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Regular Articles |
From the Departments of Pathology,*
Laboratory
Medicine,
and
Orthodontics,
Tokushima University School of
Dentistry, Tokushima, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-fodrin, in the aged salivary
gland tissues on immunoblotting, and an increase in serum
autoantibody production against 120-kd -fodrin by enzyme-linked
immunosorbent assay. An increase in the proliferative response of
splenic T cells against organ-specific autoantigen was
observed, whereas nonspecific concanavalin A
responsiveness was decreased in the aged mice. In addition, a
decrease in Fas expression was found on splenic CD4+ T
cells in the aged mice, and anti-Fas mAb-stimulated apoptosis
was down-regulated on CD4+ T cells. These results
indicate that age-associated dysregulation of CD4+ T cells
may play a crucial role on acceleration of organ-specific autoimmune
lesions in a murine model for primary SS through Fas-mediated
apoptosis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary Sjögren’s syndrome (SS) in
humans is an organ-specific autoimmune disease characterized by
lymphocytic infiltration into the salivary and lacrimal glands,
resulting in symptoms of dry mouth and dry eye due to insufficient
secretion.18,19
It is possible that individual T cells
activated by an appropriate antigen can proliferate and form a
restricted clone.20,21
Recently, we identified 120-kd
-fodrin as an important organ-specific autoantigen in both the
NFS/sld murine model for SS and in human SS
patients.22
Since it was reported that Fas
expression was observed in the salivary gland cells in human
SS,23
we speculate that Fas-mediated apoptosis may
contribute to tissue destruction with aging in the salivary and
lacrimal glands with SS. In addition, there are no published data on
the aging process in the animal model for organ-specific autoimmune
diseases including SS.
The aim of this study was to evaluate the possible relationship between the Fas-mediated apoptosis and the development and acceleration of organ-specific autoimmune lesions with aging in murine SS model of NFS/sld mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NFS/sld carrying the mutant gene sld24 were bred in our own facilities, maintained in a specific pathogen-free mouse colony, and given food and water ad libitum. An animal model for primary SS was previously established in NFS/sld mutant mice.25 Thymectomy was performed on the day 3 after birth (3d-Tx), and a total of 114 NFS/sld mice, consisting of 79 3d-Tx (females, n = 54; males n = 25) and 35 non-Tx female mice, were investigated. They were killed by cervical dislocation during time intervals of 2, 4, 6, 10, 12, 18, and 20 months of age. Five to eight mice in each age group were analyzed. The aged group consisted of 18- and 20-month-old mice and the young group was comprised of 2- and 4-month-old mice.
Histopathology
All organs were removed from the mice, fixed with 10% phosphate-buffered formalin, and embedded in paraffin. The sections (4 µm) were stained with hematoxylin and eosin. Histological grading of inflammatory lesions was done according to the modified method proposed by White and Casarett26 as follows: a score of 1 indicates that one to five foci being composed of more than 20 mononuclear cells per focus were seen; a score of 2 indicates that more than five such foci were seen but without significant parenchymal destruction; a score of 3 indicates degeneration of parenchymal tissue; a score of 4 indicates extensive infiltration of the glands with mononuclear cells and extensive parenchymal destruction; and a score of 5 indicates that severe destructive foci with focal fibrosis, ductal dilatation, and/or fatty infiltration were seen in addition to the score 4 lesions. These slides were scored by three independent, well-trained pathologists in a blinded manner.
Measurement of Fluid Secretion
Detection of tear and saliva volume in the aged and young SS animal models of NFS/sld mice was done according to a modified method as described.27 Five mice in each group were analyzed at 2- and 18-months of age.
