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Sjgren's syndrome in MRLl and MRLn mice.

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157
SJOGREN’S SYNDROME IN MRL/1 AND M R L h MICE
ROBERT W. HOFFMAN, MARGARET A. ALSPAUGH, KIM S. WAGGIE,
JAMES B. DURHAM, and SARA ELLEN WALKER
Six autoimmune murine models (MRLh, MRL/n,
NZB, NZB/NZW, PN, C57BL/6J-lpr/lpr) were compared with normal control C57BL/6J and DBM2 mice to
determine if spontaneous autoimmune disease was associated with evidence of Sjogren’s syndrome. Schirmer
tests documented dry eyes in NZB/NZW and PN mice;
other autoimmune strains and controls had normal tear
formation. All autoimmune mice had conjunctivitis, but
this abnormality was most severe in the PN strain.
Ninety-eight percent of MRL/I and MRL/n mice had
mononuclear cell infiltrates in lacrimal glands, and
salivary glands were involved to a lesser degree. New
Zealand mice and PN mice had smaller gland lesions.
The extensive gland destruction in MRL/I and MRLln
mice suggested that these substrains merit further studies as animal models of Sjogren’s syndrome.
____
From the Veterans Administration Medical Center, Columbia, Missouri and the University of MissouriXolumbia.
Supported by the Medical Research Service of the Veterans
Administration and grant 2 R 0 1 AM 25868 from the National
Institutes of Health, U.S. Public Health Service. Dr. Waggie’s
fellowship was supported by DHHS grants RR00471 and RROSO16.
Robert W. Hoffman, DO: Resident, Department of Medicine, University of Missouri-Columbia; Margaret A. Alspaugh,
PhD: Associate Professor of Medicine and Pathology, University of
Missouri-Columbia; Kim S. Waggie, DVM: Research Associate,
Department of Veterinary Pathology, University of Missouri School
of Veterinary Medicine; James B. Durham, MD: Assistant Professor
of Pathology, University of Missouri-Columbia; Sara Ellen Walker,
MD: Chief, Rheumatology Section, Harry S. Truman Memorial
Veterans Hospital and Associate Professor of Medicine, University
of Missouri-Columbia.
Address reprint requests to Sara Ellen Walker, MD, 1 IIF,
Harry S. Truman Memorial Veterans Hospital, 800 Stadium Road,
Columbia, MO 65201.
Submitted for publication March 21, 1983; accepted in
revised form August 22, 1983.
Arthritis and Rheumatism, Vol. 27, No. 2 (February 1984)
Sjogren’s syndrome was defined originally as
keratoconjunctivitis sicca and xerostomia associated
with a connective tissue disease (1). Although carefully tabulated data concerning the true incidence of
Sjogren’s syndrome are not available, several authors
have suggested that it is one of the most common
rheumatic diseases (2,3). Sjogren’s syndrome may
occur with a variety of connective tissue diseases,
including rheumatoid arthritis and systemic lupus erythematosus (2). Primary Sjogren’s syndrome, or sicca
syndrome, occurs in patients who have only ocular
and oral involvement; this disease is of special interest
because it is related to lymphoreticular malignancies
(4) *
Early attempts to induce Sjogren’s syndrome in
experimental animals were not consistently successful. Chan ( 5 ) produced mild salivary gland inflammation in 50% of guinea pigs injected with submaxillary
gland homogenate suspended in complete Freund’s
adjuvant. Other investigators found that injections of
salivary gland extract required 2 adjuvants (carbonyl
iron and Bordetelfapertussis) to produce severe sialadenitis (6).
The first spontaneous models of this disorder
were reported by Kessler (7), who described mononuclear cell infiltration in lacrimal and salivary glands of
autoimmune New Zealand black (NZB) and hybrid
New Zealand blacWNew Zealand white (NZB/NZW)
mice (8). These animals, which have been studied
extensively as models of systemic lupus erythematosus, were considered to have coexisting Sjogren’s
syndrome.
Recently, new murine models of systemic lupus
erythematosus have provided the opportunity to further examine relationships between murine lupus and
HOFFMAN ET AL
Sjogren’s syndrome. MRL/I mice and congenic
MRL/Mplpr/lpr mice develop accelerated immune
complex disease with features of lupus and rheumatoid
arthritis. These animals have anti-Sm and anti-DNA
antibodies, glomerulonephritis, vasculitis, IgM and
IgG rheumatoid factors, and erosive arthritis (9,lO).
