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* Department of Pathology, Tokushima University School of Dentistry, Tokushima, Japan; and
Center for the Development of Molecular Target Drugs, Cancer Research Institute, Kanazawa University, Ishikawa, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-fodrin as an important autoantigen in the
pathogenesis of primary Sjögren’s syndrome (SS). In the murine
primary SS model, tissue-infiltrating CD4+ T cells purified
from the salivary glands bear a large proportion of Fas ligand, and the
salivary gland duct cells constitutively possess Fas. Infiltrating
CD4+ T cells, but not CD8+ T cells, identified
significant 51Cr release against mouse salivary gland
cells. In vitro studies demonstrated that apoptotic mouse salivary
gland cells result in a specific -fodrin cleavage into 120 kDa and
that preincubation with caspase inhibitor peptides blocked -fodrin
cleavage. In vivo treatment with caspase inhibitors
N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone
and N-acetyl-Asp-Glu-Val-Asp-al-CHO into the murine model
results in dramatic inhibitory effects on the development of autoimmune
lesions and in restoration of sicca syndrome. Furthermore, we found
that immunization with recombinant -fodrin protein identical with an
autoantigen into normal recipients induced autoimmune lesions similar
to SS. These data indicate that prevention and induction of autoimmune
exocrinopathy is dependent on autoantigen cleavage via caspase cascade
and that caspase inhibitors might provide a new therapeutic option
directed at reducing tissue damage in the murine model for
SS. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We reported previously that a cleavage product of 120-kDa -fodrin
may be an important autoantigen in the pathogenesis of primary SS in
both the animal model and in humans (6). -Fodrin is a
ubiquitous, calmodulin-binding protein (7) found to be
cleaved by calcium-activated protease (calpain) in apoptotic T cells
and by calpain and/or caspase 3 (CPP32) (8) in
anti-Fas-stimulated Jurkat cells and/or neuronal apoptosis
(9, 10, 11, 12). It was demonstrated that the fodrin subunit
is cleaved in association with apoptosis and that the 120-kDa fragment
is a breakdown product of the mature form of 240-kDa fodrin subunit
(11, 12). Recent reports have shown that caspase 3 is
required for -fodrin cleavage during apoptosis
(12, 13, 14). In Jurkat cells, caspase 3-like proteases have
been reported to cleave -fodrin and poly(ADP-ribose) polymerase
(PARP) but with differential sensitivity to the caspase 3 inhibitor
N-acetyl-Asp-Glu-Val-Asp-al (DEVD)-CHO (14). In
neuroblastoma cells, treatment with staurosporin induced cleavage of
-fodrin at both caspase 3 and calpain cleavage sites
(15). Accumulating evidence indicates that the interaction
of Fas with Fas ligand (FasL) regulates a large number of
pathophysiological processes of apoptosis (16, 17). It has
been reported that both Fas and FasL are present in thyrocytes and that
their concomitant expression on thyrocytes, independent of infiltrating
T cells, is responsible for thyrocyte destruction in Hashimoto’s
thyroiditis (18). In contrast, expression of Fas by
pancreatic 
cells has been shown to have a major influence on the
susceptibility of tissue destruction in nonobese diabetic mice to
diabetes (19, 20). Thus, we speculate that Fas-mediated
cytotoxicity and caspase-mediated -fodrin proteolysis are involved
in the progression of tissue destruction in SS.
In this study, we demonstrate that an increased activity of apoptotic
proteases is required for the -fodrin proteolysis during development
of murine SS. In addition, we present evidence that a cleavege product
of autoantigen induces autoimmune exocrinopathy in normal recipients
and that treatment with caspase inhibitors
N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone
(z-VAD-fmk) and DEVD-CHO protect animals against the development
of autoimmune exocrinopathy in the SS model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female NFS/N strain mice carrying the mutant gene sld (21) were reared in our specific pathogen-free mouse colony and given food and water ad libitum. Thymectomy was performed on day 3 after birth (3d-Tx) in NFS/sld mice (22). A total of 156 mice, consisting of 74 3d-Tx and 82 nonthymectomied (non-Tx) NFS/sld mice, were investigated in the present study. C57BL/6 mice (n = 24) purchased from Charles River Japan (Atsugi, Japan) were used as controls.