In Situ End Labeling of Fragmented DNA (TUNEL)
Apoptotic cells were detected in sections using the in situ TUNEL Kit (Wako Pure Chemicals, Osaka, Japan), as described previously.28 Briefly, paraffin-embedded sections were deparaffinized and rehydrated, and washed twice in phosphate-buffered saline (PBS). Sections were incubated with proteinase K (20 mg/ml) for 10 minutes. After washing in distilled water, these sections were incubated with 2% H2O2 in PBS to block endogenous peroxidase. Sections were then presoaked in TdT buffer (0.5 mmol/L cacodylate, 1 mmol/L CoCl, 0.5 mmol/L dithiothreitol, 0.05% bovine serum albumin, 0.15 mol/L NaCl) for 10 minutes, and incubated for 2 hours at 37°C in 25 ml of TdT solution, containing 1x terminal transferase buffer, 0.5 nmol of biotin-dUTP, and 10U of TdT (Wako Pure Chemicals). After the TdT reaction, sections were soaked in TdT blocking buffer (300 nmol/L NaCl, 30 mmol/L tri-sodium citrate-2-hydrate), incubated with horseradish peroxidase-conjugated streptavidin for 30 minutes at room temperature, and developed for 10 minutes in phosphate-buffered citrate (pH 5.8) containing 0.6 mg/ml of diaminobenzidine. Nuclei were counterstained with hematoxylin. When we used DNase-I-treated and -untreated sections of submandibular glands in non-Tx mice, almost all acinar and duct cells were TUNEL+ in DNase-I-treated sections, and were TUNEL- in untreated sections (data not shown).
Flow Cytometry
Spleen cell suspensions were stained with antibodies conjugated to phycoerythrin (anti-CD4, Cedar Lane Laboratories Ltd., Ontario, Canada; B220, Pharmingen, San Diego, CA), and fluorescein isothiocyanate (anti-CD8, Cedar Lane Laboratories Ltd.; CD3, anti-I-As, anti-CD5, Pharmingen), and analyzed with an EPICS flow cytometer (Coulter, Miami, FL). Isolated mouse salivary gland (MSG) cells (described below) were stained with biotinylated anti-Fas monoclonal antibody (mAb; Pharmingen) and fluorescein isothiocyanate-conjugated avidin (Vector Laboratories, Inc., Burlingame, CA), and analyzed by EPICS. Double-labeled surface phenotypes such as CD3/B220, CD4/FasL (Santa Cruz, Santa Cruz, CA), and CD8/FasL were analyzed. Apoptotic cells were also detected by flow cytometry with an EPICS flow cytometer (Coulter) using the Annexin V-FITC Apoptosis Detection Kit (Genzyme, Cambridge, MA).
Cell Preparation
We obtained tissue infiltrating cells from salivary gland tissues as previously described.29 Briefly, affected submandibular glands from five mice were removed, cut into small pieces with scissors, passed through a 100-gauge stainless steel mesh, and suspended in RPMI 1640 containing 10% fetal calf serum, 10 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, penicillin (100 U/ml), and streptomycin(100 µg/ml). After washing twice with medium, infiltrating cells were isolated from parenchymal cells by Ficoll-Isopaque density-gradient centrifugation. Spleen cells were aseptically prepared from 8- to10-week-old, nontreated NFS/sld female mice. To purify CD4 or CD8 single-positive T cells, we used nylon fiber for T cell selection and immunomagnetic beads (Dynal, Oslo, Norway) with anti-CD4 mAb, anti-CD8 mAb, and Mac-1 mAb (Becton Dickinson, San Jose, CA) as reported previously.30,31 The purity of CD4 or CD8 single-positive cells was 90% or higher.
Primary Culture of Mouse Salivary Gland Cells
MSG epithelial cells were prepared as previously described.27,32 Briefly, MSGs were minced into 1-mm2 pieces, washed with Hanks’ balanced salt solution without Ca2+ and Mg2+, and placed in a 60-well dish containing Hanks’ balanced salt solution with 0.76 µg/ml ethylenediaminetetraacetic acid, 4.9 µg/ml L-ascorbic acid, and 4.9 µg/ml reduced glutathione. Fragments were washed with Dulbecco’s minimal essential medium/STI, and placed in a mixture of collagenase (type I, 750 U/ml) and hyaluronidase (type IV, 500 U/ml) dissolved in Dulbecco’s minimal essential medium/F12 containing 10% fetal bovine serum. The first digest suspension was passed through sterile 100-µm nylon mesh filters and were redigested for 30 minutes by the same digestion procedure and then the digest suspension was passed through a 100-µm nylon mesh filter. Adherent cells, after culture in minimal essential medium containing 10% fetal bovine serum for 24 hours at 37°C, were isolated as salivary gland epithelial cells. We confirmed that more than 95% of the cells were positively stained with anti-keratin polyclonal antibody.