The MRL/n substrain has mild autoimmune disease
with low levels of anti-DNA antibodies and late-onset
glomerulonephritis (9). Congenic C57BL/6J-lpr/lpr
mice, derived by transferring the Ipr/lpr genome to
standard inbred C57BL/6J mice, develop antinuclear
antibodies and glomerulonephritis (1 1). Palmerston
North (PN) mice have antibodies to DNA, immune
complex glomerulonephritis, and vasculitis (12).
In the current study tear formation, ocular
tissues, and salivary and lacrimal glands were evaluated in autoimmune MRLII, MRL/n, NZB, NZB/NZW,
and PN mice and results were compared with normal
C57BL/6J and DBM2 control strains. All autoimmune
strains had at least 1 manifestation of Sjogren’s syndrome but ocular dryness, conjunctivitis, and lacrimal
and salivary gland inflammation varied in different
murine models of lupus. MRL/I and MRL/n mice had
pronounced, destructive mononuclear infiltrates in
glandular tissue which resembled the lesions of Sjogren’s syndrome.
MATERIALS AND METHODS
Animals. MRLA mice, which are homozygous for the
recessive Ipr (lymphoproliferative) gene, and a closely related line (MRL/n) without Ipr genes were obtained from
Jackson Laboratories and colonies were established at the
University of Missouri-Columbia in 1977. Animals from
these colonies were donated to the study by Deborah
Wilson, MD. In 1978, the IprApr genome was transferred to
MRL/n to produce substrain MRL/Mp-lpr/lpr and MRL/n
was renamed MRL/Mp-+I+ (9). MRLIMplprllpr and
C57BL/6J-lpr/lpr mice were provided through the courtesy
of E. D. Murphy, PhD and J. B. Roths of Jackson Laboratories and housed in the Research Service of the Harry S.
Truman Memorial Veterans Hospital. New Zealand mice
descended from breeding pairs obtained in 1969 (NZB) and
1975 (NZW) from the University of Otago in Dunedin, New
Zealand. PN mice were offspring of animals donated in 1974
by Dr. Richard D. Wigley of Palmerston North, New Zealand. C57BL/6J and DBA/2 retired breeders were purchased
from Jackson Laboratories. All mice were housed in plastic
cages on hardwood bedding and fed Purina 5001 chow.
Schirmer tests. Twenty to 37 male and female MRL/I
(this group contained 4 MRL/Mplpr/lpr mice), MRL/n.
NZB, NZBNZW, PN, C57BL/6J, and DBN2 mice aged 11
to 60 weeks were examined. Mice were anesthetized lightly
with methoxyflurane, and a 0.5 x 3.0 mm strip of Whatman
# 1 filter paper was placed under the lower lid of each eye
near the medial canthus. After 2 minutes the strips were
removed, soaked areas were marked, and the length of
wetting was measured at l o x magnification using a micrometer on a dissecting microscope.
Histologic studies. Necropsies were usually performed within 8 weeks after the Schirmer tests; in several
instances, NZB and NZB/NZW males were autopsied 14 to
17 weeks after testing. Groups of 20 to 38 autoimmune and
control mice of both sexes were bled from the right orbital
vascular plexus and killed by cervical dislocation. A separate group of 20 C57BL/6J-lpr/lpr mice aged 38 weeks were
autopsied, and lacrimal and salivary glands were evaluated
for inflammation.
Autoimmune mice were killed at ages when they
were expected to have active disease. Mean ages at death
were: MRLA males 28 weeks (range 16-52) (this group
contained 2 MRL/Mp-IprApr males); MRL/I females 25
(range 16-30) (this group contained 2 MRL/Mplpr/lpr females); MRL/n males 47 (range 33-60); MRL/n females 43
(range 33-60); NZB males 47 (range 37-61); NZB females 54
(range 32-67); NZB/NZW males 33 (range 30-35);
NZBNZW females 30 (range 20-36); PN males 35 (range 2750); PN females 37 (range 26-47). Control mice were killed
when their ages were similar to autoimmune animals:
C57BL/6J males at 35 weeks; C57BL16J females at 37 (range
35-40); DBA/2 males at 27 (range 30-36); DBA/2 females at
30.
