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Two NOD Idd-associated intervals contribute synergistically to the development of autoimmune exocrinopathy Sjgren's syndrome on a healthy murine background.

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Vol. 46, No. 5, May 2002, pp. 1390–1398
DOI 10.1002/art.10258
© 2002, American College of Rheumatology
Two NOD Idd–Associated Intervals
Contribute Synergistically to the Development of
Autoimmune Exocrinopathy (Sjögren’s Syndrome) on a
Healthy Murine Background
Seunghee Cha, Hiroyuki Nagashima, Vinette B. Brown, Ammon B. Peck,
and Michael G. Humphreys-Beher†
Objective. The NOD mouse is genetically predisposed to the development of at least 2 autoimmune
diseases, autoimmune diabetes and autoimmune exocrinopathy (AEC). More than 19 chromosomal intervals
(referred to as Idd regions) that contribute to diabetes
susceptibility in the NOD mouse model have been
identified, but only 2 chromosomal intervals (associated
with Idd3 and Idd5) have been shown to control sialadenitis. In the present study, we bred the Idd3 and Idd5
chromosomal intervals from NOD mice into nonautoimmune C57BL/6 mice to determine if these intervals recreate a Sjögren’s syndrome (SS)–like phenotype.
Methods. C57BL/6.NODc3 mice carrying Idd3 and
C57BL/6.NODc1t mice carrying Idd5 were crossed and
intercrossed to generate a C57BL/6.NODc3.NODc1t
mouse line homozygous for the Idd3 and Idd5 chromosomal intervals on an otherwise disease-resistant genetic
background. C57BL/6.NODc3.NODc1t mice were evaluated for biochemical, pathophysiologic, and immunologic
markers characteristic of the SS-like phenotype present in
the NOD mouse.
Results. C57BL/6.NODc3.NODc1t mice fully
manifested the SS-like phenotype of the NOD mouse,
including decreased salivary and lacrimal gland secretory flow rates, increased salivary protein content due in
part to less fluid, aberrant proteolytic enzyme activity,
decline in amylase activity, appearance of autoantibodies to exocrine gland proteins, and glandular lymphocytic focal infiltrates. Loss of secretory function occurred more rapidly in C57BL/6.NODc3.NODc1t mice
(by 12 weeks of age) than in NOD mice (by 16 weeks of
age). No signs of insulitis or autoimmune (type 1)
diabetes were observed in the C57BL/6.NODc3.NODc1t
Conclusion. Genes located within the 2 chromosomal intervals Idd3 and Idd5 appear necessary and
sufficient for manifestation of AEC. We propose that
this murine model of SS-like disease be designated
C57BL/6.NOD-Aec1Aec2. Identification of specific genes
within the Aec1 and Aec2 genetic regions should help
elucidate the mechanism(s) underlying SS-like disease.
Sjögren’s syndrome (SS) (also referred to as
“autoimmune exocrinopathy” [AEC]) is an autoimmune
disease characterized by exocrine gland dysfunction resulting from an immunologic attack primarily against the
salivary and lacrimal glands. Primary SS defines a
chronic autoimmune attack restricted to the exocrine
glands, while secondary SS is accompanied by other
autoimmune diseases, often involving the connective
tissues (1–4). The loss of protective exocrine secretions
results in increased bacterial and fungal infections of the
ocular and oral mucosa, tissue injury, and periodontal
disease (2–4). In addition to the salivary and lacrimal
glands, other exocrine tissues, including skin, lungs, the
gastrointestinal tract, and the vaginal region, may be
involved, with concomitant irritation due to excessive
dryness of the mucosal surface (5).
Supported by grants from the NIH (DE-13769 and DE13290) and by funds from the College of Dentistry, University of
Florida, Gainesville. Dr. Cha’s work was supported by a graduate
student fellowship from the Department of Oral Biology, University of
Florida, Gainesville.
Seunghee Cha, DDS, PhD, Hiroyuki Nagashima, DDS, PhD,
Vinette B. Brown, BS, Ammon B. Peck, PhD: University of Florida,
† Dr. Humphreys-Beher is deceased.
