Fas Ligand-Mediated Exocrinopathy
Resembling Sjögren's Syndrome in Mice
Transgenic for IL-10
This information is current as
of June 14, 2017.
Ichiro Saito, Kumiko Haruta, Misa Shimuta, Hiroko Inoue,
Hiroshi Sakurai, Koichi Yamada, Naozumi Ishimaru,
Hiroyuki Higashiyama, Takayuki Sumida, Hiroshi Ishida,
Takashi Suda, Tetsuo Noda, Yoshio Hayashi and Kazuo
Tsubota
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 1999; 162:2488-2494; ;
http://www.jimmunol.org/content/162/5/2488
Fas Ligand-Mediated Exocrinopathy Resembling Sjögren’s
Syndrome in Mice Transgenic for IL-101
Ichiro Saito,2* Kumiko Haruta,† Misa Shimuta,† Hiroko Inoue,† Hiroshi Sakurai,‡
Koichi Yamada,* Naozumi Ishimaru,* Hiroyuki Higashiyama,* Takayuki Sumida,§
Hiroshi Ishida,¶ Takashi Suda,i Tetsuo Noda,# Yoshio Hayashi,* and Kazuo Tsubota†
A
lthough little is known about the contribution of IL-10 to
the inflammatory response in vivo, several studies have
implicated IL-10 in processes that may contribute to inflammation and pathogenesis in general and, in particular, in autoimmune diseases (1– 4). IL-10 exhibits a strong DNA and amino
acid sequence homology to an open reading frame of the EBV
genome called BCRF1 or viral IL-10 (5). Viral IL-10 produced by
EBV-infected cells is expressed during the late stage of the virus
cycle and exhibits most of the activities of human and mouse IL-10
(6). IL-10 was recently shown to enhance MHC class II Ag expression (7) and to induce proliferation and differentiation of B
cells (8). In addition, IL-10 induces expression of cell adhesion
molecules on endothelial cells (9, 10) and apoptotic cell death (11).
Sjögren’s syndrome (SS)3 is an organ-specific autoimmune disease caused by the progressive loss of exocrine glands and is associated with several autoimmune phenomena (12). Although particular alleles closely linked to the MHC class II locus increase the
risk of developing SS (13), studies of identical twins have implicated environmental factors in the initiation of this disease (14).
Several reports have suggested that viral infection could be the
*Department of Pathology, Tokushima University School of Dentistry, Kuramotocho,
Tokushima, Japan; †Department of Ophthalmology, Tokyo Dental College, Ichikawa,
Chiba, Japan; ‡Takasago Research Laboratories, Research Institute, Kaneka Co,
Takasago, Hyogo, Japan; §Department of Internal Medicine, Institute of Clinical
Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan; ¶Clinical Research Center, National Utano Hospital, Kyoto, Japan; iDepartment of Molecular Biology, Osaka
Bioscience Institute, Osaka, Japan; and #Department of Cell Biology, Cancer Institute, Toshima-ku, Tokyo, Japan
Received for publication August 17, 1998. Accepted for publication October
29, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Science and Culture of Japan.
2
Address correspondence and reprint requests to Dr. Ichiro Saito, Department of
Pathology, Tokushima University School of Dentistry, 3-18-15, Kuramotocho, Tokushima 770, Japan. E-mail address: [email protected]
3
Abbreviations used in this paper: SS, Sjögren’s syndrome; FasL, Fas ligand; TG,
transgenic mice; IL-10TG, IL-10 transgenic mice; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; MSG, mouse salivary gland; SSCP, single-strand conformation polymorphism.
Copyright © 1999 by The American Association of Immunologists
environmental causative agent of SS (15, 16), and evidence for an
association between EBV infection and SS has been accumulating
(17–20). These previous reports suggest that a reactivated EBV
infection may play a role in SS, contributing to the initiation or
perpetuation of an immune response in the target organs. However,
the pathologic role of the virus remains obscure.
Since IL-10 may environmentally stimulated inflammatory responses, we have investigated whether IL-10 could initiate and
maintain a response sufficient to destroy the exocrine glands. We
tested this possibility by developing a transgenic mouse model in
which the glandular epithelial cells express active IL-10. Furthermore, the experiments described in this report were undertaken to
determine whether IL-10 is sufficient for Fas/Fas-ligand (FasL)mediated tissue destruction in the glands.
Materials and Methods
Generation of transgenic mice (TG)
C57BL/6 mice were used to obtain fertilized eggs, and an EcoRI-BstI fragment containing a mouse IL-10 cDNA (provided by American Type Culture Collection, Manassas, VA) was microinjected into the pronucleus of
fertilized eggs using the standard method. When the mice were 4 wk of
age, DNA was extracted from a piece of the tail of each mouse and used
for Southern blot analysis. The EcoRI-BstI fragment, a mouse IL-10
cDNA, and mouse b-actin cDNA were used as probes. The transgene expression was detected by Northern blot analysis and organ culture. Total
RNA was isolated from all organs, including exocrine glands, by guanidine
isothiocyanate extraction. Twenty micrograms per lane of RNA was separated on 1.2% agarose gel and transferred to nylon membranes. Hybridization was conducted using a 32P-labeled cDNA probe for mouse IL-10.
The glands for the organ culture were isolated from five IL-10 TG (IL10TG) and five controls. One gland from each mouse was cultured in a
single well of a 98-well plate in RPMI 1640 supplemented with 20 mM
HEPES, 300 mg/ml L-glutamine, 100 U/ml penicillin, 100 mg streptomycin, and 10% FCS. IL-10 production was measured at 72 h of culture. The
IL-10 level in the culture supernatant was assayed using an ELISA kit
(PharMingen, San Diego, CA).
