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Increased expression of human thioredoxinadult T cell leukemiaderived factor in Sjgren's syndrome.

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Vol. 39, No. 5 , May 1996. pp 773-782
0 1996, American College of Rheumatology
Objective. To determine the involvement of human thioredoxidadult T cell leukemia-derived factor
(TRWADF) in Sjogren’s syndrome (SS) and the correlation with Epstein-Barr virus (EBV).
Methods. Indirect immunohistochemical techniques and reverse transcriptase polymerase chain reaction were utilized to analyze TRX/ADF expression and
the presence of EBV, using 6 normal tissues and 23
surgical specimens. The kinetics of expression of TRW
ADF induced by EBV was examined in vitro with
peripheral blood B cells from EBV-seronegative donors.
Results. Marked expression of TRWADF was
found in the infiltrating B cells and the epithelial cells of
salivary gland tissues from patients with SS (11 of 12
cases), but not in those from patients with other salivary
gland inflammatory conditions (0 of 11cases) or those of
normal individuals (0 of 6 cases). In immunohistologic
analyses, a striking topographic correlation between
TRWADF and EBV was found. The coexistence of
TRWADF messenger RNA and EBV DNA was detected
by polymerase chain reaction (r = 0.75, P < 0.01).
Peripheral blood B cells from EBV-seronegativedonors
showed de novo synthesis of TRX/ADF following in vitro
infection with EBV. EBV-infected B cell lines all expressed TRX/ADF. TRWADF was not detected in nonEBV-infected cells. Tumors in SCID mice reconstituted
Supported by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Science, and Culture of Japan.
Ichiro Saito, DDS, PhD, Misa Shimuta, BS, Kumiko Terauchi, MS, Nobuyuki Miyasaka, MD: Tokyo Medical and Dental
University, Tokyo, Japan; Kazuo Tsubota, MD: Tokyo Dental
College, Chiba, Japan; Junji Yodoi, MD: Kyoto University, Kyoto,
Address reprint requests to Ichiro Saito, DDS, PhD, Ilivision of Immunological Diseases, Medical Research Institute, Tokyo
Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113, Japan.
Submitted for publication May 3 1, 1995; accepted in revised
form November 27, 1995.
with mononuclear cells of salivary glands from SS
patients, which were composed of human B cells carrying EBV DNA, were positive for TRWADF.
Conclusion. These findings suggest that TRX/
ADF expression closely reflects the intracellular event of
EBV reactivation in SS. This is also the first report to
show the ectopic in vivo expression of THWADF in
human autoimmune disease.
Infiltration of activated lymphocytes is commonly observed in the lesions of organ-specific autoimmune diseases. Sjogren’s syndrome (SS) is an autoimmune disease in which both T cells and B cells
infiltrate and progressively destroy the salivary and
lacrimal glands, leading to dry mouth and dry eyes. SS
can also manifest as a generalized systemic lymphoproliferative disorder, with a predisposition to terminate as malignant lymphoma (1). The etiology of SS is
obscure, with evidence implicating both environmental factors and a genetic predisposition (2-4).
Epstein-Ban virus (EBV) is a widely occumng
virus of the herpes family that infects the epithelial
cells of the salivary glands and oropharyngeal tissue,
and B cells. After the primary infection, the virus
remains latent in the host, and occasionally becomes
reactivated (5-1 1). Evidence for an association between EBV infection and SS has been accumulating.
EBV antigen and its DNA have been found in salivary
gland tissues of SS patients at levels well above the
background levels for latent infection (12-14). Infectious EBV is present in both the saliva of SS patients
(15) and culture supernatants of B cell lines established
from SS patients (16). SS patients have elevated levels
of antibodies against EBV RNA-protein complexes
and against EBV antigens (I 7,lS). Taken together,
these observations suggest that a reactivated EBV
infection may play a role in SS, contributing to the
initiation or perpetuation of an autoimmune response
in the target organs.
