The role of caspase
cascade on the development of primary Sjögren's
syndrome
Yoshio Hayashi, Rieko Arakaki,
and Naozumi Ishimaru
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Department of Pathology, Tokushima University
School of Dentistry, Tokushima, Japan
Abstract:Primary Sjogren syndrome (SS) is an autoimmune disease characterized by diffuse lymphoid cell infiltrates in the salivary and lacrimal glands, resulting in symptoms of dry eye and dry mouth due to insufficient secretion. Previously, we have identified the 120 kDa α-fodrin as an important autoantigen on the development of SS in both animal model and SS patients, but the mechanism of α-fodrin cleavage leading to tissue destruction in SS remains unclear. In murine primary SS model, tissue-infiltrating CD4+ T cells purified from the salivary glands bear a large proportion of Fas ligand (FasL), and the salivary gland duct cells constitutively possess Fas. Infiltrating CD4+ T cells identified significant 51Cr release against mouse salivary gland (MSG) cells. In vitro studies demonstrated that apoptotic MSG cells result in a specific α-fodrin cleavage into 120 kDa, and preincubation with caspase-inhibitor peptides blocked α-fodrin cleavage. The treatment with ca spase-inhibitors in vivo prevented the development of autoimmune lesions in the salivary and lacrimal glands. Thus, an increased activity in caspase cascade may be involved in the progression of α-fodrin proteolysis and tissue destruction on the development of SS.
J. Med. Invest. 50:32-38, 2003
Keywords:Sjögren's syndrome;autoantigen;caspase;apoptosis
INTRODUCTION
Primary Sjogren's syndrome (SS) is an autoimmune disorder characterized
by lymphocytic infiltrates and destruction of the salivary and lacrimal
glands, and systemic production of autoantibodies to the ribonucleoprotein
(RNP) particles SS-A/Ro and SS-B/La (1-4). The spectrum of presentation
of the disease is broad, ranging from the organ-localized dysfunction of
exocrine gland to systemic complications such as liver, kidney and lung
involvement (5). Although it has been assumed that a combination of
immunologic, genetic, and environmental factors may play a key role on
the development of autoimmune lesions, little is known about the disease
pathogenesis. Autoimmune diseases are characterized by tissue destruction
and functional decline due to autoreactive T cells that escape self-tolerance
(6, 7). Although the specificity of cytotoxic T lympyocyte (CTL) function
has been an important issue of organ-specific autoimmune response, the
mechanisms responsible for tissue destruction in SS remain to be
elucidated. The histopathological changes in the minor salivary gland
biopsy are characterized by focal and/or diffuse lymphoid cell infiltrates
and parenchymal destruction. The majority of lymphoid cells in the salivary
biopsy are CD4+ T cells with a small proportion of CD8+ T cells
(2). These T cells express the α β antigen receptor and
cell surface antigens associated with mature memory T cells. Since
it was evident a preferential use of specific variable region segments
of the antigen receptor β chain by salivary gland T cells (8),
it has been assumed that a unknown organ-specific autoantigen targeted
by autoreactive T cells may be present in the salivary glands. We have
established and analyzed an animal model for primary SS in NFS/sld
mutant mice thymectomized 3 day after birth (3d-TX)(9-20). When the
repertoire of T cell receptor (TCR) Vβ genes transcribed and
expressed within the inflammatory infiltrates was analyzed in an
animal model, a preferential utilization of TCR Vβ gene was
detected in these lesions from the onset of disease (10). We have
previously identified a 120 kDa organ-specific autoantigen from
the salivary gland tissues of this animal model (21). The sequence
of the first 20 NH2-terminal residues was found to be identical
to that of cytoskeletal protein human α-fodrin (21).