Histology and immunohistology
All organs were removed from the mice, fixed with 4% phosphate-buffered formaldehyde (pH 7.2), and prepared for histologic examination. The sections were stained with H&E. Histological grading of the inflammatory lesions was done according to the method proposed by White and Casarett (23). Immunohistology was performed on freshly frozen sections (4 µm in thickness) by the biotin-avidin immunoperoxidase method using avidin-biotin immunoperoxidase complex reagent (Vector Laboratories, Burlingame, CA). The mAbs used are as follows: biotinylated rat mAbs to CD3 (Life Technologies, Grand Island, NY), B220, CD4, CD8, Mac-1 (BD Biosciences, San Jose, CA), murine Fas (clone 13; Transduction Laboratories, Lexington, KY), and murine FasL (K-10; BD PharMingen, San Diego, CA).
TUNEL
Apoptotic cells were detected in sections using the in situ TUNEL kit (Wako Pure Chemical, Osaka, Japan). Briefly, sections were incubated with proteinase K (20 µg/ml) for 10 min and then presoaked in TdT buffer (0.5 mmol/L cacodylate, 1 mmol/L CoCl, 0.5 mmol/L DTT, 0.05% BSA, 0.15 mol/L NaCl) for 10 min. Sections were incubated for 2 h at 37°C in 25 µl of TdT solution containing 1x terminal transferase buffer, 0.5 nmol of biotin-dUTP, and 10 U of TdT (Wako Pure Chemical). 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 HRP-conjugated streptavidin for 30 min at room temparature, and developed for 10 min in phosphate-buffered citrate (pH 5.8) containing 0.6 mg/ml diaminobenzidine. Nuclei were counterstained with hematoxylin.
Flow cytometric analysis
Surface markers were identified by mAbs with an EPICS flow cytometer (Beckman Coulter, Miami, FL). Rat mAbs to CD3 (Life Technologies), B220, CD4, CD8 (BD Biosciences), murine Fas (Jo2, BD PharMingen), and murine FasL (BD PharMingen) were used. Double-labeled surface phenotypes such as CD3/B220, CD4/FasL, and CD8/FasL were analyzed. FasL expressions were analyzed on tissue-infiltrating lymphocytes in 3d-Tx NFS/sld mice gated on CD4 and CD8. Apoptotic cells were detected with an EPICS flow cytometer (Beckman Coulter) using the Annexin VFITC Apoptosis Detection Kit (Genzyme, Cambridge, MA). For detection of T cell activation makers, single cell suspensions were stained with Abs conjugated to PE (anti-CD3, Life Technologies; anti-CD4, Cedarlane Laboratories, Hornby, Ontario, Canada; B220, BD PharMingen) and FITC (anti-CD8, Cedarlane Laboratories; Thy1.2, anti-CD44, anti-CD45RB, anti-Mel-14, BD PharMingen) and were analyzed with an EPICS flow cytometer (Beckman Coulter).
RT-PCR
Total RNA was extracted from homogenized tissues with TRIzol
reagent (Life Technologies), and cDNA was prepared from RNA with 50
pmol oligo (dT) 18 and 200 U of murine leukemia virus reverse
transcriptase (Life Technologies). Two microliters of the cDNA mixture
was used in a PCR with 10 pmol of forward and reverse primers and 2.5 U
of Taq DNA polymerase (PerkinElmer/Cetus, Norwalk, CT). The
sequences of the specific sense and anti-sense oligonucleotide
primer pairs were as follows: Fas,
5'-ATCCGAGCTCTGAGGAGGCGGGGTTCATGAAAC-3' and
5'-GGAGGTTCTAGATTCAGGGTCATCCTG-3'; -actin,
5'-ATGGATGACGATATCGCT-3' and 5'-ATGAGGTAGTCTGTCAGGT-3'. Samples
were amplified through 30 cycles at an annealing temperature of 58°C
in a PCR Thermal Cycler (PerkinElmer/Cetus). PCR products were blotted
onto nylon membrane and hybridized with
32P-labeled cDNA probes.