Histologic analysis
Sections were stained with hematoxylin and eosin using the standard
method. Histologic grading of the inflammatory lesions in the salivary and
lacrimal glands was done according to the method proposed by White and
Casarett (21). Freshly frozen sections were stained by the avidin-biotin
immunoperoxidase complex method with commercially available mAbs.
0022-1767/99/$02.00
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Although IL-10 has been implicated in the pathogenesis of several autoimmune diseases, the mechanisms by which this cytokine
mediates inflammatory lesions remain to be elucidated. Exocrine gland destruction is an important early step in the development
of Sjögren’s syndrome. To better understand the role of IL-10 in Sjögren’s syndrome, we made transgenic mice in which the mouse
IL-10 gene was regulated by the human salivary amylase promoter. Transgenic expression of IL-10 induced apoptosis of glandular
tissue destruction and lymphocyte infiltration consisting primarily of Fas-ligand (FasL)1 CD41 T cells, as well as in vitro upregulation of FasL expression on T cells. These data suggest that overexpression of IL-10 in the glands and their subsequent
Fas/FasL-mediated bystander tissue destruction is a causal factor in the development of this disease. The Journal of Immunology,
1999, 162: 2488 –2494.
The Journal of Immunology
2489
Briefly, frozen sections were fixed in acetone for 10 min, rinsed in PBS,
and incubated with an appropriate blocking agent (Vector Laboratories,
Burlingame, CA) for 20 min. They were then incubated for 1 h with following Abs: biotinylated rat mAbs to Thy-1.2, CD3, L3T4 (CD4), Ly-2
(CD8), and Mac-1 (Becton Dickinson, Sunnyvale, CA) and MHC class II
I-Ak (Becton Dickinson). They were then incubated with anti-rat IgM and
IgG (Vector Laboratories) as the second Ab for 30 min, washed with cold
PBS for 30 min, and incubated with the avidin-biotin immunoperoxidase
complex reagent (Vector Laboratories) for 30 min. After washing with
PBS, the sections were treated with a freshly prepared solution of 0.05%
3,39-diaminobenzidine and 0.005% H2O2 in Tris-HCl buffer (0.05 M, pH
7.6) for 5 min, washed with distilled water, and counterstained with methyl
green. All controls treated with normal rat serum (Cappel Laboratories,
Cochranville, PA) or PBS instead of the first Abs gave negative results.
Measurement of fluid secretion
Detection of tear and saliva volume of IL-10TG was done according to a
modified method as described (22, 23). Five mice in each group were
analyzed at 8 and 20 wk of age.
Detection of Ig levels and autoantibodies
In situ terminal deoxynucleotidyl transferase-mediated nick end
labeling (TUNEL) staining
Tissues were fixed in formalin and processed for 24 h for paraffin sectioning. Sections were mounted onto microscope slides and incubated overnight at 55°C. Sections were then deparaffinized for 5 min in xylene, 5 min
in ethanol, 3 min in 95% ethanol, 3 min in 70% ethanol, and 5 min in PBS.
Following four washes in distilled water, endogenous peroxidase was
quenched with 2% H2O2 for 5 min at room temperature, and sections were
washed two times in PBS. Labeling of 39-OH fragmented DNA ends was
performed with an in situ apoptosis detection kit (Apoptag, Oncor, Gaithersburg, MD) following the instructions of the manufacturer. Detection of
labeled ends was done with anti-digoxigenin-peroxidase Ab and 3,39diaminobenzidine substrate kit (Vector Laboratories).
Cell preparation and flow cytometric analysis
To obtain tissue-infiltrating mononuclear cells in the gland, the inflamed
glands from mice were removed, cut into small pieces with scissors
through 100-gauge stainless steel mesh, and suspended in RPMI 1640 containing 10% FCS, 10 mM HEPES buffer, penicillin (100 U/ml), and streptomycin (100 mg/ml). After washing twice with the medium, infiltrating
mononuclear cells were isolated from parenchyma cells by Ficoll-Isopaque
density (1090) gradient centrifugation. Surface markers were identified by
mAb in conjunction with the two-color immunofluorescence analysis conventionally. Double-labeled surface phenotypes such as FasL/CD4 were
analyzed. Spleen cell suspensions were stained using anti-CD4, B220,
CD8, Thy1.2, CD44, CD45RB, and Mel-14 Abs and analyzed. Cells were
gated according to size and scatter to eliminate cells and debris from
analysis.
Effects of IL-10 to FasL expression in vitro
To examine the effect of IL-10 to FasL expression on lymphocytes, spleen
cells were cultured with or without each IL-10 in three separate experiments. Recombinant mouse purified IL-10 (PharMingen) was added at various concentrations in culture medium to yield the required concentration.
Surface Ags of FasL/CD4 on stimulated spleen cells were detected with
flow cytometric analysis.
Primary mouse salivary gland (MSG) cells
Primary MSG cell cultures were prepared from B6 mouse at 3–5 wk by
enzymatic digestion with 0.76 mg/ml EDTA and a mixture of collagenase
(type I, 750 U/ml) and hyaluronidase (type IV, 500 U/ml), plated in 24-well
plates (250,000 cells per well), and maintained in DMEM containing 10%
calf serum for 10 –14 days before FACS analysis and cytotoxic assay.
These primary cultures contained a mixture of epithelial cells (85–95%)
and fibroblasts (1–5%).
