se-C-2
(MIP-la, MIP-ly, MCP-5)
Cytotokine Se-C-4
(IL-10)
Growth factor Se-C-2
(M-CSF, Growth Hormone)
CRP, SGOT, IgA


Salivary flow


SVCAM-1 SSB Serum
Cytokine Se-C-a-M3RgG1

Growth factor Se-C-13R igG2b
(VEGF-A, SCF) --- IG

a-M3R IgG3 4

Chemokine Se-C-3
(MIP-30, LTN, MDC) Autoantibody Sa-C-1
Growth factor Se-C-3 (anti-RNP, Insulin.
(EGF) Mitochondrial,
Coagulation factor Se-C-1 SSB, SCL70)
(Factor VII, Factor III)


Saliva
CCL; XCL Sa-C-2
(Eotaxin, MDC)
Cytokine Sa-C-3 ,
(CD40; CD40L; IL 18)
igA
Acute-phase reac. Sa-C-1
(CRP, SAP, SGOT)
Coagulation factor Sa-C-2
(vWF, Fibrinogen)
Cytokine Sa-C-2
(LIF, IL-10, IL-1p)


Cytokine Sa-C-1
(IFN-y, IL-7, -17, -11, -5,
-18, OSM, GM-CSF)

CXCL Sa-C-2
(GCP-2)
MMP-9


CCL; XCL Sa-C-1
(LTN, MCP-3, MIP-1P, MCP-1, RANTES)
CXCL Sa-C-1
(GRO, MIP-2, IP-10, GCP-2)
Growth factor Sa-C-1
(SCF, FGF-9, TPO)


Chemokine Se-C-1 Autoantibody Sa-C-2
(MCP-1, MCP-3, MIP-2, IP-10) (anti-beta 2GPI)

Schematic map of principal component associations. Model of principal component associations and selected original variables with autoimmune
manifestations. Red arrows mark significant positive correlations and green arrows significant negative correlations. Orange arrows represent signif-
icant associations of components with significant positive and negative loadings. The defining variables of the components are given in parentheses.
Dotted lines and grey lettering mark borderline significance. The associations involving circulating serum amyloid P (SAP), endothelin-1 and insulin
are not shown in the figure; please refer to Figure 2. Abbreviations not defined in the text: beta 2GPI, p2-glycoprotein; CRP, C-reactive protein; EGF,
epidermal growth factor; FGF, fibroblast growth factor; GM-CSF, granulocyte macrophage colony-stimulating factor; GRO, melanoma growth stim-
ulatory activity protein; IFN, interferon; LIF, leukaemia inhibitory factor; LTN, lymphotactin; M-CSF, macrophage-colony stimulating factor; OSM,
oncostatin M; SCF, stem cell factor; SCL, scleroderoderma; TPO, thrombopoietin; VEGF, vascular endothelial cell growth factor.


and growth hormone) were all positively correlated with sali-
vary flow (Figure 3 and Additional file 1 [Supplementary table
3]). The same applied for the following serum analytes, for
which no PCA-based solution could be computed: C-reactive
protein, SGOT, vascular cell adhesion molecule (VCAM)-1
and IgA.


In saliva, C-C chemokine ligand/C chemokine ligand Sa-C-2
(eotaxin and macrophage-derived chemokine [MDC]) and
cytokine Sa-C-3 (CD40 and CD40L [IL-18 borderline]) corre-
lated with decreased salivary flow. Not reflected by their cor-
responding component, salivary IL-5 (r=-0.708, P= 0.010),
thrombopoietin (r = -0.766, P = 0.004) and factor III (r = -
0.614, P= 0.034) also correlated with salivary flow.


Glandular inflammation
Glandular inflammation was negatively correlated with
cytokine Se-C-2 (negative loading for IL-1la [-0.94] and posi-
tive loading for CD40 ligand [CD40L; 0.68]). Interestingly, this
component was correlated with SSB in serum.