Serum was stored in plugged capillary tubes at
-20°C. The left eye was dissected carefully from the orbit,
fixed in one piece in 10% buffered formalin, and embedded in
paraffin. Twenty to 100 serial lop sections were stained with
hematoxylin and eosin. These tissue sections were in part
the basis of an earlier study of band keratopathy and
posterior uveitis in autoimmune and normal mice (13). The
left lacrimal gland was removed separately and the parotid,
submandibular, and sublingual salivary glands were taken en
bloc.
Complete autopsies were performed and samples of
superficial cervical lymph nodes, lung, heart, liver, pancreas, spleen, and kidney were preserved in formalin. A section
through each of these tissues was examined for infiltrates of
mononuclear cells.
In 139 animals, serial sections of ocular tissue contained samples of conjunctiva which were adequate for
evaluation of conjunctivitis. Conjunctival inflammation was
graded on a scale of 0-3: 0 = no inflammation; 1 = minimal
inflammation; 2 = intermediate inflammation; 3 = diffuse
infiltration of tissue with inflammatory cells.
Cellular infiltrations in cross sections through lacrimal glands from 183 mice and salivary glands from 178 mice
were graded on a scale of 0-4 based on a modification of the
system of Chisholm and Mason (14). In this system, 0 = no
inflammation; 1 = focal infiltration with mononuclear cells;
2 = one-fourth of gland replaced by mononuclear cells; 3 =
one-third of gland replaced by mononuclear cells; 4 = more
than half of gland replaced by mononuclear cells.
To establish the reproducibility of both grading systems, representative slides of conjunctiva, lacrimal glands,
and salivary glands were scored independently by 2 observers who were not aware of the strain being examined. Mean
scores between both observers differed by less than 1 grade.
Electron microscopy. Submandibular salivary glands
159
SJOGREN’S SYNDROME
Table 1. Schirmer tests in autoimmune MRL/I, MRL/n, NZE), NZB/NZW, and PN mice, and in normal control C57BL/6J and DBAR mice
Sex
MRLA
MRLh
NZB
NZBINZW
PN
C57BL/6J
DBA/2
M
F
M
F
M
F
M
F
M
F
M
F
M
F
Number
of mice
examined
Filter paper wetting
(mm)+
2.8 C 0.2 (1.0-4.7)
3.1 f 0.3 (1.1-6.8)
3.9 2 0.2 (2.0-5.5)$
2.7 f 0.3 (1.0-5.4)
3.8 2 0.3 (1.7-6.5)
3.7 5 0.3 (1.2-6.3)
2.2 2 0.2 (1.2-4.1)§
1.8 C 0.1 (1.1-3.1)§
2.2 5 0.2 (1.1-4.9)§
2.0 t 0.2 (0.6-3.8)s
2.8 5 0.2 (1.8-4.4)
3.3 -t 0.2 (2.0-4.6)
3.1 5 0.2 (2.2-4.6)
3.3 It 0.2 (2.1-5.6)
Age (weeks)*
30 (29-30)
28 (21-31)
36 (35-36)
33 (31-37)
44 (36-.58)
48 (28-66)
25 ( 16-34)
26 ( I 1-34)
32 (28-49)
34 (25-47)
34 (34-35)
34 (34)
30 (29-30)
30 (30)
11
12
10
11
14
23
12
12
12
12
10
10
10
10
* Mean (range).
t Mean f SEM (range).
$ Mean wetting in MRLln males was significantly greater compared with mean wetting in females ( P < 0.025).
8 In NZBINZW males, NZBlNZW females, PN males, and PN females mean Schirmer test results were significantly smaller compared with
control C57BL/6J and D B N 2 mice of the same sex. In each instance. P < 0.001.
from 2 adult MRLh mice were cut into I-mm cubes in cold
(4°C) 3% Millonigs phosphate buffered glutaraldehyde. After
fixation for 3 hours at 4”C, the tissue was washed twice in
cold Millonigs buffer and post-fixed for 1 hour in 1.5% cold
Millonigs buffered osmic acid. The tissue was dehydrated in
graded dilutions of ethanol, placed in 2 changes of propythene oxide, and embedded in Epon 812. The blocks were
heated at 60°C for 16 hours, and thin sections stained with
uranyl acetate and lead citrate were examined in a Philips
EM 300 transmission electron microscope (15,16).