FIGURE 1. Transgene construct and expression. A, To direct IL-10 expression to the exocrine glands that secrete amylase, we linked the mouse
IL-10 cDNA to the human salivary amylase promoter. The plasmid containing the 828-bp BamHI-BamHI human salivary amylase promoter
(AMY1C) fragment was ligated with the 640-bp fragment that contains the
rabbit b-globin splicing site. An EcoRI-BstI fragment containing a mouse
IL-10 cDNA was located directly 39 of the b-globin splicing site. B, IL-10
expression in the lacrimal gland (lanes 1) was detected by Northern blot
analysis, parotid gland (lanes 2), and submandibular gland (lanes 3), but
not in the brain (lanes 4), heart (lanes 5), lung (lanes 6), liver (lanes 7),
kidney (lanes 8), pancreas (lanes 9), and spleen (lanes 10). C, Detection of
IL-10 production from transgenic exocrine glands by organ culture. Organcultured glands from IL-10TG indicated a marked increase in IL-10 levels.
Cytotoxicity assay
MSG cells (2 3 106) in 7.5 ml of RPMI 1640 supplemented with 5% FBS
were labeled overnight at 37°C in 5% CO2 with 300 mCi of sodium
[51Cr]chromate. CD41 and CD81 T cells purified from splenocytes using
magnetic beads (2–3 3 106; Dynal, Great Neck, NY) in 0.2 ml of RPMI
1640 supplemented with 10% FBS were incubated with Con A (EY Laboratories, San Matei, CA) and recombinant human IL-2 (Genzyme). Each
well of 96-well microtiter plates received, in a total volume of 200 ml,
target cells, effector cells in the indicated ratios, and either medium. Microplates were centrifuged for 1 min at 1500 rpm and incubated for 4 h at
37°C. After another centrifugation, 100-ml aliquots of the supernatants
were assayed for radioactivity. The fraction of the total radioactivity released was then calculated, and the results, averaged from triplicates, were
expressed as percentage specific 51Cr release (% experimental 51Cr release 2 % 51Cr release from target cells alone). Anti-mouse FasL neutralizing mAb (FLIM58) was established from an Armenian hamster immunized with the WR19L mouse lymphoma expressing recombinant mouse
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Serum Ig levels were assessed by ELISA by using isotype-specific Abs.
Determination of serum autoantibody activity was assessed by ELISA.
Briefly, Ag was added to microtiter wells at a concentration of 1 mg/ml of
PBS for 16 h. Nonspecific sites were absorbed with 1% BSA in PBS.
Serum samples (1:250) were added to the wells for another 16-h incubation. Ag studies included bovine thymic and splenic Sm, SSA/Ro, and
SSB/La (Immunovision, Springdale, AR) and mouse IgG Fc fragment
(Rockland, Gilbertville, PA). For anti-dsDNA and anti-ssDNA Abs,
ELISA microtiter plates were purchased from Immunovision.
2490
IL-10 TRANSGENIC MICE FOR SS
FasL (detail will be described elsewhere by T. Suda). FLIM58 neutralizes
mouse but not human FasL activity.
blotted onto a nylon membrane and hybridized with biotinylated labeled
Cb probe (TTG ATG GCT CAA ACA AGG AGA CC).
Treatment with neutralizing Ab to IL-10
Analysis of single-strand conformation polymorphisms (SSCP)
A study to determine the preventive effect of treatment with neutralizing
anti-IL-10 Ab in vivo was performed, as described (24). Rat neutralizing
mAb to mouse IL-10 (JES 2A5, IgG1) and isotype mouse control IgG1
mAb (PharMingen, San Diego, CA) were used the in vivo study. Abs were
injected i.p. with a dose of 0.1 mg twice per wk into IL-10TG (n 5 5) from
4 to 8 wk of age. These mice were sacrificed and examined for histologic
examination. Mice were examined histopathologically at 8 wk and compared with untreated mice (n 5 5). Mean grade of inflammatory lesions
was expressed as described (21). Data represent the mean grade of lesions 6 SD (Mann-Whitney U test).
Amplified DNA was diluted at 1:20 in a denaturing solution (95% formamide, 10 mM EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol)
and held at 90°C for 2 min. The diluted sample (2 ml) was electrophoresed
in nondenaturing 5% polyacrylamide gels containing 10% glycerol. The
gel was run at 35 W constant power for 2 h. After electrophoresis, the DNA
was transferred to Immobilon-S (Millipore Intertech, Bedford, MA) and
hybridized with biotinylated Cb probe and visualized by subsequent incubation with streptavidin, biotinylated alkaline phosphatase, and a chemiluminescent substrate system (Plex Luminescence kit, Millipore Intertech).
PCR analysis for TCR Vb usage
To analyze TCR Vb1-Vb19 gene expression by the RT-PCR method,
RNA was transcribed into cDNA. To perform the PCR assay, the cDNA
reaction mixture was diluted with 90 ml of PCR buffer; we then added 50
pmol of the 59 and 39 primers, 1.25 mM deoxynucleotide triphosphates, 20
mM MgCl2, and 2 units of thermostable Taq polymerase (Perkin-Elmer/
Cetus, Norwalk, CT). To prevent evaporation, 150 ml of mineral oil was
added, and the reaction was started by denaturing the RNA-cDNA hybrid
by heating at 94°C for 30 s, annealing the primers at 55°C for 30 s, and
extending the primers at 72°C for 1 min. Heat denaturation started the cycle
over again, and the cycle was repeated 35 times by a DNA thermal cycler
(Perkin-Elmer Cetus). A 10-ml aliquot of the amplified DNA reaction mixture was fractionated by 1.7% agarose gel electrophoresis, and the amplified product was visualized by UV fluorescence after staining with
ethidium bromide. The sequences of TCR Vb1-Vb19 and Cb primers were
obtained from previously published data (25, 26). PCR products were
Results
Generation of IL-10TG
We constructed several lines of C57BL/6 background TG in which
the expression of mouse IL-10 cDNA (5) was regulated by the
human salivary amylase promoter (27) (Fig. 1A). Of 100 progeny
screened by Southern blot analysis of tail DNA, two (AM01 and
AM03) were positive for the amylase promoter-IL-10 transgene.