Isolation of tissue-infiltrating cells from salivary glands
We isolated tissue-infiltrating mononuclear cells from affected salivary glands, as described previously (24, 25). In brief, 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% FCS, 10 mM 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. CD4+ and CD8+ T cells were purified from infiltrating cells using magnetic beads (Dynal Biotech, Oslo, Norway).
Primary culture of mouse salivary gland (MSG) cells
Primary cultures of MSG cells were prepared as reported previously (26). Briefly, the salivary gland cells were isolated from NFS/sld mice at 3–5 wk by enzymatic digestion with 0.76 mg/ml EDTA and mixture of collagenase (type I, 750 U/ml) and hyaluronidase (type IV, 500 U/ml), plated in 24-well plates, and maintained in MEM containing 10% FCS for 5–7 days before flow cytometric analysis and cytotoxic assay. Apoptosis was induced in MSG cells by anti-Fas Ab (Jo2, 300 ng ml-1; BD PharMingen). On Western blotting, cytosolic extracts were prepared from MSG cells (1 x 107 cells), which were treated at 37°C with apoptotic stimuli for various times.
Western blot analysis
Western blot analysis with mouse mAb to -fodrin
(Affiniti, Mamhead, U.K.) and PARP (Transduction Laboratories)
were performed. Briefly, the cells were homogenated in 20 mM Tris-HCl
buffer (pH 7.4) containing 5 mM diisopropylfluorophosphate, 5 mM EDTA,
5 mM benzamidine, 2 mM PMSF, and 2 mM N-ethylmaleimide.
After centrifugation for 20 min at 12,000 rpm at 4°C, supernatant was
extracted and used for cytoplasmic protein. Pellets were homogenized in
20 mM Tris-HCl buffer containing 2% Triton X-100. Protein binding was
visualized with ECL Western blotting reagent (Amersham, Arlington
Heights, IL). Protease inhibitors included mixtures (Sigma-Aldrich, St.
Louis, MO), leupeptin (Wako Pure Chemical), E64 (Wako Pure Chemical),
and caspase inhibitors (z-VAD-fmk and Ac-DEVD-CHO; ICN Pharmaceuticals,
Costa Mesa, CA). Control for protein loading was provided by actin,
tubulin, and myosin. To detect serum autoantibodies against 120-kDa
-fodrin Ag (6), mouse IgG was isolated from serum
samples collected from 3d-Tx NFS/sld mice. Samples were
solubilized by heating and separated by 10% SDS-PAGE. The autoantigen
was electrotransferred to nitrocellulose and then quenched with 1%
powdered milk in borate-buffered saline. Nitrocellulose membranes were
incubated with testing serum at a 1/200 dilution in borate-buffered
saline and then incubated with peroxidase-conjugated horse
anti-mouse IgG (Vector Laboratories) at a 1/1000 dilution.
Cytotoxic assay
MSG cells (2 x 106) in 7.5 ml of MEM, 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 isolated from salivary gland tissues and spleen (2–3 x 106) in 0.2 ml of RPMI 1640 were supplemented with 10% FBS. 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 percent specific 51Cr release (percent experimental 51Cr release minus percent 51Cr release from target cells alone). To examine the role of FasL for cytotoxic activities, anti-murine FasL inhibitory mAb (FLIM58) (27) was used. FLIM58 neutralizes mouse but not human FasL activity (27).
Sequential activation of caspase 1-like and caspase 3-like proteases
The caspase 1 (IL-1-converting enzyme)- and caspase 3 (CPP32)-like activity in anti-Fas Ab treated with MSG cell extracts was determined using fluorescent substrate (28). Cell lysates were diluted with 0.5 ml of IL-1-converting enzyme standard buffer and incubated at 30°C for 30 min with 1 µM fluorescent substrate. The caspase inhibitors z-VAD-fmk or Ac-DEVD-CHO were added to the reaction mixture at a concentration of 1 µM. Specific caspase 1- and caspase 3-like activities were determined by subtracting the values obtained in the presence of inhibitors. The fluorescent substrates, MOCAc-YVAD(dnp)-NH2 and MOCAc-DEVD(dnp)-NH2 were custom-synthesized at the Peptide Institute (Osaka, Japan). The fluorescence of the cleaved substrates was determined using a spectrofluorometer set at an excitation wavelength of 328 nm and an emission wavelength of 393 nm.