Immunodiffusion tests. Terminal sera from 10 male
and 10 female MRL/I, MRL/n, NZB, NZB/NZW, and PN
mice were tested for precipitating antibodies to SS-A (Ro)
and SS-B (La) by a modification of the Ouchterlony double
diffusion method (17). The antigen for SS-A was extracted
from human spleen and the antigen for SS-B was prepared
from rabbit thymus (18). Prototype human sera known to
possess SS-A or SS-B antibodies were used as reference sera
at dilutions that gave optimal precipitin lines with antigen.
Unpooled samples of serum from individual mice were
placed in wells adjacent to antigen and to reference sera and
examined for prccipitin lines of identity or nonidentity with
the reference sera. Reactions were allowed to proceed at
room temperature and plates were observed for precipitin
lines at 24, 48, and 72 hours.
Statistics. Student’s I-test was calculated using the
method described by Snedecor and Cochran (19).
RESULTS
Schirmer tests. Table 1 lists mean values for
Schirmer tests in autoimmune and control strains. In
the MRL/n substrain, tear formation in males was
significantly greater compared with females. In the
other groups of mice, gender did not influence tear
formation. Furthermore, severity of autoimmune dis-
ease did not correlate with results of Schirmer tests.
MRL/l mice, which develop early and severe immune
complex disease, resembled the MRL/n substrain
which has mild late-onset disease. Ocular wetting was
diminished significantly in NZB/NZW and PN mice
compared with control C57BL/6J and DBA/2 mice of
the same sex.
Conjunctivitis. Inflammation of the conjunctiva,
a clinical and histologic manifestation of Sjogren’s
syndrome in humans (20), was found in autoimmune
mice (Figure 1). Conjunctivitis was most common in
w
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MRL/I
MRL/n
NZB
NZB/NZW
PN
Control
Figure 1. Conjunctivitis, a clinical and histologic manifestation of
Sjogren’s syndrome, was graded on a scale of 0 to 3+. Inflammation
of the conjunctiva was most common in MRLll mice; the most
severe involvement was observed in PN mice. 0 = male; 0 =
female. In the control column, solid symbols represent DBAl2 mice
and open symbols represent CS7BL/6J mice.
160
HOFFMAN ET AL
OJ
------MRL/I
MRL/n
NZB
NZB/NZW
PN
C571pr
Control
Figure 3. Cellular infiltration in lacrimal glands was graded 0 to 4in a classification adapted from Chisholrn and Mason (14). All
autoimmune strains had lacrimal gland inflammation. The most
extensive lesions were found in MRLA, MRL/n, and NZB mice. See
Figure 1 for explanations.
Figure 2. Severe inflammatory changes in a section through the
superior palpebral conjunctiva of a female MRL/n mouse aged 35
weeks. Conjunctival tissue contains diffuse infiltrates of mononuclear and polymorphonuclear cells. Overlying epithelium (arrow) is
hyperplastic. On the left, the sebaceous glands are infiltrated with
inflammatory cells (hematoxylin and eosin, original magnification
x
Lacrimal glands. Figure 3 illustrates grades of
severity of inflammatory changes in lacrimal glands
from autoimmune and control mice. The most extensive involvement was found in the MRL/l and MRLin
substrains in which 100% and 95% of mice, respective-
loo).
the MRLA substrain, in which 85% of mice were
affected. Conjunctival inflammation was generally
characterized by scattered foci of mononuclear cells
infiltrating the bulbar and palpebral conjunctiva. Occasional neutrophils were present with scattered cellular
debris. MRL/I, MRL/n, NZB, and NZB/NZW mice
commonly had extensive mononuclear cell infiltrates
in the palpebral conjunctiva surrounding the tarsal
plate. Typical conjunctivitis in a female MRWn mouse
is illustrated in Figure 2.
The most severe conjunctival involvement was
observed in PN mice; 40% of these animals had grade
3 conjunctivitis. In the P N strain, infiltrates had a
vasculitic component and mononuclear cells clustered
around blood vessels and extended into surrounding
tissue. Several PN mice with grade 3 conjunctivitis
had extensive vessel wall destruction and lysis of
infiltrating cells. The inflammatory process extended
into adjacent extraocular muscles, where fiber necrosis and degeneration were observed.
N o inflammation was found in the conjunctiva
in normal control C57BL/6J and D B N 2 mice.
Figure 4. Characteristic destructive changes in a lacrimal gland
from a 16-week-old MRL/I male. Mononuclear cells infiltrate the
adjacent parenchyma, resulting in focal destruction of secretory
alveoli (hematoxylin and eosin, original magnification x 250).