Adult T cell leukemia-derived factor (ADF)
was initially described as a factor in the induction of
interleukin-2 receptor (IL-2R)/p55, which in turn, is a
factor in the activation of CDCpositive T cells (19). It
has since been identified in T cells of patients with
adult T cell leukemia (ATL) who have evidence of
infection by human T lymphotropic virus type I
(HTLV-I) (19), and in HTLV-I-transformed T cells in
vitro (20), as well as in EBV-immortalized B cells (21).
ADF expression can also be induced by a variety of
stresses, including x-ray and ultraviolet irradiation,
hydrogen peroxide, mitogens, and phorbol myristate
acetate (22). The recombinant form of (rADF) enhances the activation and proliferation of lymphocytes
(23). IL-2Wp55 and the low-affinity Fc receptor for
IgE (CD23/Fc&II) are induced by rADF in sensitive
cell lines (21,24). As with the IL-2R system in ATL,
CD23/Fc&II is associated with a tyrosine kinase (25)
and is overexpressed in B cell lines infected with EBV
(21,25,26). Previous data indicate that the transfection
of ADF complementary DNA (cDNA) into YT cells, a
line of human large granular lymphocytes, enhances
promoter activity in the IL-2Wp55 system, perhaps via
interaction with nuclear factor KB (NF-KB) (22). ADF
demonstrates a significant homology to thioredoxin
(TRX), a prokaryotic thiol-reducing coenzyme whose
activities include the activation and proliferation of
lymphocytes (23, 27,28).
Based on these data, we investigated the expression of TRX/ADF in normal subjects and SS
patients and compared these results with EBV infection in the subjects. We also conducted studies in
which it was demonstrated that in vitro exposure to
EBV induces the expression of ADF.
Study subjects. All patients were seen at the Tokyo
Medical and Dental University Hospital or Ichikawa General
Hospital of Tokyo Dental College. Twelve patients with SS
underwent minor salivary gland biopsies for the differential
diagnosis of SS (29). Informed consent was obtained from all
patients. The biopsies revealed a focus score of >2, but did
not show evidence of lymphoma in routine histologic and
immunohistologicanalyses. Each of these patients exhibited
keratoconjunctivitis sicca with decreased tear production
(<5 mm on a Schirmer test with anesthesia, and <10 mm on
another Schirmer test with nasal stimulation 1301). Corneal
and conjunctival epithelia showed positive rose-bengal and
fluorescein staining. Decreased secretion of saliva was confirmed by gum test results of <5 ml. These patients had not
received glucocorticoids or immunosuppressive agents for at
least 6 months prior to biopsy. All patients exhibited elevated titers of rheumatoid factor (>1:160 by latex agglutination) and serum antinuclear antibodies, including anti-SS-A
or anti-SS-B. All patients were women, and their mean age
was 45 years (range 2145 years).
Normal salivary gland tissue was obtained from 3
individuals without sicca symptoms who were undergoing
minor salivary gland biopsies as part of the diagnostic
evaluation for other oral conditions. Histologically normal
salivary gland tissue was also obtained from 3 subjects at the
time of autopsy. Salivary gland tissue was also obtained at
the time of surgery in patients with sialolithiasis (n = 4),
acute suppurative sialoadenitis (n = 3), and ranula (n = 4), as
disease controls.