These mice were shown to carry about 10 –50 copies of the transgene by Southern blot analysis. Both founders transmitted transgenes to half of their offspring regardless of sex. Although half of
the offspring of mouse AM03 integrated the IL-10 transgene, only
very low levels of IL-10 expression were seen in these exocrine
glands. We therefore mainly analyzed IL-10TG derived from the
AM01 mouse in this study. We examined the expression of IL-10
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FIGURE 2. A, Histologic analysis. Sections were
stained with hematoxylin and eosin. Inflammatory lesions in the salivary and lacrimal glands were characterized by focal mononuclear cell infiltration. B,
Immunohistochemical analysis to identify cell populations revealed that a major proportion of infiltrating
mononuclear cells were CD41 T cells. Five mice in
each group were analyzed at 8 and 20 wk of age.
The Journal of Immunology
2491
Clinical onset
We then sought to determine whether the inflammation of the
glands of IL-10TG was associated with decreased salivary and
lacrimal fluid secretion. At 8 wk, the average saliva and tear volume of IL-10TG was significantly lower than that of non-TG, and
the total amount of fluid decreased gradually with advancing age
(Fig. 3). There were no significant differences with respect to sex.
Serum Ig levels in these TG were examined by ELISA. The levels
of IgG, IgM, and IgA were found to be within the control range at
8 wk. In 20-wk-old mice, the IgG1 levels in sera of TG were
1.8-fold higher than those of age-matched control mice but were
not statistically significant. Circulating autoantibodies were not
detected.
Induction of FasL-mediated tissue destruction by IL-10
by Northern blotting in the various organs of IL-10TG and found
IL-10 expression in the lacrimal and salivary glands (Fig. 1B).
Detection of IL-10 protein in organ-cultured glands indicated a
marked increase in IL-10 levels in the glands in IL-10TG compared with non-TG (Fig. 1C).
Characterization of IL-10TG
Histologic findings and disease indicated that some 8-wk-old mice
had inflammatory infiltration in the glands. Fig. 2A shows the histologic findings in glands with inflammatory infiltration. Most infiltrating lymphocytes were positive for CD4 (Fig. 2B), and a
lesser proportion (,10%) were positive for CD8. The total numbers of lymphocytes in the glands increased gradually with advancing age. In contrast, no remarkable infiltration of mononuclear
cells nor histologic changes were seen in the other organs. AntiIL-10-positive cells were detected in the glandular epithelial cells
but not in the negative littermate controls. These cells appeared to
be located in the periphery of the inflammatory lesions. The local
expression of MHC class II I-Ak was detected in the glands of
IL-10TG (data not shown).
TCR analysis of tissue-infiltrating T cells
To characterize the repertoire of TCR Vb genes of tissue- infiltrating lymphocytes in the glands, we have analyzed TCR Vb gene
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FIGURE 3. Secretion of tears and saliva. The average tear and saliva
volume of IL-10TG was significantly lower than non-TG, and the total
amount of fluid decreased gradually with advancing age. Results are
mean 6 SEM. The differences between IL-10TG and negative littermates
were statistically significant by the Student t test. p, p , 0.01; pp, p , 0.05.
Since the high prevalence of glandular hypofunction in IL-10TG
was accompanied with CD41 T cell infiltration of the glands, we
tested whether IL-10 induced apoptotic tissue destruction via the
Fas/FasL system. We found apoptotic epithelial duct/acinar cells
by TUNEL staining of the glands of IL-10TG compared with control mice (Fig. 4A). Fas was constitutively expressed on epithelial
cells in the glands of both IL-10TG (Fig. 4A) and littermate control
mice (data not shown) as demonstrated by immunohistochemical
analysis of frozen sections. In contrast, immunohistochemical
analysis revealed that FasL expression was completely absent on
salivary gland epithelial cells in IL-10TG and control mice (data
not shown). Flow cytometric analysis of the isolated mononuclear
cells from glands of 8-wk-old IL-10TG showed a large proportion
of CD41 T cells bearing FasL (Fig. 4B), whereas this large proportion of T cells in spleen and lymph node was not found in
IL-10TG (Fig. 4B) and control mice (spleen, 3.5%; lymph node,
5.4%). FasL works as an effector in CTL-mediated cytotoxicity,
and CTL cell lines selectively kill Fas-expressing target cells (28).
Taken together with our data, these results suggest that Fas/FasL
system-mediated apoptosis may be involved in the glandular tissue
destruction in IL-10TG. Therefore, we conducted in vitro experiments to determine whether IL-10 is able to induce FasL expression on CD41 T cells. To investigate whether CD41 T cells express FasL, spleen cells stimulated with rIL-10 were analyzed for
the CD41/FasL1 phenotype by two-color flow cytometric analysis. Dose-dependent FasL expression was detected in CD41 T
cells treated with rIL-10 (Fig. 4C). We next examined whether
IL-10-stimulated T cells acquire cytotoxic activity for glandular
epithelial cells in vitro. The CTL activity of splenic CD41 T cells
from B6 normal mice was investigated using primary cultured
MSG epithelial cells as target cells. CD41 T cells were stimulated
with rIL-10 alone and/or with the presence of Con A1rIL-2 for 6 h
before cytotoxicity assays. As shown in Fig. 4D, cytotoxic activities were enhanced by rIL-10 and Con A1rIL-2. These cytotoxic
activities were almost entirely inhibited by incubation with antimouse neutralizing FasL Ab. This result indicated that the FasL
expressed in these T cells stimulated with IL-10 is functional. We
next investigated whether the i.p. injection of neutralizing antiIL-10 Ab protects animals against the development of lesions. The
treatment with i.p. injection of neutralizing anti-IL-10 Ab prevented the development of tissue destruction by lymphocytic infiltration (Fig. 4E). These findings demonstrate a role for IL-10 as
an inducer of Fas/FasL-mediated apoptosis leading to glandular
tissue destruction.