Furthermore, sera from patients with SS reacted positively with
purified 120 kDa antigen, and proliferative response of peripheral
blood lymphocytes (PBMC) from SS patients to the purified
autoantigen was detected, but not from SLE or RA patients, and
healthy controls. These results indicate that the anti-120 kDa
α-fodrin immune response plays an essential role on the
development of primary SS. Recent reports have demonstrated
evidences that caspase 3 is required for α-fodrin cleavage
during apoptosis (22-24). In Jurkat cells, caspase 3-like proteases
have been reported to cleave α-fodrin and poly (ADP-ribose)
polymerase (PARP) but with differential sensitivity to the caspase
3 inhibitor, DEVD-CHO (24). We speculate that an increase in the
enzymatic activity of apoptotic proteases is involved in the
progression of α-fodrin proteolysis during development of SS.
Involvement of Fas and FasL in tissue destruction
It is now clear that the interaction of Fas with FasL regulates
a large number of pathophysiological process of apoptosis
(25, 26). We speculate that an increase in the enzymatic activity
of apoptotic proteases is involved in the progression of α-fodrin
proteolysis during development of SS. To determine the possible
involvement of Fas and FasL in tissue destruction of SS, we
first analyzed Fas expression in the salivary gland specimens
of 3d-thymectomized (3d-Tx) NFS/sld mouse model (10) and in
the mouse salivary gland cells (MSG) isolated from non-thymectomized
(non-Tx) NFS/sld mice. Immunohistology revealed that the majority
of tissue-infiltrating lymphoid cells in the salivary glands
bear FasL in SS model, and epithelial duct cells express Fas
antigen on their cell surface. 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. 1A). 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. 1B). Immunohistochemically,
epithelial duct cells in non-Tx NFS/sld and C57BL/6 salivary
glands are positive for Fas. RT-PCR analysis demonstrated
that Fas mRNA was constitutively present in the salivary glands
of SS model, non-Tx NFS/sld, and normal C57BL/6 mice. MSG
cells isolated from these mice did not express FasL on flow
cytometric analysis. 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. 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. These cytotoxic activities
were almost entirely inhibited by incubation with anti-murine
neutralizing FasL mAb (FLIM58:1µg/ml), indicating that
the cytotoxicity by activated CD4+ T cells towards salivary
gland epithelial cells was Fas-based.
Participation of caspases in α-fodrin cleavage
To confirm the organ-specificity of a cleavage product of
α-fodrin, we investigated various strains of mice with
salivary gland destruction, such as MRL/lpr, nonobese diabetic
(NOD) mice, in addition to 3d-TX NFS/sld mice. Protein immunoblot
analysis demonstrated that the120 kDa α-fodrin was
detected in these affected glands, but not in normal mice.
We examined the in vitro cleavage of α-fodrin using 240
KDa α-fodrin in MSG cells. Anti-Fas Ab-induced apoptosis
was confirmed by FACS analysis using in situ TUNEL procedure,
and DNA laddering and formation. We could detect the 120 kDa
α-fodrin in apoptotic MSG cells on immunoblotting. We
examined the in vitro cleavage of α-fodrin in MSG cells
induced by anti-Fas mAb (Jo2:300 ngml-1). Anti-Fas mAb-stimulated
apoptosis in MSG cells was confirmed by flow cytometry of DNA
content of nuclei with PI and Annexin V. Western blot analysis
demonstrated that the 240 kDa α-fodrin in apoptotic MSG
cells was cleaved to smaller fragments into 120 kDa on
time-dependent manner, and the cleavage was entirely blocked
by preincubation with caspase inhibitors (z-VAD-fmk, DEVD-CHO)
(Fig. 2A). Protease inhibitor cocktails, cysteine protease
inhibitors (E64), and serine protease inhibitor (Leupeptin)
had no significant effect on 120 kDa α-fodrin cleavage
in apoptotic MSG cells (Fig. 2A). The 113 kDa PARP in apoptotic
MSG cells was not cleaved to smaller fragments. We next
investigated whether cysteine proteases are involved in
α-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
(27), and caspase inhibitors (z-VAD-fmk, DEVD-CHO) inhibited
these activities at different dose (0.2, 2, and 20 µM)
(Fig. 2B).
Preventive effect of caspase inhibitors in vivo
We next examined whether α-fodrin cleavage to120 kDa
fragment on apoptotic human salivary gland cells (HSG)(28)
could be blocked by preincubation with specific protease inhibitors.