Immunohistologic studies. Unfixed tissue blocks were
placed in OCT compound (Miles, Naperville, IL), snapfrozen in liquid nitrogen, and stored at -80°C. Cryostat
sections (4 pm) from each block were transferred onto glass
slides for immunohistochemical analysis. Consecutive sections were subsequently placed in a sterile Eppendorf tube
for RNA extraction. Sections cut before and after the
sections obtained for RNA extraction were stained with
hematoxylin and eosin using the standard method. Acetonefixed (-2O"C, 10 minutes) sections placed on gelatin-coated
slides were incubated with murine monoclonal antibodies
directed against CD3, CD4, CD8, CD14, CD20, CD21, HLADR (Coulter, Hialeah, FL), cytokeratin (Becton Dickinson,
Mountain View, CA), factor VIII (Cedarlane, Ontario, Canada) or EBV-encoded latent membrane protein (LMP; Dakopatts, Glostrup, Denmark). Antibodies of the same isotype with irrelevant antibody activity were used as negative
controls. After rinsing, the sections were reacted with biotinylated goat anti-mouse (IgG + IgM) antibody (Tago, Burlingame, CA), followed by peroxidase-conjugated a v i d b
biotin complex (Vector Laboratories, Burlingame, CA) and
then the substrate 3,3'-diaminobenzidine. When rabbit antibody to ADF C-terminal peptide was used, the sections were
reacted with biotinylated goat anti-rabbit antibody (IgG,
heavy and light chain; Vector Laboratories), followed by
avidiwbiotin complex.
Preparation of antibody against ADF peptides. The
preparation of rabbit antibodies against synthetic T W A D F
C-terminal peptide has been described previously (31). This
C-terminal peptide corresponds to residues 76105 of the
complete ADF molecule, coded by the cDNA cloned by
Tagaya et al. (23). In order to define the specificity of the
antiserum against T W A D F , we screened a database of
protein primary structures to search for any that were
homologous to T W A D F C-terminal 29-mer peptide. As
expected, there was no significant homology to known
sequences, including the viral-encoded form of TRX (24).
The specific anti-ADF peptide was affinity purified using
Sepharose CL-4B coupled with a C-terminal 10-mer peptide
conjugated with bovine serum albumin (BSA), after absorption by BSA-Sepharose.
Cell separation and cell culture. Peripheral blood
mononuclear cells (PBMC) from SS patients and seronegative donors were obtained by centrifugation of heparinized
blood over Ficoll-Hypaque gradients. For some experiments, monocytes were depleted by 2 cycles of treatment
with carbonyl iron. To separate B cells, the cells were mixed
with 5% sheep erythrocytes in 20% newborn calf serum and
3% dextran T70 (Pharmacia, Uppsala, Sweden) to form
rosettes. After 45 minutes on ice, the rosetting and nonrosetting cells were separated by Ficoll-Hypaque gradient
centrifugation. The nonrosetting B cell fraction was incubated for 1-2 hours in horizontally placed tissue culture
bottles to remove adherent cells. Cells were placed in RPMI
1640 containing penicillin (100 unitdml), streptomycin (100
d m l ) , glutamine (10 mg/ml), and 10% fetal calf serum
(Gibco, Grand Island, NY). The isolated population expressed CD20 (>98.5% B cells), CD2 (<1% T cells), and
CD14 (<0.5% monocytes). Burkitt lymphoma Daudi and
BJAB cell lines were obtained from Dr. K. Yamamoto
(Tokyo Medical and Dental University). These cell lines
were cultured in RPMI 1640 supplemented with 10% fetal
calf serum (FCS; Gibco), 100 units/ml of penicillin, and 100
mglpl of streptomycin. Cells were isolated from salivary
gland tissues as previously reported (15). Briefly, the minced
tissues were treated with 0.5 mg/ml collagenase (Sigma,
St. Louis, MO) and 0.15 mg/ml DNase I (Sigma) for 2 hours
at 37°C.
EBV infection. The procedures followed for the preparation and titration of cell-free EBV (strain B95-8) were as
described previously (32). Purified B cells (1 X lo5) were
infected with EBV preparation with a titer of 2 x 10’
Epstein-Barr nuclear antigen-inducing units/ml. EBV preparations and culture medium tested for the presence of
endotoxin by the Limulus amebocyte assay (Sigma) were
found to contain <20 pg/ml of contaminating endotoxin.