In saliva multiple factors exhibited a linear interrelationship with
measures of glandular inflammation. Acute phase reactants
Sa-C-1 (C-reactive protein, SGOT and serum amyloid P) and
coagulation factor Sa-C-2 (von Willebrand factor [vWF] and
fibrinogen) correlated negatively with FS and RI. vWF was
negatively loaded on coagulation factor Sa-C-2, consistent
with its initial positive correlation with increased IR (r = 0.792,
P= 0.002). In addition, secretary IgA was inversely correlated
with FS. Components extracted from protein families involved
in specific immune reactions revealed C-X-C chemokine lig-
and (CXCL) Sa-C-2 (granulocyte chemotactic protein [GCP]-
2) to be positively correlated with IR (r = 0.664, P= 0.018),
and cytokine Sa-C-1, combining eight cytokines, exhibited a
negative association with IR. In contrast, cytokine Sa-C-2 (leu-
kaemia inhibitory factor, IL-10 and IL-1p3) showed an almost
significant positive correlation with FS and IR (both r = 0.581,
P= 0.061). Correlation patterns compared with other compo-
nents further supports its connection with glandular inflamma-
tion (Figure 2).


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Arthritis Research & Therapy Vol 10 No 1 Delaleu et al


Figure 4


Serum


Saliva
CCL; XCL Sa-C-2
Cytokine Sa-C-3


Schematic map of principal component associations. Model of bidirectional inter-component associations. Red arrows mark significant positive cor-
relations and green arrows significant negative correlations. Orange arrows represent interrelations of components of which at least one had signifi-
cant positive and negative loadings.


Autoantibodies
We found all isotypes of M3R autoantibodies that we investi-
gated to be significantly increased in NOD mice compared
with Balb/c mice. Circulating SSB was the only autoantibody
that significantly correlated with any SS disease manifestation.
PCA failed to extract components from circulating autoanti-
body levels, and therefore individual associations are reported
in Figures 2 and 3. Interestingly, SSB levels were associated
with cytokine Se-C-2 (r = -0.590, P = 0.043) and VCAM-1 (r
= 0.587, P = 0.045) and anti-M3R IgG1 correlated with
cytokine Se-C-5 (IL-18; r = 0.649, P = 0.023). Levels of anti-
M3R IgG3 correlated negatively with MIP-1ia (r =-0.626, P=
0.029) and MCP-5 (r= -0.580, P= 0.048), which in turn were
associated with salivary flow (r = 0.735, P = 0.006 and r =
0.742, P= 0.006, respectively). Consistent with this observa-
tion, levels of anti-M3R IgG3 correlated with cytokine Sa-C-3
(CD40, CD40L and borderline IL-18; r= 0.724, P= 0.012) on
its part associated with decreased salivary flow. In addition,
the positive association between M3R IgG3 and CXCL Sa-C-
2 (GCP-2; r= 0.789, P= 0.002) in its turn correlating with RI,
suggests that anti-M3R IgG3 has a role in connecting different
SS-related disease manifestations. In addition, salivary matrix
metalloproteinase (MMP)-9 also correlated positively with
increased anti-M3R IgG3.

Autoantibodies in saliva were grouped into autoantibody Sa-
C-1 (anti-RNP, anti-insulin, anti-mitochondrial, SSB and anti-
scleroderma-70 antibodies) and autoantibody Sa-C-2 (anti-P2
glycoprotein). Components positively correlated with autoanti-


body Sa-C-1 were chemokine Se-C-3 (MIP-3p, lymphotactin
and MDC), C-C chemokine ligand/XCL Sa-C-1 (lymphotactin,
MCP-3, MIP-1 P, MCP-1 and RANTES [regulated upon activa-
tion, normal T-cell expressed and secreted]), CXCL Sa-C-1
(melanoma growth stimulatory-activity protein, MIP-2, induci-
ble protein [IP]-10 and GCP-2) and growth factor Sa-C-1
(stem-cell factor, fibroblast growth factor-9 and thrombopoie-
tin). In contrast, chemokine Se-C-2 and cytokine Se-C-4, in
turn also associated with salivary flow, exhibited a significant
negative correlation with autoantibody Sa-C-1. Growth factor
Se-C-3 (epidermal growth factor) and coagulation factor Se-
C-1 (factors VII and III), together with endothelin-1, correlated
negatively with autoantibody Sa-C-1 as well. Autoantibody Sa-
C-2 correlated positively with chemokine Se-C-1 (MCP-1,
MCP-3, MIP-2 and IP-10) and circulating insulin.