SJOGREN’S SYNDROME
161
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Control
Figure 5. Inflammation in submandibular salivary glands was graded 0 to 4 + . Sialadenitis was most severe in NZB/NZW mice. Foci of
inflammation were present in 38% of control mice. See Figure 1 for
explanations.
ly, had lacrimal gland infiltrates. Lacrimal glands from
the MRL substrains typically contained large multifocal confluent infiltrates of lymphocytes, with occasional histiocytcs and plasma cells. These lesions were
characterized by perivascular and periductal aggregates of cells that infiltrated the adjacent parenchyma
and resulted in focal destruction of secretory alveoli.
Fibrosis was present in areas diffusely infiltrated with
inflammatory cells. A lesion in an MRL/I male is
illustrated in Figure 4.
Lacrimal gland infiltrates were found in 85% of
NZB, 57% of NZB/NZW, and 75% of PN mice.
Inflammation in these animals was less severe compared with MRL mice. In congenic C57BL/6J-lpr/lpr
mice, 28% of animals had grade 1 and 2 infiltrates. No
control mice had a histologic score greater than grade
1, and most controls had no evidence of inflammation.
Submandibular glands Grading of inflammation
in the submandibular salivary gland is shown in Figure
5. Eighty-six percent of MRL/l mice and 94% of
MRL/n mice had grade I and 2 infiltrates. Sialadenitis
in the submandibular glands resembled lacrimal gland
involvement, with collections of mononuclear cells
around vessels and ducts. Lymphocytes and plasma
cells extended into and destroyed adjacent glandular
tissue. Figure 6 illustrates a characteristic lesion resulting in destruction of a duct and acinar tissue.
Moderately severe involvement was found in 2
NZB/NZW mice, which had grade 3 lesions; the
incidence of submandibular gland inflammation in this
group was 40%. Minimal inflammation was found in
submandibular glands from NZB and PN mice, and
C57BL/6J-lpr/lpr mice had normal submandibular
glands. Focal grade 1 infiltrates were found in 38% of
C57BL/6J and DBAR mice.
Parotid glands. Inflammation of grade 1 and 2
severity involved 23% of MRL/I and 26% of MRL/n
parotid glands. Eighteen percent of NZB mice, 12% of
NZB/NZW mice, and 10% of PN mice had grade 1
infiltrates. Mononuclear cell infiltrates were not found
in parotid glands from C57BL/6J-lpr/lpr. C57BL/6J,
and DBAR mice.
Sublingual glands. Focal grade 1 sublingual
gland lesions occurred in 4-19% of autoimmune
MRL/l, MRL/n, NZB, NZB/NZW, and PN mice;
C57BL/6J-lpr/lpr mice had no inflammatory changes.
Unexpectedly, 4 female control C57BL/6J mice had
infiltrates in sublingual glands. Two glands were classified as grade 1 , and 2 glands were grade 2. The
overall incidence of sublingual inflammation in controls was 13%.
Examination of other tissues. As expected from
our experience and the work of others (8-12), glomerulonephritis and vasculitis were common in renal
Figure 6 . Submandibular gland infiltrated with mononuclear cells,
35-week-old MRL/I female. A duct and acini are invaded by
inflammatory cells (hematoxylin and eosin, original magnification
x 250)
162
tissue from autoimmune mice. Variable numbers of
lymphocytes and plasma cells surrounded the renal
pelvis and renal arteries in the autoimmune strains; in
some instances focal collections of mononuclear cells
were scattered throughout the renal parenchyma. Hyperplasia of lymphoid tissue was observed frequently
in the lupus strains. Several animals had lymphomas
and in 2 instances, metastases to salivary glands were
identified. These lesions were clearly distinct from the
cellular infiltrates that resembled Sjogren’s syndrome.
Lymphocytic infiltrates were found commonly around
bronchioles and pulmonary arteries in the autoimmune
strains. These lesions were most severe in the MKL/l
substrain. Pulmonary and renal infiltrates were not
present in the normal strains. The heart, liver, and
pancreas were not infiltrated with lymphocytes in
autoimmune or control mice.
Electron microscopy. In 2 MRL/n females, cellular infiltrates in lacrimal glands were composed of
small and medium lymphocytes. These cells were
observed infiltrating acini and ductal epithelium (Figure 7).