RNA extraction. Briefly, aliquots of cells or frozen
sections from each block were placed in an Eppendorf tube
(2 ml), to which was added 400 pl of ice-cold lysis solution D
containing 4M guanidine thiocyanate, 25 mM sodium citrate,
pH 7.0, 0.1M 2-mercaptoethanol, 0.5% (weight/volume) sarcosyl, and diethyl pyrocarbonate (Aldrich Chemical, Milwaukee, WIbtreated water (DEPC-W). This was followed
by the addition of 400 pl of phenol, 40 pl of chloroform-isoamyl alcohol (49: 1, volume/volume), and 80 pl of 2N Naacetate, pH 4.0. The mixture was chilled on ice for 15
minutes and centrifuged at 12,000 revolutions per minute for
20 minutes at 4°C. The aqueous phase was treated with 400
pl of chloroform and centrifuged at 12,000 rpm for 10
minutes. The aqueous phase was recovered and mixed with
an equal volume of isopropanol at -20°C for at least 60
minutes. After centrifugation at 12,000 rpm for 20 minutes at
4”C, solution D and isopropanol were added to the pellets,
and incubation was carried out at -20°C for 60 minutes.
After repelleting by centrifugation, the RNA was washed
with 75% ethanol in DEPC-W and centrifuged at 12,000 rpm
for 30 minutes. The RNA was stored in DEPC-W at -20°C
until further processing.
Polymerase chain reaction (PCR) amplification. After
incubation of 100 ng of RNA at 65°C for 5 minutes followed
immediately by chilling on ice, the reaction mixture (20 units
of ribonuclease inhibitor; lakara, Kyoto, Japan), l o x PCR
buffer (500 mM KCI, 200 mM Tris HCI buffer, pH 8.4, 25
mM MgCl,, 1 mg/ml BSA), 1.25 mM dNTPs (dATP, dCTP,
dGTP, dTTP; Pharmacia LKB Biotechnology, Uppsala,
Sweden), lox hexanucleotide mixture (Boehringer, Mannheim, Germany), 0.1% dithiothreitol (Aldrich Chemical),
and 3 units reverse transcriptase (RT)-derived Rousassociated virus 2 (Takara) was added to the RNA solution,
and the mixture was then incubated at 42°C for 30 minutes.
Following incubation, it was heated at 94°C for 5 minutes
and chilled on ice. Synthesized cDNAs were diluted with
autoclaved distilled water to 100 pl and stored at -20°C. The
PCR assay was performed as described previously (33). The
sequences of the primers were specific, as confirmed by a
computer-assisted search of updated versions of GenBank,
and were chosen with balanced nucleotide compositions
ranging from 40% to 60% of the GC contents.
The following primers were used: TRX/ADF (sense
5’-TGGTGAAGCAGATCGAGAG-3’, antisense 5’-GACTAATTCATTAATGGTGG-3‘), generating a 40-basepair
PCR product, and pactin (sense 5‘-CCTTCCTGGGCATGGACiTCCTG-3‘, antisense 5‘-GGAGCAATGATCTTGAT
CTTC-3‘), generating a 202-bp PCR product. PCR products
were resolved on a 1.7% agarose gel and transferred to a
nylon membrane. Hybridization under high-stringency conditions was done with a TRX/ADF internal oligonucleotide
probe (5’-GGCTCCAGAAAATTCACCCA-3’). Blots were
then stripped and reprobed with an internal oligonucleotide
probe for pactin (5’-AAAGACCTGTACGCCAACA-3’) to
ascertain the amount of the total PCR product. The intensity
of signals was determined by laser densitometry. For each
sample, the amount of T W A D F messenger RNA (mRNA)
was quantitated relative to the respective level of pactin
mRNA. Data were obtained utilizing the following formula:
Optical density (OD) of TRXIADF
mRNA transcripts of
EBV-infected cells/OD of p-actin
mRNA transcripts of EBV-infected cells
Kelative mRNA =
OD of TRX/ADF mRNA transcripts of
EBV-uninfected cells/OI) of
p-actin mRNA transcripts
of EBV-uninfected cells
Semiquantitative PCR assay to detect viral genes.