2492
IL-10 TRANSGENIC MICE FOR SS
expression (Vb1-Vb19) in the isolated cells from salivary and lacrimal gland tissues and spleen cells at each age using RT-PCR. We
found no significant Vb gene-biased expression in the gland tissues at age 8 wk in the onset of inflammatory infiltrates (Fig. 5A).
Multiple Vb gene usage was also detected in mice examined during the late stage up to 20 wk of age (data not shown). No overexpression of unique Vb genes was observed in any of the mice.
To investigate the clonotypes of T cells, we selected the Vb families, Vb5, Vb11, Vb12, and Vb13, which were overexpressed in
the glands from mice, for the combination of RT-PCR and subsequent SSCP clonality analysis, as reported previously (26). A few
dominant bands were detected in the PCR products of the glands
from mice. However, identical bands were commonly observed in
spleen cells (Fig. 5B). These results indicate that the T cells infiltrating the glands were polyclonal. These data are consistent with
nonrestricted clonal T cell expansion in these sites of IL-10TG,
enabling the recognition of unknown multiple Ags. Since Rouvier
et al. have demonstrated that the Ag-nonspecific cytotoxic lymphocytes kills Fas-expressing target cells (29), these findings sug-
gest that tissue destruction in the glands may occur as a result of
bystander killing by nonspecific T cells expressing FasL.
Discussion
Because this IL-10TG mouse model reproduces the IL-10 expression seen in human autoimmune disease and leads to the pathology
that has many similarities to human organ-specific autoimmune
disease, overexpression of IL-10 in glands may induce the development of this disease in humans. In the comparable transgenic SS
model, mice expressing the human T-cell lymphotrophic virus type
I tax gene (30) or hepatitis C virus envelop genes (31), perturbation
of protein synthesis or transport may explain the onset of disease.
By using viral Ags as transgenes, these reports have demonstrated
lymphocyte infiltration into the exocrine glands, but the effector
cell population and the mechanisms that trigger the tissue destruction were not characterized in detail. Studies from other animal
models of SS, such as the MRL/lpr mouse (32) and the NFS/sld
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FIGURE 4. A, Immunohistochemical analysis of in situ TUNEL detection and Fas expression on salivary glands in IL-10TG. A significant increase of
apoptotic epithelial duct/acinar cells was observed in the glands from IL-10TG, compared with control mice. The percentage of apoptotic epithelial
duct/acinar cells staining positively with TUNEL was enumerated using a 10 3 20-grid net micrometer disc covering an objective of area 0.16 mm. Data
were analyzed in 10 fields per section and were expressed as mean percentage 6 SD in five mice examined per each group. p, p , 0.01; pp, p , 0.05.
Epithelial duct cells were intensely stained with anti-Fas Ab in both IL-10TG and control mice. Five mice in each group were analyzed at 8 and 20 wk
of age. B, Flow cytometric analysis of FasL expression on the tissue-infiltrating lymphocytes purified from affected glands gated on CD4. A significantly
high proportion of FasL-positive infiltrating CD41 T cells was observed. The dark-shaded histograms are isotype control. Histograms show a representative
experiment from four different mice. C, In vitro induction of FasL on spleen cells by IL-10. CD41/FasL1 by two-color flow cytometric analysis was used.
Dose-dependent FasL expression was detected in CD41 T cells treated with rIL-10. Data are expressed as mean fluorescence intensity. D, Cytotoxic activity
of IL-10-stimulated CD41 T cells from spleen toward Fas-sensitive MSG primary culture cells. This activity was almost entirely inhibited by anti-mouse
neutralizing FasL Ab. E, Preventive effect of i.p. injection of anti-mouse IL-10 neutralizing Ab. A 0.1 mg/ml IL-10 neutralizing Ab (n 5 5) or a 0.1 mg/ml
isotype control Ab (n 5 5) diluted with PBS was injected i.p. twice per wk into IL-10TG mice from 4 wk to 8 wk of age.
The Journal of Immunology
mouse (33, 34), have shown the importance of T cells in autoimmune disease. However, in these mice the precise mechanisms in
causing tissue destruction of the glands were not clarified.
Although IL-10 has been implicated in the in vitro regulation of
the functions of lymphoid cells, based on its ability to suppress the
synthesis of proinflammatory cytokines from T cells and monocytes/macrophages, the possibility that FasL expression of local
CD41 T cells induced by IL-10 is a primary cause of glandular
epithelial cell loss would be consistent with reports that demonstrated that continuous administration of IL-10 to NZB/W F1 mice
caused a significant acceleration in the development of autoimmunity in these mice, whereas treatment with anti-IL-10 Ab delays
onset of autoimmunity (24). Furthermore, transgenic expression of
IL-10 accelerates the prevalence and onset of diabetes (10, 35–37),
indicating that IL-10 is not a general inhibitor in certain autoimmune diseases in vivo.