In vivo administration of caspase inhibitors
To examine the therapeutic effects of i.v. injection of caspase
inhibitors, a 1 mg/ml (50 µg/head) z-VAD-fmk (n = 11)
and Ac-DEVD-CHO (n = 9) in DMSO were injected i.v.
three times per week into 3d-Tx NFS/sld mice from 4 to 7 wk,
because autoimmune lesions in the salivary and lacrimal glands start to
develop at 3 wk of age. Dose of caspase inhibitor was determined
according to the previous report (29). Mice were examined
histopathologically at 8 wk and compared with 3d-Tx NFS/sld
mice injected with DMSO alone (n = 6). In addition,
autoantibody production against the 120-kDa -fodrin in serum was
tested in both treated and nontreated mice. Detection of tear and
saliva volume of the treated mice and control SS animal model of 3d-Tx
NFS/sld mice was done according to a modified method as
described (26).
In vivo immunization with autoantigen
Recombinant -fodrin protein identical with a 120-kDa cleavage
product (JS-1, 5 µg/ml) (6) emulsified with CFA (Difco,
Detroit, MI) was administerred twice s.c. into syngeneic normal
NFS/sld mice at 4 and 6 wk (n = 12). At 4
and 8 wk after the s.c. injection, mice were examined on
histopathological and immunological analysis. For controls, mice
injected with rat brain -fodrin (5 µl/ml; n = 10),
recombinant -fodrin protein encoding full-length -fodrin (JS-1,
1–1784 bp; 2.7A, 2258–4884 bp; 3'DA, 3963–7083 bp; a mixture of 5
µg/ml) (n = 10) (6), lysozyme (5
µg/ml; n = 10), GST emulsified with CFA (50
µl/head; n = 10), and CFA alone (50 µl/head;
n = 10) were examined. Rat brain -fodrin was
purified according to the method described previously
(30).
Proliferative T cell responses
Single cell suspensions of spleen cells from treated and control
mice were cultured in 96-well flat-bottom microtiter plates (5 x
105 cells/well) in RPMI 1640 containing 10% FCS,
penicillin/streptomycin, and 2-ME. Cells were cultured with recombinant
-fodrin (JS-1, 5 µg/ml), rat brain -fodrin (5 µg/ml),
lysozyme (5 µg/ml, Sigma-Aldrich), and OVA (5 µg/ml,
Sigma-Aldrich). During the last 8 h of the 72-h culture period, 1
µCi of [3H]thymidine was added per well, and
the incorporated radioactivity was determined using an automated beta
liquid scintillation counter.
ELISA
Serum autoantibodies were detected using recombinant -fodrin
(JS-1). After coating with the recombinant -fodrin in 96-well ELISA
plates, biotinylated anti-mouse IgG (Vector Laboratories) was added
as a second Ab. Measurements of specific autoantibodies were read by
automatic ELISA reader (Flow Laboratories, McLean, VA) at 492
nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histological and immunohistological characteristics of autoimmune
exocrinopathy in 3d-Tx NFS/sld SS model mice were described
in detail (22, 31, 32). To determine the possible
involvement of the apoptotic cascade in tissue destruction, we examined
apoptotic cells in the salivary gland specimens from 3d-Tx and non-Tx
NFS/sld mice. Immunohistology revealed that the majority of
tissue-infiltrating lymphoid cells in the salivary glands bear FasL in
the SS model (Fig. 1
A) and
that epithelial duct cells express Fas Ag on their cell surface (Fig. 1B). We found that tissue-infiltrating
CD4+ T cells isolated from the affected glands
bear a large proportion of FasL (>85%), compared with
CD8+ T cells bearing FasL on flow cytometry
(<23%; p < 0.01; Fig. 1E). A minor
proportion of infiltrating CD4+ T cells express
Fas (<31%), and CD8+ T cells bearing Fas were
negligible (<5%). Primarily cultured MSG cells isolated from 3d-Tx,
non-Tx NFS/sld and C57BL/6 mice constitutively express Fas
with high proportion (51–60%) on flow cytometry (Fig. 2A). Immunohistochemically,
epithelial duct cells in non-Tx NFS/sld and C57BL/6 salivary
glands are positive for Fas (Fig. 2B). RT-PCR analysis
demonstrated that Fas mRNA was constitutively present in the salivary
glands of the SS model, non-Tx NFS/sld, and normal C57BL/6
mice (Fig. 2C). MSG cells isolated from these mice did not
express FasL on flow cytometric analysis (Fig. 2D). A
significant increase of TUNEL+-apoptotic
epithelial duct cells in the salivary glands was observed in SS model
mice, compared with those in non-Tx NFS/sld and C57BL/6 mice
at all ages (Fig. 3, A–C). We next investigated whether
tissue-infiltrating T cells are responsible for tissue destruction as
judged by in vitro 51Cr release cytotoxic assay
against MSG cells. Infiltrating CD4+ T cells, but
not CD8+ T cells, identified significant
51Cr release against MSG cells (Fig. 3D). These cytotoxic activities were almost entirely
inhibited by incubation with anti-murine neutralizing FasL mAb
(FLIM58, 1 µg/ml) (Fig. 3D). No significant cytotoxicities
were found in splenic CD4+ and
CD8+ T cells toward MSG cells.