DNA was prepared from these samples by lyophilization for
16 hours, followed by digestion with RNase for 2 hours and
proteinase K for 16 hours before phenol extraction and
ethanol precipitation. This method of DNA preparation
avoids the use of tissue homogenizers, a possible source of
cross-contamination of tissue sample DNA (13). Total RNA
for detecting HTLV-I and human immunodeficiency virus
(HIV) from TRX/ADF-positive biopsy specimens was subjected to RT-PCR analysis. The following primers were
antisense 5‘-GACCGGTGCCTTCTTAGG-3’), HTLV-I (sense
CEM cells, HTLV-I-infected T cell line (MT-2), and EBVinfected Burkitt lymphoma cell line (Daudi) served as positive controls. Constant amplification in the reaction tube was
assessed by coamplification with HLA-DQa primers (13).
PCR products were transferred to nylon membranes using a
slot-blot apparatus (Hoefer Scientific Instruments, San Francisco, CA). The intensity of the signal obtained with each
Figure 1. Immunostaining of salivary gland specimens from patients with Sjogren’s syndrome (SS) and control specimens. Cryostat-frozen
serial sections were fixed in cold acetone, stained with rabbit anti-thioredoxin/aduIt T cell leukemia-derived factor ( a n t i - W A D F ) peptide
antibody and cell type-specific monoclonal antibodies, and examined by immunohistologic techniques. Numerous anti-TRWADF-positive
epithelial cells (acini and ductal structures) and lymphoid cells are seen in salivary gland biopsy specimens from patients with SS (A, D, and
G). The localization of TWADF-positive cells is almost the same as that of CD20-positive cells (C) and cytokeratin-positive cells (E).No cells
stained with an isotype-matched rabbit IgG (B). A normal salivary gland biopsy specimen (F) and anti-TRWADF antibody preabsorbed with
T W A D F peptides do not show cytoplasmic staining (H).(Original magnification X 150 in A and B, x 180 in C-H.)
internal oligonucleotide probe (EBV 5’-TTCTGCTAAGCCCAAC3’, HTLV-I 5’-TGTCCAGAGCATCAGATCA-3’,
and HIV 5’-ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC-3’) was determined by densitometric tracing of the autoradiograph.
Flow cytometric analysis. Five thousand cells were
analyzed on an Epics-Profile flow cytometer (Coulter). To
assess TRXlADF cytoplasmic staining, cells fixed with 1%
parafomaldehyde and 50% ethanol were incubated first with
10 ,ug/rnl of anti-ADF polyclonal antibody and then with
Figure 2. Immunohistologic staining for T W A D F and Epstein-Barr virus-encoded latent membrane protein (EBV-LMP)
in SS salivary gland, demonstrating W A D F staining in the lymphocytic infiltrate and salivary gland epithelial cells. The
localiration of EBV-LMP-positive cells (A) is almost the same as that of TRX/ADF positive cells (B). (Original
magnification X 150.) See Figure I for other definitions.
fluorescein-conjugated goat anti-rabbit antibody. Rabbit IgG
with irrelevant antibody activity was used as a specificity
SCID mice. C.B.-17 SCID mice, ages 7-9 weeks,
were provided from a breeding colony maintained at Clea
Japan, Inc. (Tokyo, Japan). Disease was reproduced in
SCID mice by intraperitoneal injection of 1 x lo’ mononuclear cells from salivary gland surgical specimens from
subjects with SS (n = 3) or other salivary inflammatory
conditions (n = 4) or peripheral blood EBV-seronegative
donors (n = 2). Animals were checked daily for the presence
of tumors. When a palpable tumor was observed, the animal
was killed by cervical dislocation and autopsied under sterile
conditions. The tumors were analyzed by immunohistologic
staining, PCR assay, and flow cytometry.
Statistical analysis. The statistical significance of relationships between the amounts of T W A D F mRNA and
EBV DNA in the corresponding tissues was evaluated by
linear regression analysis.