Drs. Cha and Nagashima contributed equally to this work.
Address correspondence and reprint requests to Ammon B.
Peck, PhD, Department of Oral Biology, PO Box 100424, University of
Florida, Gainesville, FL 32610. E-mail: [email protected]
Submitted for publication August 31, 2001; accepted in
revised form January 11, 2002.
Diagnosis of SS often involves the detection of
infiltrating lymphocytic populations in the minor salivary
glands, by histopathologic analyses of a labial gland
biopsy specimen (2,3). Foci of lymphocytes infiltrating
the lacrimal and salivary glands are predominantly
CD4⫹, with smaller CD8⫹ and B cell components,
and are defined as foci if there are ⬎50 lymphocytes
per 4 mm2 on histologic sections. A preferential T
cell receptor repertoire has been reported, along with
up-regulation of numerous cytokines, including
interleukin-1␤ (IL-1␤), IL-6, IL-10, tumor necrosis factor ␣, and interferon-␥ (1–3). The significance of such
factors in the development of the autoimmune pathology has yet to be determined.
Serologic evaluations are used to identify the
presence of rheumatoid factor, hypergammaglobulinemia, and specific antinuclear antibodies (ANAs) in SS,
especially antibodies to SSA/Ro and SSB/La (1–4).
Additional autoantibodies which are reactive with the
cellular components of the exocrine glands, including
␣-fodrin and the type 3 muscarinic acetylcholine receptor (M3R), are present (6–8). Xerophthalmia and xerostomia are assessed by specific tests for changes in
exocrine gland flow rates and biochemical changes in
protein composition. While the mechanisms responsible
for the loss of secretory function in SS are not yet
known, the disruption of normal neurosecretory processes is a critical factor contributing to the development
of dry mouth and dry eye complications (9).
As with other autoimmune diseases, SS both
shows a genetic predisposition with familial clustering
and has an unknown environmental trigger (10,11). The
most revealing hereditary markers of autoimmune disease susceptibilities have been those encoded by genes
of the major histocompatibility complex (MHC). However, studies of populations of various ethnicities have
yielded inconsistent results, suggesting a weak class II
MHC association for SS, an observation also reported
for the NOD (nonobese diabetic) mouse model of SS
(12–15). High levels of circulating autoantibodies further suggest that there may be immunoglobulin genes
relevant to the disease itself, while family studies indicate that autosomal genes not linked to either HLA or
immunoglobulin genes are involved in the underlying
genetic susceptibility to AEC (11). Environmental triggers, ranging from viral infections to hormonal and/or
X-linked factors, have all been advanced as potential
inducers of disease.
While certain disease characteristics have been
elucidated in studies using human tissues, the underlying
mechanisms of the disease have been difficult to assess,
since patients commonly present with end-stage disease.
To circumvent this, much research has turned to the
study of animal models of SS, especially the NOD mouse
model (12,15–19). Similar to findings in human disease,
the broad range of immunologic defects in NOD mice
are under polygenic control, and isolation of the genetic
intervals involved in the autoimmune pathology in the
NOD mouse has been an area of intense investigation
that has led to the development of the “threshold”
model of autoimmunity (13,14). Generation of numerous congenic partner strains of the NOD mouse has
been helpful in understanding both the genetic and the
immunologic components involved not only in diabetes,
but also in sicca syndrome, exhibited by these mice
(13,14,20). Studies using NOD-scid and NOD.Ig␮null
mice have played a role in showing the dependence of
secretory dysfunction on the presence of autoantibodies
in SS (7,12,15).
Functional change in the differentiated exocrine
glands appears to be dependent on genetically determined nonimmunity factors that lead to tissue targeting
and immune activation against the exocrine tissue (21–
23). The exocrine gland changes include morphologic
abnormalities (e.g., delayed organogenesis), aberrant
tissue-specific gene expression of various proteins (e.g.,
parotid secretory protein expression in adult submandibular gland tissues), increased apoptotic protease activity, and novel matrix metalloproteinase (MMP) (e.g.,
MMP-2 and MMP-9) activity (21–24). In the present
study, we showed that these pathophysiologic abnormalities, together with the appearance of lymphocytic infiltrates within the exocrine glands, are under the additive
and hierarchic control of genes associated with a 43-cM
region of chromosome 3 (Idd3) and a 47-cM region of
chromosome 1 (Idd5) (18). We propose the designation
of Aec1 for the genetic interval corresponding to Idd3
and Aec2 for the genetic interval corresponding to Idd5.