Proliferation Assay
Proliferative T cell response to the 120-kd -fodrin antigen was
examined in spleen cells from 3d-Tx NFS/sld mice in a
defined chronological order. Antigen-stimulated blastogenesis was
measured in spleen cells from young and aged NFS/sld mice.
Spleen cells were plated at 1 x 106
cells
per well in 96-well microtiter plates in 200 µl HL-1 medium (Ventrex,
Portland, ME) containing 2 mmol/L glutamine and 10
µg/ml-1 antigen. During the last 8 hours of
the 72-hour culture period, 1 µCi of
[3H]-thymidine was added per well, and the
incorporated radioactivity was determined. Data are expressed as counts
per minute per culture in triplicate.
Western Blot Analysis
Autoantigen cleavage of the 120-kd -fodrin in the tissue
homogenates from salivary glands was analyzed by immunoblotting with
polyclonal antibodies to synthetic -fodrin of the purified 120-kd
antigen.22
Tissue samples were homogenized in 20 mmol/L
Tris-HCl buffer (pH 7.4), containing 5 mmol/L diisopropyl
fluorophosphate, 5 mmol/L ethylenediaminetetraacetic acid, 5 mmol/L
benzamidine, 2 mmol/L phenylmethyl sulfonyl fluoride, and 2 mmol/L
N-ethylmaleimide. After centrifugation for 20 minutes
at 12,000 rpm at 4°C, the supernatant was extracted and used for
cytoplasmic protein. Pellets were homogenized in 20 mmol/L Tris-HCl
buffer containing 2% Triton X-100. After centrifugation for 20 minutes
at 12,000 rpm at 4°C, the supernatant was used for membrane protein.
Enzyme-Linked Immunosorbent Assay
Serum autoantibodies were detected using recombinant -fodrin
protein (JS-1).22
After coating with the recombinant
-fodrin protein in a 96-well ELISA plate, biotinylated anti-mouse
immunoglobulin G (Vector Laboratories) was added as second antibody.
Measurements of JS-1 specific autoantibodies were read by automatic
enzyme-linked immunosorbent assay (ELISA) reader (Flow Laboratories,
McLean, VA). To detect serum antibody to DNA, the relative avidity of
the serum antibodies for binding to ssDNA was measured in a solid phase
ELISA. Purified ssDNA was prepared by boiling calf thymus DNA (Sigma
Chemical Co., St. Louis, MO) for 10 minutes followed immediately by
dilution in ice-cold borate-buffered saline. Flat bottom 96-well
microtiter plates (Nunc, Roskilde, Denmark) were coated with ssDNA (50
mg/ml) overnight at 4°C in PBS, blocked at room temperature with 5%
skim milk, and incubated with sera. Plates were read on an automatic
ELISA reader (Flow Laboratories) at 492 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Severe destructive autoimmune lesions developed in the major
salivary (submandibular, parotid, and sublingual) and lacrimal glands
in aged NFS/sld mice of 12 months of age or more, compared
with those in the young SS model mice (Figure 1)
. A higher incidence of autoimmune
lesions was observed in females than in males at all ages. The severity
of the lesions correlates with sex difference and aging. Inflammatory
infiltrations were more marked and were accompanied by a more severe
periductal fibrosis with advance of age. Multiple confluent foci of
mononuclear cell infiltration and extensive parenchymal destruction
were seen in the aged mice older than 12 months of age. In particular,
severe inflammatory lesions with focal fibrosis, irregular
dilatation of ducts, and/or fatty degeneration were frequently observed
only in the aged groups of 18 and 20 months of age (Figure 2
, A and B).