In summary, proteins were mostly associated exclusively with
either salivary flow, parameters of glandular inflammation, or
autoantibody levels. Dual associations were only apparent for
the following: chemokine Se-C-2, cytokine Se-C-4, cytokine
Se-C-2, cytokine Sa-C-3 and CXCL Sa-C-2. From variables
not included in PCA, only VCAM-1 in serum correlated with
two autoimmune parameters.

Associations among components
Correlations among components associated with autoimmune
manifestations are presented in Figures 2 and 4. Between
components associated with salivary flow, an antagonistic
interplay between cytokine Sa-C-3 (associated with


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decreased salivary flow) and all three components extracted
from serum (positively associated with salivary flow) was
apparent. Components associated with FS and/or RI corre-
lated strongly with each other, indicating a process in which
cytokine Sa-C-2 and CXCL Sa-C-2 oppose acute-phase reac-
tant Sa-C-1 and secreted IgA. In addition, cytokine Se-C-2
and coagulation factor Sa-C-2 played a dual role by combining
the positively associated IL-1la and vWF with the negatively
associated CD40L and fibrinogen, respectively. Regarding
components associated with autoantibodies, the components
related to anti-M3R IgG1 correlated negatively with autoanti-
body Sa-C-1, whereas all components that were positively
associated with autoantibody Sa-C-1 correlated with each
other.

We observed a complete absence of original variables or com-
ponents that correlated with salivary flow and either FS or RI.
This absence was as absolute when analyzing inter-compo-
nent correlations (Figure 2). In contrast, some components
were associated with either salivary flow or glandular inflam-
mation, having positive or negative inter-relationships with
components related to autoantibody levels, such as cytokine
Se-C-4 (IL-10) and cytokine Sa-C-1 (interferon-y, IL-7, oncos-
tatin M, granulocyte macrophage-colony stimulating factor, IL-
17, IL-11, IL-5 and IL-18). CXCL Sa-C-2 (GCP-2) exhibited
negative associations with the proteins related to increased
anti-M3R IgG1 and positive associations with anti-M3R IgG3
and MMP-9. Interestingly, MMP-9 expression correlated posi-
tively with components associated with increased glandular
inflammation.

Discussion
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 sys-
tem 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 immuno-
logically 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 sam-
ple volume required and enabled us to investigate the analytes'
connectivity through correlation networks. Such a study can-
not 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


Available online http://arthritis-research.com/content/10/1/R22



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 electrophore-
sis 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 spectrom-
etry. Nevertheless, the two approaches of antibody-based
biomarker identification and mass spectrometry-based global
proteome profiling may well complement one another in delin-
eating SS-specific disease signatures. Nevertheless, further
technological advances are required in both fields to achieve
more complete coverage of crucial immune mediators. Unfor-
tunately, 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 inte-
grated model of SS.

By accounting for SS-related extraglandular autoimmune man-
ifestations 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 [28-30]. 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 [13,31]. This divergence
of these models from clinical SS may lead one to draw errone-
ous 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 situ-
ation in the salivary glands. The presence and local production
of some inflammatory mediators and autoantibodies in the sal-
ivary glands were previously described (Additional file 1 [Sup-
plementary 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 sit-
uation (represented by Balb/c mice), the average correlation
coefficient of analytes included in the model did not show a


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Arthritis Research & Therapy Vol 10 No 1 Delaleu et al.



negative association with salivary flow (r = -0.076). The nonin-
vasive collection method and the lack of extraction procedures
predispose biomarkers, measured in saliva, as potential surro-
gate markers of disease and disease activity [25,32]. In addi-
tion, 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 secretary 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 [5,6]. 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-1a and MCP-5, which we found to be associ-
ated with higher salivary flow, are Thl -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 sal-
ivary 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 meas-
ured to be upregulated, with (perhaps most importantly) posi-
tive associations between M3R IgG3 and secretary CD40,
CD40L, IL-18, GCP-2 and MMP-9. Previous studies identified
IgG, as the crucial isotype in anti-M3R antibody mediated inhi-
bition of salivary flow in STAT6-/- [20] and IL4-/- [23] deficient
NOD mice. Nevertheless, IgG2 subclasses and IgG3 are gen-
erally 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 com-
plement activation; the latter component of the immune sys-
tem was recently related to SS pathology [39]. In addition,
IgG3 can form complexes through self-association and gener-
ate cryoglobulins [38], a feature observed in SS [6]. With
regard to the quality of the antibody response, we found total