Recent studies with animal models of organ-specific autoimmune diseases, such as the nonobese diabetic mouse (38), and with
human SS (39, 40), have characterized the pathogenic role of
CD41 Th cells such as Th1 and Th2. However, relatively little is
known about the functional activity of CD41 Th cells at the site of
damage in autoimmune diseases. The perforin/granzyme and Fas/
FasL systems are the two major mechanisms of cytotoxicity of
CTL, but the specific role of each seems largely to depend upon the
target cells (41). On the other hand, the CD41 CTL, which often
lack perforin (42, 43), mainly use FasL as an effector. In fact, it has
been reported that Th1, Th0, and some Th2 cells have Fas/FasL
system-dependent cytotoxicity (28). Moreover, CD41 CTL activities, which have been implicated in the pathogenesis of SS, have
been detected against various viruses (44 – 46). Taken together, our
results show that IL-10-induced FasL expression on CD41 T cells
may play an important role in tissue destruction.
Clonally expanded T cell populations using a restricted usage of
TCR gene segments may be essential in the pathogenesis of autoimmune diseases (47). However, we found no preferential utilization of restricted TCR Vb gene in the glands of IL-10TG. The
diverse TCR b gene usage of infiltrating T cells was observed in
the lacrimal glands of patients with SS (48). It was also reported
that the restricted Vb-bearing T cells were detected on the analyzed CD41 T cells during the early stage of the disease, which
could initiate the destruction of the salivary glands, and the polyclonal nature of Vb gene usage was found during the late stage of
the disease (49). SS is a chronic disease, the onset of which is not
clear. The patients are usually asymptomatic at the early stage. By
the time clinical symptoms manifest themselves, TCR repertoires
could have already become diverse, as observed in our transgenic
model mice. Another possible mechanism underlying the diverse
TCR in the glands is that most of the T cells infiltrating in the
inflammatory sites might be recruited nonspecifically. In this regard, only ,5% of T cells at the inflammation sites are specific to
myelin basic protein in experimental autoimmune encephalomyelitis (50). In the SS glands, cell adhesion molecules on vascular
endothelial cells, such as ICAM-1 and VCAM-1, are up-regulated
(51). Furthermore, it has been reported that IL-10 induces cell
adhesion molecule expression on endothelial cells (9, 10). These
adhesion molecules can readily promote nonspecifically activated
lymphocytes to adhere to local vessels and to migrate into the
inflamed tissues.
In conclusion, these results are strongly suggestive of a role for
IL-10 in the functional FasL activation on nonspecific bystander T
cells and could be consistent with a role for Fas/FasL-mediated
apoptosis in the development of tissue destruction. Moreover, preventive effects against lesions treated with their neutralizing Abs
have important implications for testing useful therapies.
References
1. Katsikis, P. D., C.-Q. Chu, F. M. Brennan, R. N. Maini, and M. Feldmann. 1994.
Immunoregulatory role of interleukin 10 in rheumatoid arthritis. J. Exp. Med.
179:1517.
2. Llorente, L., Y. Richaud-Patin, R. Fior, J. Alcocer-Varela, J. Wijdenes,
B. M. Fourrier, P. Galanaud, and D. Emilie. 1994. In vivo production of interleukin-10 by non-T cells in rheumatoid arthritis, Sjoegren’s syndrome, and systemic lupus erythematosus. Arthritis Rheum. 37:1647.
3. Cush, J. J., J. B. Splawski, R. Thomas, J. E. McFarlin, H. Schulze-Koops,
L. S. Davis, K. Fujita, and P. E. Lipsky. 1995. Elevated interleukin-10 levels in
patients with rheumatoid arthritis. Arthritis Rheum. 38:96.
4. Cohen, S. B. A., P. D. Katsikis, C.-Q. Chu, H. Thomssen, L. M. C. Webb,
R. N. Maini, M. Londei, and M. Feldmann. 1995. High level of interleukin-10
production by the activated T cell population within the rheumatoid synovial
membrane. Arthritis Rheum. 38:946.
5. Moore, K. W., P. Vieira, D. F. Fiorentino, M. L. Trounstine, T. A. Khan, and
T. R. Mosmann. 1990. Homology of cytokine synthesis inhibitory factor (IL-10)
to the Epstein-Barr virus gene BCRF1. Science 248:1230.
6. Burdin, N., C. Peronne, J. Banchereau, and F. Rousset. 1993. Epstein-Barr virus
transformation induces B lymphocytes to produce human interleukin 10. J. Exp.
Med. 177:295.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 5. A, Expression of TCR Vb genes (Vb1–Vb19) mRNA in
the tissue-infiltrating mononuclear cells into the lacrimal (LG) and salivary
glands (SG) of IL-10TG mice. a, Lacrimal gland of negative littermates; b,
lacrimal gland of IL-10TG; c, salivary gland of negative littermates; d,
salivary gland of IL-10TG. B, SSCP analysis of PCR-amplified Vb-chain
products including Vb5, Vb11, Vb12, and Vb13 in the lacrimal gland and
salivary gland from IL-10TG mice, including IL-10(1) TG and negative
littermates (IL-10(2)). Sp, spleen cells.
2493
2494
29. Rouvier, E., M.-F. Luciani, and P. Golstein. 1993. Fas involvement in Ca21independent T cell-mediated cytotoxicity. J. Exp. Med. 177:195–202.
30. Green, J. E., S. H. Hinrichs, S. H. Vogel, and G. Jay. 1989. Exocrinopathy
resembling Sjogren’s syndrome in HTLV-1 tax transgenic mice. Nature 341:72.
31. Koike, K., K. Moriya, K. Ishibashi, H. Yotsuyanagi, Y. Shintani, H. Fujie,
K. Kurokawa, Y. Matsuura, and T. Miyamura. 1997. Sialadenitis histologically
resembling Sjoegren syndrome in mice transgenic for hepatitis C virus envelope
genes. Proc. Natl. Acad. Sci. USA 94:233.