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The average saliva and tear volume of the aged SS animal model was
significantly lower than that of the young group (Figure 3)
. The total amount of fluid decreased
gradually with advance of age. Decrease in saliva and tear secretion
probably correlates with severe inflammatory changes.
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To determine the possible involvement of apoptotic cascade in
tissue destruction in the SS animal model, we examined apoptotic cells
in the salivary gland specimens from the aged and young SS model mice.
A significant increase of TUNEL+-apoptotic
epithelial duct cells in the salivary glands was detected in the aged
3d-Tx NFS/sld mice, compared with those in the young group
at all ages (Figure 4)
. A lesser number
of TUNEL+-apoptotic acinar cells was observed,
but no significant difference of TUNEL+-acinar
cells was detected in between the aged and young NFS/sld
mice at all ages. A higher proportion of cultured MSG cells expressing
Fas in the aged mice was detected than that in the young mice on flow
cytometry (Figure 5A)
. No significant
changes were found in the tissue-infiltrating
CD4+ T cells bearing FasL in the salivary gland
tissues both from the aged and the young SS model mice (Figure 5B)
.
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Western blot analysis demonstrated a more intense band of
anti-120-kd -fodrin in the aged salivary gland tissues than that in
the young group (Figure 6A)
, indicating
that age-associated acceleration of autoantigen cleavage was detected
in the organ. In addition, a higher titer of serum autoantibodies
against 120-kd -fodrin was detected in the aged mice as compared
with those in the young mice by ELISA (Figure 6B)
. When we analyzed the
proliferative response of splenic T cells against organ-specific
autoantigen, we found a significant increase in blastogenesis, compared
with that in the young mice (Figure 6C)
. In contrast, a significant
decrease in nonspecific concanavalin A responsiveness of splenic T
cells was detected in the aged SS model mice (Figure 6D)
.
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To examine whether the autoimmune responses in the aged mice was
affected by the phenotypic changes of peripheral T cells, we
analyzed the surface phenotype in splenic T cells expressing Fas and
FasL. A significant decrease of CD4+ T cells
expressing Fas in the aged mice was observed as compared with those in
the young mice, whereas no difference in CD8+ T
cells expressing Fas was found (Figure 7A)
. We observed no difference in
FasL expression on both splenic CD4+ and
CD8+ T cells (data not shown). We next
investigated Fas-mediated apoptosis in freshly isolated splenic
CD4+ T cells in vitro from the young
and aged mice. Anti-Fas mAb-stimulated apoptosis was significantly
decreased in the aged CD4+ T cells in spleen than
those in the young mice (Figure 7B)
suggesting that there may be a
dysregulation of Fas-mediated apoptosis on activation-induced cell
death in the aged SS animal model.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Fas-mediated apoptosis is recognized as a major pathway for the induction of the tissue damage in autoimmune diseases.33,34 Expression of Fas by pancreatic ß cells has been shown to have a major influence on the susceptibility of tissue destruction in nonobese diabetic mice to diabetes.35 Recently, we reported that estrogen deficiency induces severe destructive organ-specific autoimmune lesions in a murine SS model, and demonstrated an important role of Fas-mediated apoptosis toward target tissue destruction.32 We detected a significant increase in TUNEL+-apoptotic epithelial duct cells in the salivary glands in the aged SS animal model than those in the young model. We found Fas expression on the cultured MSG cells from the aged SS model mice was significantly more augmented than those in the young model. An increased expression of Fas by MSG cells has been shown to have a major influence on the susceptibility of tissue destruction in the aged salivary glands. These results indicate that the age-associated acceleration of apoptotic cascade developed in the organ-specific autoimmune lesions in the salivary and lacrimal glands in this animal model. Indeed, we detected a significant decrease in saliva and tear flow secretion in the aged group.