In apoptotic HSG cells, calpain inhibitor peptide and caspase
inhibitor (Z-VAD-fmk) had partially blocked 120 kDa α-fodrin
formation. Moreover, a combination of calpain inhibitor peptide
and caspase inhibitors (Z-VAD-fmk and Z-DEVD-fmk) almost entirely
inhibited the formation of 120 kDa α-fodrin. Protease
inhibitor cocktails, other cysteine protease inhibitors (E64),
and serine protease inhibitor (Leupeptin) had no effect on
120 kDa α-fodrin cleavage in apoptotic HSG cells. By
immunohistochemistry using polyclonal Ab against synthetic
120 kDa α-fodrin, a cleavage product of α-fodrin
was present exclusively in epithelial duct cells of the labial
salivary gland biopsies from SS patients, but not in control
individuals. Protein immunoblot analysis confirmed the same
results. This indicates that a cleavage product of 120 kDa
α-fodrin is present in the diseased glands with human
SS, but not in control glands. We further investigated whether
the i.v. injection of caspase-inhibitors protects SS animal
model against the development of autoimmune lesions. The both
treatment with i.v. injection of z-VAD-fmk and DEVD-CHO (3
times per week) (P<0.005) prevented the development of
autoimmune lesions in the salivary and lacrimal glands. The
average saliva and tear volume of the treated SS animal model
was significantly higher than that of the control group. A
significant decrease of autoantigen-specific T cell proliferation
was observed in spleen cells from treated mice. In addition,
serum autoantibody production against 120 kDa α-fodrin
was clearly inhibited by the treatment with caspase-inhibitors.
The treatment of 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 suggst that increased activity of caspase cascade
is involved in the progression of α-fodrin proteolysis
during the initial stages on the development of primary SS.
Autoimmune lesions induced by immunization with autoantigen
To examine the autoimmune nature of 120 kDa α-fodrin, recombinant
α-fodrin protein identical to an autoantigen was administerred
subcutaneously (s.c.) into normal NFS/sld mice at 4 wks. Organ-specific
autoimmune lesions similar to SS developed at 8 wks after the injection
in almost all mice immunized with autoantigen, but not in all groups of
control (19). 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. A specific cleavage of
α-fodrin into 120 kDa was detected in the salivary glands of
immunized mice, but not in controls. Mice injected with recombinant
autoantigen showed a significant increase of autoantigen-specific T
cell proliferation in spleen cells. A high titer of serum autoantibodies
against 120 kDa α-fodrin was detected in immunized mice, compared
with control mice by ELISA. These data demonstrated evidences that a
cleavage product of 120 kDa α-fodrin is pathogenic autoantigen
on the development of murine primary SS.
Concluding remarks
There is increasing evidences that the cascade of caspases is a critical
component of the cell death pathway (29-31), and a few proteins have been
found to be cleaved during apoptosis. We provided evidence that α-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
cytoskeltal rearrangement, and it is of interest to point out that α-fodrin
binds to ankylin, which contains a cell death domain (32). It has been shown
that cleavage products of α-fodrin inhibit ATP-dependent glutamate and
&ganma;-aminobutyric acid accumulation into synaptic vesicles (33), supposing
that a cleavage product of 120 kDa α-fodrin could be a novel component
of an unknown immunoregulatory networks such as cytolinker proteins (34).
These results are strongly suggestive of essential roles of caspase cascade
for α-fodrin cleavage leading to tissue destruction in autoimmune
exocrinopathy of primary SS.
REFERENCES
1.Bloch KJ, Buchanan WW, Wohl MJ, Bunim J:Sjögen's syndrome.
A clinical, pathological and serological study of sixty-two
cases. Medicine 44:187-231, 1965.
2.Fox RI, Saito I:Sjögren's syndrome. Clinical Immunology-Principles
and Practice. Mosby, 1145-1153, 1995.
3.Fox RI, Robinson CA, Curd JG, Kozin F, Howell FV:Sjögren's
syndrome. Proposed criteria for classification. Arthritis
Rheum 29:577-585, 1986
4.Chan EK, Hamel JC, Buyon JP, Tan ET:Molecular definition
and sequence motifs of the 52-kD component of human SS-A/Ro
autoantigen. J Clin Invest 87:68-76, 1991.