32. Hamano, H., I. Saito, N. Haneji, Y. Mitsuhashi, N. Miyasaka, and Y. Hayashi.
1993. Expression of cytokine genes during development of autoimmune sialadenitis in MRL/lpr mice. Eur. J. Immunol. 23:2387.
33. Haneji, N., H. Hamano, K. Yanagi, and Y. Hayashi. 1994. A new animal model
for primary Sjogren’s syndrome in NFS/sld mutant mice. J. Immunol. 153:2769.
34. Haneji, N., T. Nakamura, K. Takio, K. Yanagi, H. Higashiyama, I. Saito, S. Noji,
H. Sugino, and Y. Hayashi. 1997. Identification of a-Fodrin as a candidate autoantigen in primary Sjogren’s syndrome. Science 276:604.
35. Lee, M.-S., L. Wogensen, J. Shizuru, M. B. A. Oldstone, and N. Sarvetnick.
1994. Pancreatic islet production of murine interleukin-10 does not inhibit immune-mediated tissue destruction. J. Clin. Invest. 93:1332.
36. Lee, M.-S., R. Mueller, L. S. Wicker, L. B. Peterson, and N. Sarvetnick. 1996.
IL-10 is necessary and sufficient for autoimmune diabetes in conjunction with
NOD MHC homozygosity. J. Exp. Med. 183:2663.
37. Wogensen, L., M.-S. Lee, and N. Sarvetnick. 1994. Production of interleukin 10
by islet cells accelerates immune-mediated destruction of b cells in nonobese
diabetic mice. J. Exp. Med. 179:1379.
38. Kagi, D., B. Odermatt, P. Seiler, R. M. Zinkernagel, T. M. Mak, and
H. Hengartner. 1997. Reduced incidence and delayed onset of diabetes in perforin-deficient nonobese diabetic mice. J. Exp. Med. 186:989.
39. Ohyama, Y., S. Nakamura, G. Matsuzaki, M. Shinohara, A. Hiroki, T. Fujimura,
A. Yamada, K. Itoh, and K. Nomoto. 1996. Cytokine messenger RNA expression
in the labial salivary glands of patients with Sjoegren’s syndrome. Arthritis
Rheum. 39:1376.
40. Fox, R. I., H. I. Kang, D. Ando, J. Abrams, and E. Pisa. 1994. Cytokine mRNA
expression in salivary gland biopsies of Sjogren’s syndrome. J. Immunol. 152:
5532.
41. Kagi, D., F. Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata,
H. Hengartner, and P. Golstein. 1994. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 265:528.
42. Strack, P., C. Martin, S. Saito, R. H. Dekruyff, and S.-T. Ju. 1990. Metabolic
inhibitors distinguish cytolytic activity of CD4 and CD8 clones. Eur. J. Immunol.
20:179.
43. Apasov, S., F. Redegeld, and M. Sitkovsky. 1993. Cell-mediated cytotoxicity:
contact and secreted factors. Curr. Opin. Immunol. 5:404.
44. Orentas, R. J., J. E. Hildreth, E. Obah, M. Polydefkis, G. E. Smith,
M. L. Clements, and R. F. Siliciano. 1990. Induction of CD41 human cytolytic
T cells specific for HIV-infected cells by a gp160 subunit vaccine. Science 248:
1234.
45. Stanhope, P. E., A. Y. Liu, W. Pavlat, P. M. Pitha, M. L. Clements, and
R. F. Siliciano. 1993. An HIV-1 envelope protein vaccine elicits a functionally
complex human CD41 T cell response that includes cytolytic T lymphocytes.
J. Immunol. 150:4672.
46. Yasukawa, M., A. Inatsuki, and Y. Kobayashi. 1988. Helper activity in antigenspecific antibody production mediated by CD41 human cytotoxic T cell clones
directed against herpes simplex virus. J. Immunol. 140:3419.
47. Zhang, S., A. Bendelac, D. Mathis, C. Benoist, M. A. Lipes, A. Rosenzweig,
K.-N. Tan, G. Tanigawa, J. G. Seidman, and G. S. Eisenbarth. 1993. T cell
receptor specificity and diabetes in nonobese diabetic mice. Science 262:1582.
48. Mizushima, N., H. Kohsaka, K. Tsubota, I. Saito, and N. Miyasaka. 1995. Diverse T cell receptor b gene usage by infiltrating T cells in the lacrimal glands of
Sjogren’s syndrome. Clin. Exp. Immunol. 101:33.
49. Legras, F., T. Martin, A.-M. Knapp, and J.-L. Pasquali. 1994. Infiltrating T cells
from patients with primary Sjoegren’s syndrome express restricted or unrestricted
T cell receptor Vb regions depending on the stage of the disease. Eur. J. Immunol. 24:181.
50. Mor, F., and I. R. Cohen. 1992. T cells in the lesion of experimental autoimmune
encephalomyelitis: enrichment for reactivities to myelin basic protein and to heat
shock proteins. J. Clin. Invest. 90:2447.
51. Saito, I., K. Terauchi, M. Shimuta, S. Nishimura, K. Yoshino, T. Takeuchi,
K. Tsubota, and N. Miyasaka. 1993. Expression of cell adhesion molecules in the
salivary and lacrimal glands of Sjogren’s syndrome. J. Clin. Lab. Anal. 7:180.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
7. Go, N. F., B. E. Castle, R. Barrett, R. Kastelein, W. Dang, T. R. Mosmann,
K. W. Moore, and M. Howard. 1990. Interleukin 10, a novel B cell stimulatory
factor: unresponsiveness X chromosome linked immunodeficiency B cells.
J. Exp. Med. 172:1625.
8. Fluckiger, A.-C., P. Garrone, I. Durand, J.-P. Galizzi, and J. Banchereau. 1993.
Interleukin 10 (IL-10) upregulates functional high affinity IL-2 receptors on normal and leukemic B lymphocytes. J. Exp. Med. 178:1473.