Immunohistologic analysis of TRWADF-expressing cells in SS salivary glands. To analyze the expression of TRX/ADF in the salivary glands of patients
with SS, we stained frozen sections of salivary gland
tissues with rabbit anti-ADF C-terminal peptide antibody using an immunohistologic technique. In 11 of
the 12 cases, SS salivary gland tissues showed strong
reactivity with anti-TRX/ADF antibody. Positive
staining was present in the cytoplasm of many scattered lymphocytes and most epithelial cells (acini
and/or ductal structures) (Figures 1A. D, and G). The
stained cells appeared to be B cells and epithelial cells,
based on their reactivity in serial sections of tissue
stained with anti-CD20 and anti-CD21 antibodies (B
cell markers) (Figure 1C) and anticytokeratin antibody
(epithelial cell marker) (Figure lE), respectively, and
their lack of reactivity with anti-CD3 antibody (T cell
marker), anti-CD14 (macrophage marker), or antifactor VIII antibody (endothelial cell marker). No
staining with anti-ADF antibody was detected in the
normal salivary glands (n = 6) (Figure 1F) or those
from the 11 patients with other inflammatory conditions including sialolithiasis, ranula, and acute suppurative sialoadenitis. The positive staining was abolished by preincubation of the antibody with C-terminal
fragments (amino acids 76-105) of TRWADF (Figure
lH), and no cells were stained with an isotypematched rabbit IgG (Figure IB).
Since the detection of EBV-encoded EBVLMP was carried out on 1 of the serial sections,
assessment of the relationship between TRWADF
expression and EBV-LMP localization was possible. A striking correlation of EBV-LMP-positive cells
and TRX/ADF-positive cells was observed in the
salivary glands of S S patients. EBV-LMP was detected in 10 of 12 SS salivary gland biopsy specimens.
Normal salivary glands from 6 different individuals
and salivary glands from 11 patients with other salivary gland infldmmatory lesions did not show cytoplasmic staining with anti-EBV-LMP antibody. EBVLMP-positive cells were found among the many
infiltrating lymphocytes and in most epithelial cells of
the SS salivary glands. Interestingly, the localization
of EBV-LMP-positive cells was almost the same as
that of ADF-positive cells (Figure 2). All of the EBVLMP-positive areas were also positive for TRWADF,
and only 1 EBV-LMP-negative subject showed slight
positivity for TRX/ADF.
Detection of ADF mRNA and viral genes. To
confirm the immunohistologic data, we used the RT-
Figure 3. Reverse transcriptase-polymerase chain reaction (PCR)
assay of T W A D F messenger RNA (mRNA) expression in salivary
gland specimens. Lanes 1-12, Salivary gland specimens from patients with SS; lanes 13-18, specimens from normal salivary glands.
TRWADF mRNA expression was increased in SS salivary glands
compared with normal salivary glands ( T W A D F 406-basepairPCR
product; pactin 202-bp PCR product). See Figure 1 for other
PCR assay for detection of TRWADF mRNA in SS
and normal salivary gland samples. TRWADF mRNA
was present in 8 of 12 SS biopsy specimens but in only
1 sample from the 6 normal salivary glands. However,
the TRWADF mRNA in this normal salivary gland
tissue was found to be virtually negligible (Figure 3).
ADF, initially identified in HTLV-I-infected cells of
patients with ATL, can be produced in other virusinfected cells (21). We designed primers specific for
HTLV-I and HIV (which have been implicated in the
pathogenesis of SS [34,35]), in addition to EBV, in a
PCR assay to detect viral genes in TRWADF mRNApositive-salivary glands of 8 patients. Despite the
detection of increased levels of EBV DNA in TRW
ADF mRNA-positive samples, no HTLV-I or HIV
was detected (Figure 4A). EBV DNA was detected in
all of the T W A D F mRNA-positive SS biopsy specimens. A significant correlation was observed between
the level of TRWADF mRNA and the amount of EBV
DNA in each biopsy sample from SS patients (r =
0.75, P < 0.01) (Figure 5 ) . The 6 normal salivary gland
samples from healthy individuals did not show increased levels of EBV DNA (Figure 5).