5.Kruize AA, Smeenk RJT, Kater L:Diagnostic criteria and immunopathogenesis
of Sjögren's syndrome:implications for therapy. Immunol
Today 16:557-559, 1995.
6.Gianani R, Satventnick N:Virus, cytokine, antigens, and
autoimmunity. Proc Natl Acad Sci USA 93:2252-2259, 1989.
7.Feldmann M, Bennan FM, Maini, RN:Rheumatoid arthritis. Cell
85:307-310, 1996.
8.Sumida T, Yonaha F, Maeda T, Tanabe E, Koike T, Tomioka
H, Yoshida S:T cell receptor repertoire of infiltrating T
cells in lips of Sjögren's syndrome patients. J Clin
Invest 89:681-685, 1992.
9.Hayashi Y, Kojima A, Hata M, Hirokawa K:A new mutation involving
the sublingual gland in NFS/N mice. Am J Pathol 132:187-191,
1988.
10.Haneji N, Hamano H, Yanagi K, Hayashi Y:A new animal model
for primary Sjögren's syndrome in NFS/sld mutant
mice. J Immunol153:2769-2777, 1994.
11.Hayashi Y, Haneji N, Hamano H, Yanagi K, Takahashi M, Ishimaru
N:Effector mechanism of experimental autoimmune sialadenitis
in the mouse model for primary Sjogren's syndrome. Cell Immunol
171:217-225, 1996.
12.Takahashi M, Mimura Y, Hamano H, Haneji N, Yanagi K, Hayashi
Y:Mechanism of the development of autoimmune dacryoadenitis
in the mouse model for primary Sjögren's syndrome.
Cell Immunol 170:54-62, 1996.
13.Ishimaru N, Saegusa K, Yanagi K, Haneji N, Saito I, Hayashi
Y:Estrogen dediciency accelerates autoimmune exocrinopathy
in murine Sjögren's syndrome through Fas-mediated
apoptosis. Am J Pathol 155:173-181, 1999.
14.Ishimaru N, Yoneda T, Saegusa K, Yanagi K, Haneji N, Moriyama
K, Saito I, Hayashi Y:Severe destructive autoimmune lesions
with aging in murine Sjögren's syndrome through Fas-mediated
apoptosis. Am J Pathol 156:1557-1564, 2000.
15.Saegusa K, Ishimaru N, Yanagi K, Haneji N, Nishino M, Azuma
M, Saito I, Hayashi Y:Treatment with anti-CD86 costimulatory
molecule prevents the autoimmune lesions in murine Sjögren's
syndrome (SS) through up-regulated Th2 response. Clin Exp
Immunol 119:354-360, 2000.
16.Saegusa K, Ishimaru N, Haneji N, Yanagi K, Yoneda T, Saito
I, Hayashi Y:Mechanisms of neonatal tolerance induced in an
animal model for primary Sjogren's syndrome by intravenous
administration of autoantigen. Scand J Immunol 52:264-270,
2000.
17.Saegusa K, Ishimaru N, Yanagi K, Haneji N, Nishino M, Azuma
M, Saito I, Hayashi Y:Autoantigen-specific CD4+CD28low T cell
subset prevents autoimmune exocrinopathy in murine Sjogren's
syndrome. J Immunol 165:2251-2257, 2000.
18.Ishimaru N, Yanagi K, Ogawa K, Suda T, Saito I, Hayashi
Y:Possible role of organ specific autoantigen for Fas ligand-mediated
activation-induced cell death in murine Sjögren's
syndrome. J Immunol 167:6031-6037, 2001.
19.Saegusa K, Ishimaru N, Yanagi K, Mishima K, Arakaki R,
Suda T, Saito I, Hayashi Y:Prevention and induction of autoimmune
exocrinopathy is dependent on pathogenic autoantigen cleavage
in murine Sjogren's syndrome. J Immunol 169:1050-1057, 2002.