Mice. C57BL/6J, NOD/Lt, NOD.B6-Idd3.B10-Idd5,
C57BL/6.NODc3 (Idd3, Idd10, Idd17), and C57BL/6.NODc1t
(Idd5) mice were bred and maintained at the animal facility of
the Department of Pathology, University of Florida. C57BL/
6.NODc3 and C57BL/6.NODc1t mice were crossed and intercrossed, and then appropriate breeding pairs were selected for
sibling matings to produce C57BL/6.NOD-Idd3Idd5 congenic
mice. C57BL/6.NOD-Idd3Idd5 mice (to be designated C57BL/
6.NOD-Aec1Aec2) were generally healthy, showed no aberrant
weight gains or losses, bred as expected of C57BL/6J parental
mice, and showed no signs of autoimmune diabetes. Both male
and female mice of each strain were analyzed at 12 weeks and
16 weeks of age for pathophysiologic and immuno-
logic markers of autoimmunity. All experimental procedures
were approved by the University of Florida Institutional
Animal Care and Use Committee.
Histologic evaluation. Freshly excised lacrimal and
salivary glands from control and experimental mice were
placed into 10% neutral buffered formalin (pH 7.2) (no.
HT50-1; Sigma, St. Louis, MO). The fixed tissue was embedded in paraffin, sectioned, stained with hematoxylin and eosin,
and viewed by light microscopy. Lymphocyte infiltration was
quantified by determining the number of lymphocytic foci
present in sections of exocrine gland tissue. Lymphocyte
aggregations containing ⬎50 cells were considered foci, and
the number of foci in areas of 4 mm2 (referred to as focus
score) was determined. Focus scores were expressed as the
mean of the values obtained in 4 female mice per strain (18).
Analyses of salivary protein. Whole saliva samples
were collected for exactly 10 minutes from individual mice
following intraperitoneal injection of isoproterenol and pilocarpine dissolved in saline, as detailed elsewhere (21). Salivary
protein concentrations were determined using the Bradford
assay kit (no. 500-0002; Bio-Rad, Emeryville, CA). Specific
amylase activity in each saliva sample was measured by starch
hydrolysis using the ␣-Amylase kit (no. 577; Sigma).
Protease assays. Gelatinase activity in submandibular
and lacrimal glands was determined using the MMP assay kit
(no. ECM700; Chemicon, Temecula, CA). Lysates of exocrine
glands were prepared as described elsewhere (22). A unit of
activity was defined as 1.0 ␮g fluorescein isothiocyanate
(FITC)–conjugated type IV collagen substrate degraded/
minute/mg gland lysate, based on fluorescence intensity using
520 nm (emission) and 495 nm (excitation). Caspase 3 activity
was determined by enzyme activity assay using the Caspase-3
Substrate 1 kit (no. 235400; Calbiochem, San Diego, CA).
Organs from 4 mice were pooled for each group and assayed 4
times to ensure reproducibility.
Parotid secretory protein analyses. Aberrant parotid
secretory protein (PSP) expression and its proteolysis in the
submandibular gland were determined by Western blot analysis of whole gland lysates as detailed elsewhere (17). Briefly,
PSP between the normal 27-kd and NOD 24-kd protein
isoforms was used as an indicator of proteolytic activity
recognizing the NLNL cleavage site (23).
Autoantibody determinations. Purified rat M3R isolated from transfected COS-7 cells was separated on a 12%
sodium dodecyl sulfate–polyacrylamide gel (15 ␮g/lane) as
described elsewhere (6). A 1:500 dilution of sera from control
and experimental groups of mice was used as a source of
primary antibody, followed by a 1:1,000 dilution of an alkaline
phosphatase–conjugated goat anti-mouse second antibody for
the Western blot protocol. The reaction with receptor was
visualized colorimetrically using the chromogenic reagent nitroblue tetrazolium (18).