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-fodrin by apoptotic proteases
We examined the in vitro cleavage of -fodrin in MSG cells
induced by anti-Fas mAb (Jo2, 300 ng
ml-1). Anti-Fas mAb-stimulated apoptosis in MSG
cells was confirmed by flow cytometry of DNA content of nuclei
with propidium iodide and annexin V (Fig. 4A). Western blot analysis
demonstrated that the 240-kDa -fodrin in apoptotic MSG cells was
cleaved to smaller fragments into 120 kDa in a time-dependent manner,
and the cleavage was entirely blocked by preincubation with caspase
inhibitors (z-VAD-fmk and DEVD-CHO) (Fig. 4B). Protease
inhibitor mixtures, cysteine protease inhibitors (E64), and serine
protease inhibitor (leupeptin) had no significant effect on 120-kDa
-fodrin cleavage in apoptotic MSG cells (Fig. 4B).
The 113-kDa PARP in apoptotic MSG cells was not cleaved to smaller
fragments. We next investigated whether cysteine proteases are involved
in -fodrin cleavage on apoptotic MSG cells. The caspase 1- and
caspase 3-like activities in anti-Fas mAb-stimulated MSG cell extracts
were determined using fluorescent substrates, and caspase inhibitors
(z-VAD-fmk and DEVD-CHO) inhibited these activities at different doses
(0.2, 2, and 20 µM) (Fig. 4C).
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We investigated whether the i.v. injection of caspase inhibitors
protects animals against the development of autoimmune lesions.
Treatment with i.v. injection of both z-VAD-fmk and
DEVD-CHO (three times per week) (p < 0.005)
prevented the development of autoimmune lesions in the salivary and
lacrimal glands (Fig. 5, A and
B). The average saliva and tear volume of the treated SS
animal model was significantly higher than that of the control group
(Fig. 5C). A significant decrease of autoantigen-specific T
cell proliferation was observed in spleen cells from treated mice (Fig. 5D). In addition, serum autoantibody production against
120-kDa -fodrin was clearly inhibited by the treatment with caspase
inhibitors (Fig. 5E).
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To examine the autoimmune nature of 120-kDa -fodrin,
recombinant -fodrin protein identical with an autoantigen was
administerred s.c. into normal NFS/sld mice at 4 wk.