Our data showed an increased cleavage product of organ-specific
autoantigen, 120-kd -fodrin, in the aged salivary gland tissues on
immunoblotting and a significant increase in serum autoantibody
production against 120-kd -fodrin by ELISA. Moreover, an increase in
the proliferative response of splenic T cells against organ-specific
autoantigen was observed, whereas nonspecific concanavalin A
responsiveness was decreased in the aged mice. It is likely that the
autoreactive CD4+ T cell clone reactive with
tissue-specific self-peptide is expanded during aging in the SS animal
model. These results strongly suggest that age-associated dysregulation
of immune functions may play a major role on acceleration of
organ-specific autoimmune lesions. To examine whether the expression of
Fas and FasL in aging T cells correlated with a susceptibility to
apoptosis, we compared anti-Fas-induced apoptosis in T cell subsets
between the aged and young SS animal model. We found that the freshly
isolated CD4+ T cells from the aged SS model mice
are resistant to anti-Fas-induced apoptosis. Our data show a decreased
expression of Fas at basal levels in CD4+ T cell
subsets from the aged SS animal model as compared with the young model.
In contrast, studies on aging humans show an increase in Fas expression
on T cells.36
It was reported that there is an altered
expression of genes regulating apoptosis in lymphocytes from aging
humans, and that T cell subsets from aging humans show increased
susceptibility to undergo anti-Fas-induced apoptosis as compared with T
cell subsets from young humans.36
The reason for this
discrepancy in Fas expression levels in humans and mice is not known,
but could be related to species differences. The Fas receptor
cross-linking results in the death of Fas+ T
cells on ligation with FasL.37,38
This interaction plays a
central role to terminate an ongoing immune response by inducing
apoptosis in activated T cells.39
Activation-induced cell
death through Fas-mediated apoptosis is an important process that
regulates the size and the duration of the primary immune T cell
response.40
Activation-induced cell death in T cells
in vivo has been proposed to limit the expansion of an
immune response by eliminating effector cells that are no longer
needed.
Aging is generally associated with an increase in frequency of infection and increased incidence of cancer and autoimmune diseases.41 One of the questions remains unresolved as to whether the cells expressing low levels of apoptotic molecules represent a subset of T cells that are normally present in young subjects, and aging represents an expansion of such a population. An increase in this population reacting with tissue specific self-peptide would be a consequence of increased production of this population or decreased death of a subset that does not express high levels of Fas/FasL. Further studies are needed to elucidate the mechanisms that T cells with decreased apoptotic molecules may represent a prolonged in vivo activation as a reflection of aging.
In conclusion, we have demonstrated, for the first time, that age-associated dysregulation of CD4+ T cells may play a crucial role in the acceleration of organ-specific autoimmune lesions in the salivary and lacrimal glands, and aging itself seems to influence both effector and target cells through Fas-mediated apoptosis in a murine model for primary SS.
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Footnotes |
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This work was supported in part by a Grant-in-Aid for Scientific Research (No. 08407057) from the Ministry of Education, Science and Culture of Japan.
Accepted for publication January 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fodrin as a candidate autoantigen in primary Sjögren’s syndrome. Science 1997, 276:604-607This article has been cited by other articles:
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  S. Cha, A.B. Peck, and M.G. Humphreys-Beher PROGRESS IN UNDERSTANDING AUTOIMMUNE EXOCRINOPATHY USING THE NON-OBESE DIABETIC MOUSE: AN UPDATE Crit. Rev. Oral. Biol. Med., January 1, 2002; 13(1): 5 - 16. [Abstract] [Full Text] [PDF]
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 N. Ishimaru, K. Yanagi, K. Ogawa, T. Suda, I. Saito, and Y. Hayashi Possible Role of Organ-Specific Autoantigen for Fas Ligand-Mediated Activation-Induced Cell Death in Murine Sjogren's Syndrome J. Immunol., November 15, 2001; 167(10): 6031 - 6037. [Abstract] [Full Text] [PDF]
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