ANA determinations. ANAs in the sera of mice were
detected using the ANA screening test (#1000-F; Sigma).
HEp-2 substrate slides were overlaid with the appropriate
mouse serum samples (diluted 1:40) and then incubated for
30 minutes. After 3 washes with phosphate buffered saline,
the substrate slides were overlaid with FITC-conjugated
goat anti-mouse IgG (diluted 1:250) (F5897; Sigma) for 3
hours. Nuclear fluorescence was detected by fluorescence
Figure 1. Location of the Idd5 (Aec2) and Idd3 (Aec1) genetic
intervals on mouse chromosomes 1 and 3, respectively, as determined
by microsatellite mapping. Production of the C57BL/6.NOD-Aec1Aec2
mouse line was achieved by breeding the C57BL/6.c1t to the C57BL/
6.c3 congenic strains, and intercrossing to obtain the F2 generation.
Microsatellite mapping was used to follow the inheritance of the Idd3
and Idd5 chromosomal intervals. F2-generation mice homozygous for
both intervals were inbred for additional generations.
Statistical analysis. Values are presented as the
mean ⫾ SEM. One-way analysis of variance, Student t-test,
and paired comparison tests for significant differences in saliva
and tear volume, protein concentration, amylase, and protease
activities were performed with SAS computer software programs. P values less than 0.05 were considered significant.
Generation of the C57BL/6.NOD-Aec1Aec2
mouse. To determine if the chromosomal intervals defined by Idd3 and Idd5 are sufficient to permit full
development of an SS-like disease, C56BL/6.NODc3
mice were mated with C56BL/6.NODc1t mice to generate F1 mice. The F1 mice were then intercrossed to breed
an F2 generation, from which breeding pairs that were
homozygous for both the Idd3 and Idd5 chromosomal
segments were selected (Figure 1). To confirm that the
resulting mouse line contained the full genetic regions
and to reduce the probability that an internal crossover
event had occurred, each breeding mouse was screened
using the microsatellite markers D1Mit5, D1Mit15,
D3Mit132, and Tshb, as well as internal probes to these
markers (20). Since it is not possible to determine if the
Secretory dysfunction in the C57BL/6.NODAec1Aec2 mouse. AEC-susceptible NOD/Lt mice exhibit marked loss of secretory function beginning at 16
weeks of age. As shown in Figure 2, C57BL/6.NODAec1Aec2 mice, when stimulated with an injection of
isoproterenol/pilocarpine, exhibited a reduced secretory
response by the salivary glands at 16 weeks of age, equal
to that observed in NOD/Lt mice. In contrast, secretory
responses by salivary glands of control C57BL/6J mice
exhibited an increased response to stimulation with
isoproterenol/pilocarpine over time, reflecting normal
increase in size and weight. Interestingly, in the C57BL/
6.NOD-Aec1Aec2 mice the temporal loss of secretory
responses actually occurred sooner (beginning at 12
weeks of age) than in NOD/Lt mice (Figure 2). It should
be noted that NOD/Lt mice whose Idd3 and Idd5 regions
have been replaced by those chromosomal regions derived from C57BL/6J mice exhibit secretory function
similar to that observed in healthy C57BL/6J mice. Thus,
these findings reveal the importance of genes within
these 2 chromosomal intervals for the expression of
secretory function.
Expression of biochemical markers associated
with disease pathogenesis. NOD/Lt mice ectopically
express a number of differentiation marker proteins
suggestive of a loss of homeostasis in exocrine gland
epithelial cells (15,17,18,21–24), similar to patients with
SS. An analysis of these markers in C57BL/6.NODAec1Aec2 congenic mice revealed alterations similar to
those observed in autoimmune disease–expressing
NOD/Lt parental strains (Tables 1 and 2). The congenic
mice had reduced saliva flow rates leading to a concomitant increase in protein concentrations, similar to that
Figure 2. Temporal changes in saliva volume in mice following intraperitoneal injection of an isoproterenol/pilocarpine mixture. Whole
saliva was collected by micropipette from the oral cavities of 6 mice per
group. Salivary flow rates were measured at 8, 12, 16, and 20 weeks.