Immunodiffusion tests. Antibodies to SS-A and
SS-B were not detected by immunodiffusion in sera
from MRL/I, MRL/n, NZB, NZB/NZW, and PN mice.
Figure 7. Electron micrograph of lacrimal gland tissue from a
female MRLh mouse. Architecture of the gland has been destroyed
by infiltration of lymphocytes. Representative lymphocytes are
labeled (L). Prominent endoplasmic reticulum, prominent Golgi
apparatus, and cytoplasmic extrusion identify these cells as activated lymphocytes. Two fibroblasts (F) appear to be laying down
bundles of collagen (arrowheads) (transmission electron microscopy, original magnification x 3,300).
HOFFMAN ET AL
DISCUSSION
The first models of spontaneous Sjogren’s syndrome were described by Kessler (7,21), who reported
corneal changes in NZB/NZW mice and periductal and
periarteriolar infiltrates in salivary and lacrimal glands
from NZB and NZB/NZW mice. Keyes and associates
(22) confirmed the finding of histopathologic abnormalities in NZB/NZW hybrids and reported epimyoepithelial-like structures in glandular tissue. Examination of parotid and submandibular salivary glands of
NZB/NZW mice by electron microscopy showed that
the inflammatory lesions consisted of lymphocytes
surrounding vessels and extending into adjacent glandular parenchyma. Ductal cell proliferation and epimyoepithelial island formation were not found (23,24).
In the current study, we compared 6 murine
models of lupus with 2 normal strains of mice that do
not develop autoimmune disease. Modified Schirmer
tests, histologic evaluation of conjuctivae and lacrimal
and salivary glands, and serologic assays for antiSS-A and anti-SS-B antibodies were utilized to determine if lupus-like disease was invariably associated
with manifestations of Sjogren’s syndrome.
We are aware of only one earlier study which
examined tear production in animal models of autoimmune disease. Kessler (21) performed Schirmer tests
in 6 NZB/NZW mice. Mean wetting was 3.1 mm,
compared with 4.8 mm in NZW mice used as controls.
Our studies demonstrated significant ocular dryness in
NZB/NZW and PN mice, which had moderately severe lesions in the lacrimal glands. In contrast, MRL/l
and MRL/n mice did not have functionally dry eyes,
although both substrains had extensive inflammation
and destruction of lacrimal glands. Ocular wetting
appeared to correlate with body weight in the autoimmune and normal strains of mice tested, and normal
tear production in the large MRL animals may have
overestimated lacrimal gland function. Nevertheless,
the unexpected discrepancy between results of
Schirmer tests and lacrimal gland pathology may simply reflect the inadequacy of this test as a sensitive
measure of ocular wetting in small animals.
In the current study we found varying degrees
of inflammation of the conjunctiva in autoimmune
strains. Conjunctivitis was most severe in PN mice,
which exhibited focal and diffuse inflammation with
perivascular infiltrates of lymphocytes and polymorphonuclear leukocytes. The other autoimmune strains
had less severe involvement of conjunctivae, and
conjunctivitis was not found in C57BL/6J and DBA/2
controls. These findings suggested that individual mu-
SJOGREN’S SYNDROME
rine models of lupus exhibited selective manifestations
of ocular pathology and did not have diffuse inflammatory eye disease.
A unique feature of this study was the use of a
grading system to compare severity of inflammation in
glands from a series of autoimmune models. In autoimmune mice, the most severe inflammatory changes
were found in MRL/I and M R L h lacrimal glands. In
25% of MRL mice, at least one-third of the area in
lacrimal gland cross sections was replaced by mononuclear cell infiltrates. The most severely involved salivary glands were submaxillary glands. Parotid glands
were involved to a mild degree, and sublingual salivary
glands resembled controls. Although infiltrates of
grade 2+ severity were observed in lacrimal glands
from 3 of 18 C57BL/6J-lpr/lpr mice, salivary glands
from this strain were normal. It was concluded that the
lpr/lpr genome, which accelerates autoimmunity in
mice with the MRL genetic background, was not
linked with severe or consistent inflammatory lesions
in lacrimal and salivary glands.
Although severity of inflammation differed in
various strains of autoimmune mice, the pattern of
mononuclear cell infiltration was the same. Characteristically, lymphocytes and plasma cells surrounded
arterioles and ducts, and cells invaded and destroyed
acinar tissue. The distribution of inflammatory cells in
these animals resembled the abnormalities in lacrimal
and salivary glands of NZB and NZB/NZW mice
which were described by other investigators (7,23,24).