Organ-specific autoimmune lesions similar to SS developed at 8 wk after
the injection in almost all mice immunized with autoantigen, but not in
all groups of control (Fig. 6A; Table I). No inflammatory lesions were observed
in other organs. A majority of infiltrating cells were
CD4+ and FasL+, and the
epithelial duct cells express Fas on their cell surface (Fig. 6A). A minor proportion of CD8+ and
Fas+ infiltrating cells was observed (data not
shown). A specific cleavage of -fodrin into 120 kDa was detected in
the salivary glands of immunized mice, but not in controls (Fig. 6B). The activation markers (CD44high,
CD45RBlow, Mel-14low) were
up-regulated in spleen cells gated on CD4 from immunized mice, compared
with controls (Fig. 6C). Mice injected with recombinant
autoantigen showed a significant increase of autoantigen-specific T
cell proliferation in spleen cells (Fig. 6D). A high titer
of serum autoantibodies against 120-kDa -fodrin was detected in
immunized mice, compared with control mice, by ELISA (Fig. 6E). These data strongly suggest that a cleavage product of
120-kDa -fodrin is a pathogenic autoantigen on the development of
murine primary SS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We reported previously that a cleavage product of 120-kDa -fodrin
may be an important autoantigen in the development of primary SS, and
anti-120-kDa -fodrin Abs have been frequently detected in sera
from patients (6). Because it was shown that the fodrin
subunit is cleaved in association with apoptosis and the 120-kDa
fragment is a breakdown product of the mature form of 240-kDa fodrin
subunit (11, 12), we examined the in vitro cleavage of
-fodrin using primarily cultured MSG cells. We clearly detected
120-kDa -fodrin in anti-Fas-induced apoptotic MSG cells by
Western blotting. We found that a significant increase of
TUNEL+-apoptotic epithelial duct cells in the
salivary glands was detected in the 3d-Tx NFS/sld SS model
compared with those in non-Tx NFS/sld mice at all ages. MSG
cells constitutively express Fas with high proportion, and
tissue-infiltrating CD4+ T cells isolated from
the salivary gland tissues of the SS model mice bear a large proportion
of FasL. Moreover, we confirmed that tissue-infiltrating
CD4+ T cells, but not CD8+
T cells, are responsible for tissue destruction as judged by in vitro
51Cr release cytotoxic assay against MSG cells in
vitro. These cytotoxic activities were inhibited by incubation with
anti-murine neutralizing FasL mAb. Although it has been reported
that Fas-induced apoptosis seems to be the major killing pathway of the
CD4+ cytotoxic T cells (41), our
data suggest that one mechanism by which activated
CD4+ T cells induce cytotoxicity toward salivary
gland cells in the murine SS model is Fas based. When we investigated
whether cysteine proteases are involved in -fodrin cleavage, the
caspase 1- and caspase 3-like activities in anti-Fas Ab-treated MSG
cell extracts were determined using fluorescent substrates. In
apoptotic MSG cells, caspase inhibitors (z-VAD-fmk and DEVD-CHO)
inhibited the formation of 120-kDa -fodrin, whereas protease
inhibitor mixtures, other cysteine protease inhibitors (E64), and
serine protease inhibitor (leupeptin) had no effect on 120-kDa
-fodrin cleavage in apoptotic MSG cells. Furthermore, the treatment
of the murine SS model with i.v. injection of z-VAD-fmk and DEVD-CHO
prevented the development of autoimmune conditions, resulting in
restoration of saliva and tear secretion. These results suggest that
increased activity of the caspase cascade is involved in the
progression of -fodrin proteolysis during the initial stages of the
development of primary SS. Moreover, we obtained experimental evidence
that immunization with recombinant -fodrin protein identical with an
autoantigen is sufficient to induce autoimmune SS lesions in normal
recipients, but not with rat brain -fodrin or with recombinant
full-length -fodrin. These results indicate that caspase-mediated
-fodrin cleavage into the 120-kDa fragment plays a critical role in
the development of autoimmmune exocrinopathy in primary SS and suggest
that the primary mediators of the disease are autoantigen-driven T cell
responses.
When human T cell leukemia CEM cells were induced to undergo
apoptosis, the 240-kDa -fodrin was cleaved to a single detectable
fragment of 120 kDa (42). It is plausible that the 120-kDa
fragment is a breakdown product of the 150-kDa -fodrin cleavage
(9). There is increasing evidence that the cascade of caspases
is a critical component of the cell death pathway
(43, 44, 45), and a few proteins have been found to be cleaved
during apoptosis. These include PARP, a small U1 nuclear
ribonucleoprotein, and -fodrin, which were subsequently identified
as substrates for caspases (35, 36, 37). We provided evidence
that -fodrin is cleaved by one or more members of caspases during
apoptotic cell death in SS salivary glands. Fodrin cleavage by caspases
can potentially lead to cytoskeletal rearrangement, and it is of
interest to point out that -fodrin binds to ankylin, which contains
a cell death domain (46). In contrast, it has been
shown that cleavage products of -fodrin inhibit ATP-dependent
glutamate and 
-aminobutyric acid accumulation into synaptic vesicles
(47), supposing that a cleavage product of 120-kDa
-fodrin could be a novel component of an unknown immunoregulatory
network such as cytolinker proteins (48).