Values are the mean and SEM.
chromosomal regions in this new line are exactly those of
the Idd3 and Idd5 intervals defined in the NOD mouse,
we have designated this new line C57BL/6.NODAec1Aec2, where Aec1 corresponds to Idd3 and Aec2
corresponds to Idd5. Characterization of the disease
phenotype was carried out on offspring after 3 additional brother-sister intercrosses.
Table 1. Sjögren’s syndrome–like pathophysiology in the C57BL/6.NOD-Aec1Aec2 mouse strain compared with other strains*
Whole saliva
Strain, age
C57BL/6J, 16 weeks
12 weeks
16 weeks
NOD/Lt, 16 weeks
16 weeks
No. of
Amylase activity,
units/mg protein
Submandibular gland lysate
caspase 3 activity, pmoles/
minute/␮g protein
3.1 ⫾ 0.1
210 ⫾ 6.9
26.1 ⫾ 5.3
4.8 ⫾ 0.8†
5.7 ⫾ 0.3‡
5.0 ⫾ 0.2
3.4 ⫾ 0.1§
101 ⫾ 6.5‡
137 ⫾ 9.9†
117 ⫾ 4.6
210 ⫾ 2.1§
29.5 ⫾ 7.3
31.8 ⫾ 11.3
* Values are the mean ⫾ SEM from 3 experiments. ND ⫽ not determined.
† P ⬍ 0.05 versus parental C57BL/6J mice, by Student’s t-test (analysis of variance).
‡ P ⬍ 0.01 versus parental C57BL/6J mice, by Student’s t-test (analysis of variance).
§ P ⬍ 0.05 versus parental NOD/Lt mice, by Student’s t-test (analysis of variance).
Table 2. Analysis of MMP-9 activity in the C57BL/6.NOD-Aec1Aec2
mouse strain compared with other strains*
Gland lysate tested
0.413 ⫾ 0.06
0.900 ⫾ 0.32†
0.890 ⫾ 0.03
0.495 ⫾ 0.01‡
0.020 ⫾ 0.01
0.030 ⫾ 0.01
0.050 ⫾ 0.01
0.023 ⫾ 0.01
* Matrix metalloproteinase 9 (MMP-9) activity was determined when
mice were 16 weeks old. Values are the mean ⫾ SEM from 3
† P ⬍ 0.01 versus parental C57BL/6J mice, by Student’s t-test (analysis
of variance).
‡ P ⬍ 0.01 versus parental NOD/Lt mice, by Student’s t-test (analysis
of variance).
observed in the NOD/Lt strain expressing SS-like pathophysiology (P ⬍ 0.01 versus C57BL/6 parental strain).
Amylase activity in the saliva of C57BL/6.NODAec1Aec2 mice at 12 weeks was ⬃50% of the enzyme
activity in the parental healthy strain (P ⬍ 0.01) (Table 1).
PSP, a salivary protein not normally synthesized
by acinar cells of the submandibular gland, is aberrantly
expressed in the submandibular gland of adult C57BL/
6.NOD-Aec1Aec2 congenic mice, similar to findings in
NOD/Lt mice (results not shown). The PSP that is
secreted into whole saliva, primarily by acinar cells of the
parotid gland, has been shown to undergo enzymatic
cleavage at an NLNL site located in the N-terminus
region in mice that develop AEC, but not in healthy
mice (25). This difference is shown in Figure 3, comparing PSP profiles from whole saliva of 20-week-old
C57BL/6J and NOD/Lt mice and 8–16-week-old C57BL/
6.NOD-Aec1Aec2 mice. As expected, with development
of AEC, PSP in whole saliva of C57BL/6.NODAec1Aec2 mice exhibited a temporal change from a
27-kd to a 24-kd isoform associated with the expression
of a proteolytic enzyme. However, the fact that the
alteration in PSP occurred between 8 weeks and 12
weeks of age indicates that the disease phenotype probably appears earlier in C57BL/6.NOD-Aec1Aec2 mice
than in NOD/Lt mice.
