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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;
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Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan;
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Clinical Research Center, National Utano Hospital, Kyoto, Japan;
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Department of Molecular Biology, Osaka Bioscience Institute, Osaka, Japan; and
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Department of Cell Biology, Cancer Institute, Toshima-ku, Tokyo, Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 environmental causative agent of SS 15, 16 , and evidence for an association between EBV infection and SS has been accumulating 17, 18, 19, 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 ß-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 (IL-10TG) 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 µg/ml L-glutamine, 100 U/ml penicillin, 100 µg 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. 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% 3,3'-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
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 µg/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.
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 3'-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 3,3'-diaminobenzidine 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 µg/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%).
Cytotoxicity assay
MSG cells (2 x 106) in 7.5 ml of RPMI 1640 supplemented with 5% FBS were labeled overnight at 37°C in 5% CO2 with 300 µCi of sodium [51Cr]chromate. CD4+ and CD8+ T cells purified from splenocytes using magnetic beads (2–3 x 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 µl, 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-µl 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 - % 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 FasL (detail will be described elsewhere by T. Suda). FLIM58 neutralizes mouse but not human FasL activity.
Treatment with neutralizing Ab to IL-10
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) 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). Mean grade of inflammatory lesions was expressed as described 21 . Data represent the mean grade of lesions ± SD (Mann-Whitney U test).
PCR analysis for TCR Vß usage
To analyze TCR Vß1-Vß19 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 µl of PCR buffer; we then added 50 pmol of the 5' and 3' 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 µl 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-µl 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 Vß1-Vß19 and Cß primers were obtained from previously published data 25, 26 . PCR products were blotted onto a nylon membrane and hybridized with biotinylated labeled Cß probe (TTG ATG GCT CAA ACA AGG AGA CC).
Analysis of single-strand conformation polymorphisms (SSCP)
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 µl) 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 Cß probe and visualized by subsequent incubation with streptavidin, biotinylated alkaline phosphatase, and a chemiluminescent substrate system (Plex Luminescence kit, Millipore Intertech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
A). 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 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).
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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. Anti-IL-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).
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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.
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Since the high prevalence of glandular hypofunction in IL-10TG was
accompanied with CD4+ 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 CD4+ 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 CD4+ T cells. To investigate whether
CD4+ T cells express FasL, spleen cells stimulated with
rIL-10 were analyzed for the CD4+/FasL+
phenotype by two-color flow cytometric analysis. Dose-dependent FasL
expression was detected in CD4+ 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 CD4+ T cells from B6 normal mice
was investigated using primary cultured MSG epithelial cells as target
cells. CD4+ T cells were stimulated with rIL-10 alone
and/or with the presence of Con A+rIL-2 for 6 h before
cytotoxicity assays. As shown in Fig. 4D, cytotoxic
activities were enhanced by rIL-10 and Con A+rIL-2. These cytotoxic
activities were almost entirely inhibited by incubation with
anti-mouse 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
anti-IL-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.
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To characterize the repertoire of TCR Vß genes of tissue-
infiltrating lymphocytes in the glands, we have analyzed TCR Vß gene
expression (Vß1-Vß19) in the isolated cells from salivary and
lacrimal gland tissues and spleen cells at each age using RT-PCR. We
found no significant Vß gene-biased expression in the gland tissues
at age 8 wk in the onset of inflammatory infiltrates (Fig. 5A). Multiple Vß 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 Vß genes was
observed in any of the mice. To investigate the clonotypes of T cells,
we selected the Vß families, Vß5, Vß11, Vß12, and Vß13, 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
suggest that tissue destruction in the glands may occur as a result of
bystander killing by nonspecific T cells expressing FasL.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 CD4+ 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, 36, 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 CD4+ Th cells such as Th1 and Th2. However, relatively little is known about the functional activity of CD4+ 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 CD4+ 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, CD4+ CTL activities, which have been implicated in the pathogenesis of SS, have been detected against various viruses 44, 45, 46 . Taken together, our results show that IL-10-induced FasL expression on CD4+ 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 Vß gene in the glands of IL-10TG. The diverse TCR ß gene usage of infiltrating T cells was observed in the lacrimal glands of patients with SS 48 . It was also reported that the restricted Vß-bearing T cells were detected on the analyzed CD4+ T cells during the early stage of the disease, which could initiate the destruction of the salivary glands, and the polyclonal nature of Vß 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.
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Footnotes |
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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:
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.
Received for publication August 17, 1998. Accepted for publication October 29, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Fodrin as a candidate autoantigen in primary Sjogren’s syndrome. Science 276:604.This article has been cited by other articles:
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