EBV induction of ADF expression in human B
cells in vitro. Fresh peripheral blood-B cells (purity
98.5%) obtained from EBV-seronegative donors were
infected in vitro with cell-free EBV (strain B95-8).
TRWADF expression was measured by RT-PCR and
flow cytometric analysis. As expected, the purified B
cells expressed no detectable TRWADF at any time
before EBV infection (Figures 6 and 7A). The lethally
heat-inactivated B95-8 showed a decline in T W A D F
expression over 10 days of culture (Figure 6). In the B
1 2 3 4 5 6 7 8 9 101112131415161718
Figure 4. Polymerase chain reaction assay to detect viral gene in
salivary gland specimens from patients with SS. Slot 1, Epstein-Barr
virus (EBV)-, human T lymphotropic virus type I (HTLV-I)-, and
human immunodeficiency virus (HIV)-positive cell lines as positive
controls. Slots 2 and 3, TRWADF-positive salivary gland biopsy
samples from SS patients. No HTLV-1 or HIV was detected. See
Figure 1 for other definitions.
cells infected with EBV, increasing levels of TRW
ADF expression were observed within 5 days (Figures
6 and 7B), and increased steadily thereafter. The
resulting EBV-immortalized B lymphoblastoid cell
lines continued to express TRWADF constitutively
(Figures 6 and 7C). We next screened B cell lines
derived from Burkitt lymphoma for the expression of
T W A D F , by flow cytometric analysis. In contrast to
EBV-negative Burkitt lymphoma BJAB, the EBV-
0 Normal SO
4 -
a .
Relative ADF mRNA level
Figure 5. Relationship between TRX/ADF messenger RNA
(mRNA) and Epstein-Barr virus (EBV) DNA in salivary gland (SG)
biopsy specimens from patients with SS. A significant positive
correlation was found (r = 0.75, P < 0.01). See Figure I for other
positive B cell line derived from Burkitt lymphoma
Daudi cells expressed TRWADF (Figures 6 and 7E).
Expression of ADF in SCID mice reconstituted
with SS salivary gland mononuclear cells. Six SCID
mice were injected with isolated mononuclear cells
from salivary gland biopsy specimens from patients
with SS (n = 3). Within 62-172 days after injection, all
mice developed abdominal tumors. Isolated mononuclear cells from surgical specimens (2 cases of sialolithiasis and 2 cases of ranula) and from peripheral
blood EBV-negative donors (n = 2) were injected into
18 SClD mice. None of the mice in this group showed
evidence of tumors within 250 days, except for I that
had died just before pathologic examination. Immunohistologic studies revealed that the tumors primarily
comprised human EBV-LMP-positive B cells (Figures
8A and B). The B cells were polyclonal, based on
staining for surface immunoglobulin heavy and light
chains (results not shown). RT-PCR, as well as flow
cytometry, showed that a majority of the tumor cells
consistently expressed TRWADF (Figures 6 and 7D).
Previous evidence for EBV reactivation in SS
patients includes the presence of EBV antigens (12)
and elevated levels of EBV DNA (12-14) in SS salivary gland tissues and active production of virus in the
saliva (15) or from B cell lines established from PBMC
1 2 3 4 5 6 7
P-Actin b
Figure 6. Analysis of TRWADF messenger RNA expression in
purified peripheral blood B cells after infection with Epstein-Barr
virus (EBV), B cell tumors obtained from SCID mice reconstituted
with SS salivary gland lymphocytes, and EBV-negative B cell line
derived from Burkitt lymphoma BJAB. Lane I , B cells isolated from
peripheral blood mononuclear cells of an EBV-seronegative donor.