Markers indicating onset of AEC. Salivary and
lacrimal tissues targeted in AEC in humans and in the
NOD/Lt mouse demonstrate up-regulated activities of
MMPs (MMP-9) (24,26,27) and caspases (17). We found
that MMP-9 activity in the submandibular gland of mice
from a congenic C57BL/6J background was similar to
the activity detected in NOD/Lt mice (Table 2). Although elevated over the level in the parental C57BL/6J
background, the level of MMP activity in the lacrimal
gland of C57BL/6.NOD-Aec1Aec2 mice did not reach
that observed in the NOD/Lt mice (P ⬍ 0.05). Reciprocally, the replacement of the Idd3 and Idd5 genetic
intervals in the NOD/Lt background with those intervals
derived from C57BL/6J mice resulted in NOD/Lt mice
with MMP activity similar to that observed in C57BL/6J
mice (Table 2). Caspase 3 activity in 16-week-old
C57BL/6.NOD-Aec1Aec2 mice was elevated over that in
C57BL/6J control mice but did not achieve the level in
NOD/Lt mice. It may be possible that, as a consequence
of the early onset of disease in these mice, maximum
caspase 3 expression occurs at an earlier age.
The autoimmune response in C57BL/6.NODAec1Aec2 mice. NOD/Lt mice develop focal lymphocytic
infiltrates in the salivary and lacrimal glands, which are
accompanied by increased cytokine production and production of autoantibodies (28–30). Histologic evaluation
of the submandibular and lacrimal glands indicated the
presence of focal immune cell infiltrates detected at 12
weeks in the submandibular gland and at 16 weeks in the
lacrimal gland of these mice (Figure 4). According to the
standard grading system for determining the number of
foci of lymphocytes infiltrating the exocrine tissues, the
Figure 3. Temporal changes in the sodium dodecyl sulfate–
polyacrylamide gel electrophoresis profiles for parotid secretory protein (PSP) in the saliva of C57BL/6.NOD-Aec1Aec2 mice at 8, 12, and
16 weeks of age. Two-microgram specimens of whole saliva from
normal C57BL/6J, autoimmune exocrinopathy–susceptible NOD/Lt,
and C57BL/6.NOD-Aec1Aec2 mice were studied. PSP was detected by
Western blotting using a polyclonal rabbit anti-mouse PSP antibody
(17). Saliva samples from 5 individual mice were examined on at least
2 separate occasions.
Figure 4. Photomicrographs showing focal leukocyte infiltrates (arrows) in the submandibular (SMX) and lacrimal (LAC) glands of NOD/Lt,
C57BL/6.NOD-Aec1Aec2, and NOD.B6-Idd3.B10-Idd5 mice. Exocrine tissues were freshly explanted, fixed in 10% neutral buffered formalin,
embedded in paraffin, and sectioned (0.4␮). Sections were visualized by light microscopy (hematoxylin and eosin stained; original magnification ⫻
C57BL/6.NOD-Aec1Aec2 congenic mice had focus
scores reflective of those in NOD/Lt mice (Table 3).
C57BL/6J mice do not typically have focal infiltration
of exocrine tissues at either 12 weeks or 16 weeks of
age (31).
Flow cytometric analyses indicated that the immune cell aggregates consisted of CD4⫹,CD8⫹ T cells,
and B lymphocytes. The B cells accounted for ⬃25–30%
of the lymphocytes infiltrating the lacrimal glands, simTable 3. Lymphocyte infiltration in the exocrine glands of C57BL/
6.NOD-Aec1Aec2 mice compared with other strains as measured by
focus score*
Focus score
Strain, age
C57BL/6J, 16 weeks
12 weeks
16 weeks
NOD/Lt, 16 weeks
NOD.B6-Idd3.B10-Idd5, 16 weeks
* Foci contained ⬎50 lymphocytes/4-mm2 area of tissue. Numbers of
foci were determined by 2 individuals, under blinded conditions.