circulating and secretary 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 path-
ogenic 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 autoantibod-
ies. Furthermore, we found circulating IL-10 to correlate nega-
tively 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 autoan-
tibody 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 pro-
teins 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 proc-
esses 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-11 is also a major inducer of GCP-
2 [44] and was, similar to IL-1 p, associated with glandular
inflammation. Leukaemia inhibitory factor, inducible through IL-
1 p [45] and loaded on the same component together with IL-
lp and IL-10, has been shown to have parallels with IL-1,
tumour necrosis factor-a and IL-6 within the context of promot-
ing 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 macro-
phage dominated inflammatory sites, IP-10 is related to Thl
immune responses, and RANTES can be involved in Thl 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 neo-
vascularization 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 mole-
cules such as IP-10, tissue inhibitor of metalloproteinase-1
and endostatin-1 were also altered [48]. Based on our find-
ings, pathogenic neovascularization deserves to receive more
attention in SS research.


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Conclusion
Increased concentrations of molecules that govern adaptive
immune responses were associated with an aggravation of SS
coupled with a strong association of Th2-related proteins with
hyposalivation. In addition, we observed great independence
among the different disease manifestations of SS, which must
be considered when one is evaluating potential drug targets.
Nevertheless, CD40, CD40L, IL-18, GCP-2 and anti-M3R
IgG3 may, however, represent a pivotal point that intersects
the various aspects of SS pathology. As multiplex technology
allowed us to draw a comprehensive picture of the overt dis-
ease stage, we suggest that treatment outcome may similarly
be monitored [49]. Hypothesis-driven verification of our find-
ings, together with validation of our findings in human SS
specimens, are important goals for the future. We also believe
that discovery-driven studies, such as the one presented here,
can widen the horizon and inspire new component-focused
basic research. Nevertheless, bridging the gap between these
two approaches represents a major challenge for the years to
come, but the reward will be a more integrated perspective on
immunology and autoimmune disease.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
ND and RJ designed the study. ND carried out the animal
experiments, salivary flow measurements and collection of
organs. HI participated in preparation and carried out the qual-
itative evaluation of the organs. ND carried out the morphomet-
rical analyses. JC and ND carried out the fluorescence-
activated cell sorting analyses for anti-M3R autoanatibodies.
ND carried out the data analyses. ND and RJ wrote the
manuscript. RJ supervised the study. All authors read and
approved the final manuscript.

Additional files


The following Additional files are available online:

Additional file 1
Supplementary table 1 provides a literature overview;
Supplementary table 2 provides a comparison of disease
parameters and analyte concentrations in serum and
saliva; Supplementary table 3 provides principal
component analyses (component structure and
loadings); Supplementary table 4 shows strain
membership prediction by defining variables from
principal components; and Supplementary figure 1
shows extraglandular disease manifestations in the
kidneys and the lungs present in a subset of NOD mice.
See http://www.biomedcentral.com/content/
supplementary/ar2375-S1 .pdf


Available online http://arthritis-research.com/content/10/1/R22




Acknowledgements
Funding for this study was provided by The Meltzer Fond, Olaf and Gull-
borg Johannessens Legacy, the Broegelmann Foundation and the Stra-
tegic Research Program, and Western Norway Regional Health
Authority. We thank Ana Carina Madureira, Gry Bernes and the staff at
the Animal Facility, Department of Physiology, University of Bergen and
at the Department of Pathology, Haukeland University Hospital, Bergen,
Norway for excellent technical assistance. In addition, we thank Inge
Jonassen from the Bergen Center of Computational Biology for helpful
advice, Dr B Delaleu-Justitz for careful revision of the manuscript, and Dr
S Klister for her support.

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