20.Saegusa K, Ishimaru N, Yanagi K, Arakaki R, Ogawa K, Saito
I, Katunuma N, Hayashi Y:Cathepsin S inhibitor prevents autoantigen
presentation and autoimmunity. J Clin Invest110:361-369, 2002.
21.Haneji N, Nakamura T, Takio K, Yanagi K, Higashiyama H,
Saito I, Noji S, Sugino H, Hayashi Y:Identification of α-fodrin
as a candidate autoantigen in primary Sjögren's syndrome.
Science 276:604-607, 1997.
22.Nath R, Raser KJ, Stafford D, Hajimohammadreza I, Posner
A, Allen H, Talanian RV, Yuen P-W, Gilbertsen RB, Wang KK:Non-erythroid
α-spectrin breakdown by calpain and interleukin
1β-converting-enzyme-like protease (s) in apoptotic
cells: contributory roles of both protease families in neuronal
apoptosis. Biochem J 319:683-690, 1996.
23.Janicke RU, Sprengart ML, Porter AG:Caspase-3 is required
for alpha-fodrin cleavage but dispensable for cleavage of
other death substrates in apoptosis. J Biol Chem 273:15540-15545,
1996.
24.Cryns VL, Bergeron L, Zhu H, Li H, Yuan J:Specific cleavage
of alpha-fodrin during Fas- and tumor necrosis factor-induced
apoptosis is mediated by an interleukin-1beta-converting enzyme/Ced-3
protease distinct from the poly (ADP-ribose) polymerase protease.
J Biol Chem 271:31277-31282, 1996.
25.Brunner T, Mogll RJ, LaFace D, Yoo NJ, Mahboubl A, Echeverri
F, Martin SJ, Force WR, Lynch DH, Ware CF, Green DR:Cell-autonomous
Fas(CD95)/Fas-ligand interaction mediates activation-induced
apoptosis in T cell hybridomas. Nature 373:441-444, 1995.
26.Ju S-T, Panka DJ, Cui H, Ettinger R, El-Khatib M, Sherr
DH, Stanger BZ, Marshak-Rothstein A:Fas(CD95)/FasL interactions
required for programmed cell death after T-cell activation.
Nature 373:444-448, 1995.
27.Enari M, Talanian RV, Wong WW, Nagata S:Sequential activation
of ICE-like and CPP32-like proteases during Fas-mediated apoptosis.
Nature 380:723-726, 1996.
28.Shirasuna K, Sato M, Miyazaki T:A neoplastic epithelial
duct cell line established from an irradiated human salivary
gland. Cancer 48:745-752, 1981.
29.Holtzman DM, Deshmukh M:Caspases:A treatment target for
neurodegenerative disease? Nature Med 3:954-955, 1997.
30.Rudel T, Bokoch GM. Membrane and morphological changes
in apoptotic cells regulated by caspase-mediated activation
of PAK2. Science 276:1571-1574, 1997.
31.Huang S, Jiang Y, Li Z, Nishida E, Mathias P, Lin S, Ulevitch
RJ, Nemerow GR, Han J:Apoptosis signaling pathway in T cells
is composed of ICE/Ced-3 familiy proteases and MAP kinase
kinase 6β. Immunity 6:739-749, 1997.
32.Feinstein E, Kimchi A, Wallach D, Boldin M, Varfolomeev
E:The death domain:a module shared by proteins with diverse
cellular function. Trends Biochem Sci 20:342-344, 1995.
33.Ozkan ED, Lee FS, Ueda T:A protein factor that inhibits
ATP-dependent glutamate and &ganma;-aminobutyric acid
accumulation into synaptic vesicles:purification and initial
characterization. Proc Natl Acad Sci USA 94:4137-4142, 1997.
34.Brown MJ, Hallam JA, Yamada KM, Shaw S:Intergration of
human T lymphocyte cytoskelton by cytolinker protein. J Immunol
167:641-645, 2001.
Received for publication January 8, 2003;accepted Januay 26,
2003.
Address correspondence and reprint requests to Yoshio Hayashi,
Department of Pathology, Tokushima University School of Dentistry,
Kuramoto-cho, Tokushima 770-8504, Japan and Fax:+81-88-633-7327.
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