Values are the mean ⫾ SEM.
ilar to the findings previously reported for NOD/Lt mice
(30). The ratio of CD4⫹:CD8⫹ T cells was lower in the
Idd3Idd5 double-congenic mice compared with the
NOD parental strain (4:1 versus 2:1; P ⬎ 0.05), in both
salivary and lacrimal glands. Steady-state concentrations
of messenger RNA for various inflammatory cytokines
in the double-congenic mice were similar to those in the
parental NOD mice (data not shown), with the exception of elevated levels of transforming growth factor ␤
(TGF␤) in the double-congenic mice. In contrast to our
expectation, TGF␤ levels were 88% and 56% higher in
the submandibular and lacrimal glands, respectively, in
the C57BL/6.NOD-Aec1Aec2 mice.
The presence of ANAs was determined by immunofluorescence staining of HEp-2 cells. HEp-2 cells were
first incubated with sera obtained from parental
C57BL/6J and NOD/Lt mice as well as C57BL/6.NODAec1Aec2 mice, and then with FITC-conjugated goat
anti-mouse IgG antiserum. Distinct staining was evident
within the nucleus and slight staining evident in the
cytoplasm when the HEp-2 cells were incubated with
either NOD/Lt or C57BL/6.NOD-Aec1Aec2 sera, but
not with C57BL/6J sera (results not shown). These sera
were positive at a 1:40 dilution, a concentration known
to enable detection of ANAs in parental NOD/Lt mice.
Western blotting with purified rat M3R protein isolated
from transfected COS-7 cells (6) was used to determine
the presence of autoantibodies to M3R. Again, the sera
from both NOD/Lt and C57BL/6.NOD-Aec1Aec2 mice
reacted positively, while sera derived from the parental
C57BL/6J mice failed to react.
Previously, the NOD Idd3 and Idd5 genetic intervals have been transferred separately into the genetic
background of the disease-resistant strain C57BL/6J
(18). The NOD-derived Idd5 (Aec2) interval resulted in
elevated expression of biochemical markers of SS-like
disease associated with the asymptomatic initiation
phase in NOD/Lt mice. Idd3 (Aec1) did not appear to
cause alterations in biochemical markers of salivary
gland or immune system function. The transfer of either
of these alleles did not produce evidence of insulitis or
diabetes on a normal background. There was no impact
on exocrine tissue secretory response in either congenic
C57BL/6 strain. The transfer of individual resistance
alleles did not prevent disease progression or onset of
loss of secretory function.
However, the transfer of both the C57BL/6 Idd3
and C57BL/10 Idd5 alleles to the NOD/Lt genetic
background reduced the expression of autoimmunity
markers and partially restored secretory response. This
suggests that these 2 alleles are capable of acting in an
additive and hierarchic manner to control AEC (18,19).
From the results of the present studies, it would appear
that the Idd3 and Idd5 susceptibility alleles in the NOD
mouse are sufficient for the generation of SS-like disease
on a previously autoimmune-resistant genetic background. Thus, the intervals identified originally as contributing to diabetes onset contain genetic material
leading to AEC, with designations of Aec2 and Aec1 for
Idd5 and Idd3, respectively.
With transfer of the NOD Idd3 and Idd5 susceptibility alleles into the C57BL/6 disease-free mouse,
there was a more rapid progression to onset of SS-like
pathophysiology in terms of focal lymphocytic infiltration in the submandibular gland, and, more importantly,
loss of saliva secretory capacity, than in NOD mice. The
Idd5 interval on chromosome 1 and Idd3 interval on
chromosome 3 encompass 47 cM and 43 cM of NOD
genetic material, respectively (20). Sialadenitis in the
NOD mouse has previously been mapped to the distal
portion of chromosome 1, while diabetes segregates to a
centromeric portion of chromosome 1 (20).
Recently, the Idd5 interval has been subdivided
into 2 smaller loci whose proximal Idd5.1 includes Casp8
as well as Cd152 (Ctla-4), and whose distal Idd5.2
contains Nramp1 and Cmkar2 as candidate genes for
diabetes (32). Although murine Idd5.1 overlaps the
orthologous CTLA-4/IDDM12 locus of humans and allelic variants in Ctla-4 contribute to diabetes susceptibility, development of AEC does not appear to rely on this
genetic interval, since Aec2 is located distal to Idd5.1 but
does encompass the Idd5.2 locus and Bcl-2 gene (32).