Taken together, these results are strongly suggestive of essential
roles of caspase cascade for -fodrin cleavage leading to tissue
destruction in autoimmune exocrinopathy of primary SS. Moreover, in
vivo preventive effects against autoimmune lesions treated with caspase
inhibitor have important implications for testing useful therapies.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yoshio Hayashi, Department of Pathology, Tokushima University School of Dentistry, 3 Kuramotocho, Tokushima 770, Japan. E-mail address: hayashi{at}dent.tokushima-u.ac.jp
3 Abbreviations used in this paper: SS, Sjögren’s syndrome; PARP, poly(ADP-ribose) polymerase; FasL, Fas ligand; z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone; 3d-Tx, thymectomy performed on day 3 after birth; non-Tx, nonthymectomied; MSG, mouse salivary gland; FLIM, FasL inhibitory mAb; DEVD, N-acetyl-Asp-Glu-Val-Asp-al.
Received for publication December 6, 2001. Accepted for publication May 3, 2002.
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References |
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-fodrin as a candidate autoantigen in primary Sjögren’s syndrome. Science 276:604.-spectrin. J. Biol. Chem. 264:5826.-spectrin breakdown by calpain and interleukin 1-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochem. J. 319:683.
-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis. J. Biol. Chem. 273:15540.-fodrin during Fas- and tumor necrosis factor-induced apoptosis is mediated by an interleukin-1-converting enzyme/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. J. Biol. Chem. 271:31277.II- and II-spectrin by caspase 3 (CPP32) in apoptotic cells. J. Biol. Chem. 273:22490., a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81:801.[Medline]
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-aminobutyric acid accumulation into synaptic vesicles: purification and initial characterization. Proc. Natl. Acad. Sci. USA 94:4137.This article has been cited by other articles:
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N. Kuroda, T. Mitani, N. Takeda, N. Ishimaru, R. Arakaki, Y. Hayashi, Y. Bando, K. Izumi, T. Takahashi, T. Nomura, et al. Development of Autoimmunity against Transcriptionally Unrepressed Target Antigen in the Thymus of Aire-Deficient Mice J. Immunol., February 15, 2005; 174(4): 1862 - 1870. [Abstract] [Full Text] [PDF]
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 K. Takada, M. Takiguchi, A. Konno, and M. Inaba Autoimmunity against a Tissue Kallikrein in IQI/Jic Mice: A MODEL FOR SJOGREN'S SYNDROME J. Biol. Chem., February 4, 2005; 280(5): 3982 - 3988. [Abstract] [Full Text] [PDF]
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 T. Maruyama, I. Saito, Y. Hayashi, E. Kompfner, R. I. Fox, D. R. Burton, and H. J. Ditzel Molecular Analysis of the Human Autoantibody Response to {alpha}-Fodrin in Sjogren's Syndrome Reveals Novel Apoptosis-Induced Specificity Am. J. Pathol., July 1, 2004; 165(1): 53 - 61. [Abstract] [Full Text] [PDF]
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 A. Rosen and L. Casciola-Rosen ALTERED AUTOANTIGEN STRUCTURE IN SJoGREN'S SYNDROME: IMPLICATIONS FOR THE PATHOGENESIS OF AUTOIMMUNE TISSUE DAMAGE Critical Reviews in Oral Biology & Medicine, May 1, 2004; 15(3): 156 - 164. [Abstract] [Full Text] [PDF]
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N. Ishimaru, R. Arakaki, M. Watanabe, M. Kobayashi, K. Miyazaki, and Y. Hayashi Development of Autoimmune Exocrinopathy Resembling Sjogren's Syndrome in Estrogen-Deficient Mice of Healthy Background Am. J. Pathol., October 1, 2003; 163(4): 1481 - 1490. [Abstract] [Full Text] [PDF]
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