MRL/l mice have severe, early-onset immune
complex disease which possesses many characteristics
of systemic lupus erythematosus (9) and rheumatoid
arthritis (10) A 60% incidence of periductal mononuclear cell infiltrates in salivary glands from 5- to 6month old MRL/I females has been reported by Hang
et al(10). Because sialadenitis was not found preferentially in mice affected with destructive rheumatoid-like
arthritis, the gland infiltrates were not considered to be
related to a specific autoimmune process.
Several features of our report offer new perspectives in interpreting lacrimal and salivary inflammation in the MRLA model of lupus. By using a
detailed grading system to classify severity of inflammation and studying large numbers of mature mice of
both sexes, we were able to reliably demonstrate
extensive infiltrates resembling Sjogren’s syndrome in
a large percentage of MRL/I and MRL/n mice. Smaller
infiltrates were observed in the other autoimmune
strains and in a small number of control mice. Mononuclear infiltration followed a strict order of involve-
163
ment in glandular tissue. In almost every instance
infiltrates were most extensive in lacrimal glands, and
decreasing grades of severity of inflammation were
assigned to submandibular, parotid, and sublingual
salivary glands, respectively. Furthermore, severity of
lesions in these glands did not correlate with the extent
of pulmonary or renal infiltration in individual animals.
These patterns of involvement support our conclusion
that the mononuclear infiltrates reflect a specific,
destructive inflammatory process in lacrimal and salivary glands.
Examination at the ultrastructural level confirmed the invasive nature of lymphocytic infiltrates in
lacrimal glands from 2 MRL/n mice. Electron microscopy was also used to examine lacrimal gland tissue
for structures resembling viruses. Type C viruses have
been implicated in the pathogenesis of murine lupus
(25), and infections with Epstein-Barr virus (26) and
cytomegalovirus (27) have been associated with rheumatoid arthritis and Sjogren’s syndrome. Viral particles were not found in the limited number of animals
we examined. We also did not find tubuloreticular
structures, which have been observed in humans with
Sjogren’s syndrome (28). Nevertheless, the absence of
viral structures did not exclude the possibility that
viral infection contributed to inflammatory lesions in
lacrimal and salivary glands of autoimmune mice.
Although the precise roles for antibodies to SSA and SS-B have not been defined, they are important
markers for Sjogren’s syndrome and systemic lupus
erythematosus (2). In some instances, these antibody
systems have been implicated as participants in immune complex-mediated tissue injury (2,29-3 I). Although we could not detect anti-SS-A or anti-SS-B
using the relatively insensitive Ouchterlony technique,
we believe that these antibodics should be sought in
autoimmune mice by using more sensitive techniques.
We are currently developing an enzyme-linked immunosorbent assay for antibodies to SS-A and SS-B.
This sensitive method, which has been utilized to
detect anti-SS-B antibodies in human serum (32), will
allow sera from autoimmune mice to be reexamined
for serologic markers of Sjogren’s syndrome.
In humans with Sjogren’s syndrome, infiltrates
in labial salivary glands consisted primarily of OKT4positive (T helper-inducer) lymphocytes (33). This
finding was of interest in the context of the present
study because MRL/I mice have excessive lymph node
infiltration with theta antigen-positive cells which do
not carry surface antigens Ly-I, Ly-2, or Ly-3. It has
been postulated that excessive activity of these murine
164
HOFFMAN ET AL
T helper cells contributes to disease in autoimmune
mice b y stimulating B cells (34,35).Additional studies
of lymphocyte surface markers on infiltrating cells in
lacrimal and salivary glands from murine models of
lupus will provide insight into the pathogenesis of
Sjogren’s syndrome. Identification of subsets of lymphocytes in autoimmune mice may facilitate identification of a murine model that closely resembles Sjogren’s syndrome in humans.
ACKNOWLEDGMENTS
Harry Kessler, DDS, provided encouragement and
helpful advice. The authors acknowledge the expert technical assistance of Julia Burge and Fortune Campbell. Electron microscopy was performed with the assistance of
Wayland N. McKenzie, PhD. The Medical Media Service of
the Harry S. Truman Memorial Veterans Hospital prepared
the figures used in this manuscript.
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