Candidate genes in the Aec2 interval include fibronectin
1 (Fn1), insulin-like growth factor binding protein 2 and
5 (Igfbp2 and Igfbp5), type IV collagen (Col6a3), B cell
translocation gene 2 (Btg2), IL-10 (Il10), and laminin 1
and 2 (Lamc1 and Lamc2). A genetic association between promoter region polymorphisms in the IL-10 gene
and primary SS has been reported recently (33).
The Idd3 allele in C57BL/6.NOD-Aec1Aec2 congenic mice contains additional susceptibility intervals
Idd10 and Idd17 (20,32,34). Based on the results of a
number of studies of autoimmune diabetes and experimental autoimmune encephalomyelitis, using a 0.35-cM
Idd3 susceptibility segment from chromosome 3 (33–35),
it has been proposed that the IL-2 gene is the candidate
gene responsible for the influence on immune cell
activation in autoimmunity in these mouse models
(31,33,34,36–38). The other Idd alleles of chromosome 3
(Idd10, Idd17, Idd18) have been proposed to enhance
the contribution of the Idd3 susceptibility interval, but
are not necessary for its action in autoimmune disease
(39,40). The rapid onset of secretory dysfunction in
C57BL/6.NOD-Aec1Aec2 mice, correlating with the appearance of detectable autoantibodies to ANA and
M3R, may result from epistatic interactions of additional
susceptibility alleles. Alternatively, additional genetic
susceptibility alleles that are too weak to produce autoimmune disease in the normally healthy genetic background of this strain may reside on the C57BL/6J genetic
Our study using complementary DNA microarray technology has provided an interesting connection
between genetically programmed nonimmune components and adult onset of AEC in the NOD mouse.
Although identification of the major causative gene(s)
and critical antigen(s) is still far from complete, candidate genes can be narrowed down further by the generation of probes from C57BL/6.NOD-Aec1Aec2 mice.
This will allow us to discard a list of genes reflecting
strain differences and to find common genes in the NOD
mouse and the C57BL/6.NOD-Aec1Aec2 mouse, which
are definitely associated with the manifestation of dis-
ease phenotype. Having high synteny between humans
and mice, mouse candidate genes identified by the
microarray system can be used to screen human libraries
and examine common mutations or polymorphisms in
the orthologous human genes, in order to determine
how the candidate genes affect the organogenesis of
salivary glands and function in disease pathogenesis in
the subset of humans who are susceptible. In addition,
more reliable screening tools for diagnosis and intervention strategies to cure SS may become more readily
The C57BL/6 genome most likely harbors additional autoimmune susceptibility alleles. Besides I-Ag7,
NOD mice are negative for the expression of a second
MHC marker, I-E (31,39). The presence of an I-Enull
genotype in various mouse strains, such as C57BL/6, has
been reported to lead to the spontaneous occurrence of
focal infiltrates in the exocrine tissues of older mice
(typically ⬎1 year of age). The role of the I-E locus in
autoimmunity can now be assessed in the new genetic
model for SS by backcrossing the appropriate mouse
MHC alleles.
Generation of the C57BL/6.NOD-Aec1Aec2
mouse presents a new opportunity to elucidate the
genetic control of SS. A number of techniques, e.g.,
positional cloning, expression microarrays, or traditional
recombinant congenic analyses for Aec1 and Aec2 intervals maintaining the traits of SS-like disease, may permit
the identification of candidate genes. Identified genes
can then be used to reveal the mechanism underlying the
development of autoimmune disease in susceptible individuals. This may open new targets for gene or drug
therapy. These markers may further serve as a diagnostic
tool for early detection of primary SS, and more importantly, for screening of patients with other autoimmune
connective tissue diseases for the potential development
of secondary SS.
The authors would like to thank Joy Nanni and Janet
Cornelius for technical assistance throughout these studies.
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