9. Vora, M., L. I. Romero, and M. A. Karasek. 1996. Interleukin-10 induces Eselectin on small and large blood vessel endothelial cells. J. Exp. Med. 184:821.
10. Wogensen, L., X. Huang, and N. Sarvetnick. 1993. Leukocyte extravasation into
the pancreatic tissue in transgenic mice expressing interleukin 10 in the islets of
Langerhans. J. Exp. Med. 178:175.
11. Fluckiger, A.-C., I. Durand, and J. Banchereau. 1994. Interleukin 10 induces
apoptotic cell death of B-chronic lymphocytic leukemia cells. J. Exp. Med. 179:
91.
12. Fox, R. I., and I. Saito. 1994. Criteria for diagnosis of Sjogren’s syndrome.
Rheum. Dis. Clin. North Am. 20:391.
13. Kang, H.-I., H.-M. Fei, I. Saito, S. Sawada, S.-I. chen, D. Yi, E. Chan, C. Peebles,
T. L. Bugawan, H. A. Erlich, and R. I. Fox. 1993. Comparison of HLA class II
genes in Caucasoid, Chinese, and Japanese patients with primary Sjoegren’s syndrome. J. Immunol. 150:3615.
14. Fox, R. I., T. Chilton, S. Scott, L. Benton, F. V. Howell, and J. H. Vaughan. 1987.
Potential role of Epstein-Barr virus in Sjoegren’s syndrome. Rheum. Dis. Clin.
North Am. 13:275.
15. Garry, R. F., C. D. Fermin, D. J. Hart, S. S. Alexander, L. A. Donehower, and
H. L. Zhang. 1990. Detection of a human intracisternal A-type retroviral particle
antigenically related to HIV. Science 250:1127.
16. Mariette, X., F. Agbalika, M. T. Daniel, M. Bisson, P. Lagrange, J. C. Brouet, and
F. Morinet. 1993. Detection of human T lymphotropic virus type I tax gene in
salivary gland epithelium from two patients with Sjögren’s syndrome. Arthritis
Rheum. 36:1423.
17. Fox, R. I., G. Pearson, and J. H. Vaughan. 1986. Detection of Epstein-Barr virusassociated antigens and DNA in salivary gland biopsies from patients with
Sjoegren’s syndrome. J. Immunol. 137:3162.
18. Saito, I., B. Servenius, T. Compton, and R. I. Fox. 1989. Detection of EpsteinBarr virus DNA by polymerase chain reaction in blood and tissue biopsies from
patients with Sjogren’s syndrome. J. Exp. Med. 169:2191.
19. Tateishi, M., I. Saito, K. Yamamoto, and N. Miyasaka. 1993. Spontaneous production of Epstein-Barr virus by B lymphoblastoid cell lines obtained from patients with Sjoegren’s syndrome. Arthritis Rheum. 36:827.
20. Mariette, X., J. Gozlan, D. Clerc, M. Bisson, and F. Morinet. 1991. Detection of
Epstein-Barr virus DNA by in situ hybridization and polymerase chain reaction
in salivary gland biopsy specimens from patients with Sjogren’s syndrome.
Am. J. Med. 90:286.
21. White, S. C., and G. W. Casarett. 1974. Induction of experimental autoallergic
sialadenitis. J. Immunol. 112:178.
22. Hoffman, R. W., M. A. Alspaugh, K. S. Waggie, J. B. Durham, and S. E. Walker.
1984. Sjogren’s syndrome in MRL/1 and MRL/n mice. Arthritis Rheum. 27:157.
23. Delporte, C., B. C. O’Connell, X. He, H. E. Lancaster, A. C. O’Connell, P. Agre,
and B. J. Baum. 1997. Increased fluid secretion after adenoviral-mediated transfer
of the aquaporin-1 cDNA to irradiated rat salivary glands. Proc. Natl. Acad. Sci.
USA 94:3268.
24. Ishida, H., T. Muchamuel, S. Sakaguchi, S. Andrade, S. Menon, and M. Howard.
1994. Continuous administration of anti-interleukin 10 antibodies delays onset of
autoimmunity in NZB/W F1 mice. J. Exp. Med. 179:305.
25. Furukawa, M., A. Sakamoto, Y. Kita, Y. Ohichi, R. Matsumura, R. Tsubata,
T. Tsubata, I. Iwamoto, Y. Saito, and T. Sumida. 1996. T-cell receptor repertoire
of infiltrating T cells in lachrymal glands, salivary glands and kidneys from
alymphoplasia (ALY) mutant mice: a new model for Sjogren’s syndrome.
Br. J. Rheumatol. 35:1223.
26. Sumida, T., I. Matsumoto, H. Murata, T. Namekawa, R. Matsumura, H. Tomioka,
I. Iwamoto, Y. Saito, Y. Mizushima, T. Hasunuma, T. Maeda, and K. Nishioka.
1997. TCR in Fas-sensitive T cells from labial salivary glands of patients with
Sjogren’s syndrome. J. Immunol. 158:1020.
27. Ting, C.-N., M. P. Rosenberg, C. M. Snow, L. C. Samuelson, and M. H. Meisler.
1992. Endogenous retroviral sequences are required for tissue-specific expression
of a human salivary amylase gene. Genes Dev. 6:1457.
28. Suda, T., T. Okazaki, Y. Naito, T. Yokota, N. Arai, S. Ozaki, K. Nakao, and
S. Nagata. 1995. Expression of the Fas ligand in cells of T cell lineage. J. Immunol. 154:3806.
IL-10 TRANSGENIC MICE FOR SS