Lane 2 . 5 days after infection with EBV. Lane 3, 1 week after EBV
infection. Lane 4 , 2 weeks after infection. Lane 5 , B cell tumor from
SCID mice. Lane 6, B cells 10 days after infection with irradiated
EBV. Lane 7, EBV-negative B cell line BJAB ( T W A D F 406basepair polymerase chain reaction [PCR] product; Pactin 202-bp
PCR product). See Figure 1 for other definitions.
Relative log fluorescence
Figure 7. Flow cytofluorimetric analysis of TRX/ADF expression
in various human B cells. A, B cells isolated from peripheral blood
mononuclear cells of an Epstein-Ban virus (EBVtseronegative
donor. B, Five days after infection with EBV. C, Two weeks after
EBV infection. D,Cell suspensions of B cell tumor from SCID mice
reconstituted with SS salivary gland lymphocytes. E, EBV-negative
B cell line BJAB. Virtually all EBV-infected cells expressed TRX/
ADF (ED). In the absence of EBV infection, no TRX/ADF
expression was observed (A and E). See Figure 1 for other definitions.
(16) of patients. Antibodies against EBV antigens are
also elevated in SS sera (16-18). Another defined
manifestation of active EBV infection is the presence,
in the circulation, of infected B cells that can transform into B cell lymphomas (36-38). Mariette et a1 (14)
previously showed that, by the in situ hybridization
method, EBV DNA could be in a substantial proportion of epithelial cells and lymphoid cells in salivary
glands from patients with SS, which is consistent with
our data from irnmunohistochemical and PCR analyses
that EBV antigens and EBV DNA were present in SS
salivary glands. Furthermore, we have developed
EBV-induced lymphomas in SCID mice reconstituted
with salivary gland mononuclear cells from SS patients, but not in SCID mice reconstituted with mononuclear cells from patients with other salivary gland
Figure 8. Immunohistologic staining of tumor obtained from SCID mice reconstituted with salivary gland lymphocytes
from patients with Sjogren’s syndrome. A major proportion of the tumor cells were CD20 positive (A) and Epstein-Barr
virus-encoded latent membrane protein positive (B). (Original magnification x 180 in A, x 250 in B.)
inflammatory conditions or EBV-negative individuals.
Thus, SS patients exhibit many signs of active, uncontrolled EBV reactivation.
Investigation of the relationship between T W
ADF and EBV in closely parallel sections showed that
EBV-positive sampleswere also positive for T W
ADF. A close topographic association of EBV antigen
and TRWADF expression in SS salivary glands was
indicated. EBV-infected human B cell lines expressed
TRWADF. Tumors in SCID mice reconstituted with
mononuclear cells from the salivary glands of SS
patients, which were composed of human B cells
carrying EBV DNA, were also positive for TRWADF.
These findings suggest that there is a close relationship
between T W A D F production and EBV infection. In
addition, peripheral blood B cells from EBVseronegative donors showed de novo synthesis of
T W A D F following in vitro infection of EBV. These
data suggest that T W A D F expression closely reflects
the intracellular environment elicited by EBV infection. Previous studies have shown that T W A D F is
widely distributed in a variety of virus-infected cells
(22). Overexpression of TRWADF is detected not only
in HTLV-I-transformed T cells, but also in EBVtransformed B cells and human papillomavirusinfected cells (20,21,39), suggesting that T W A D F is
an essential protein for the symbiosis of viruses and
host cells.
Diffuse TRWADF staining was demonstrated in
the SS salivary glands. Moreover, EBV-infected lymphoma cells showed strong positivity for TRWADF in
SCID mice tumors reconstituted with mononuclear
cells of the salivary glands from SS patients. Recently,
mammalian TRX was reported to be involved in the
regulation of Fos and Jun DNA-binding activity by a
unique reducto-oxidation mechanism (40). In addition,
Okamoto et a1 (41) reported that TRWADF could
regulate the DNA binding of NF-KB protein by a
similar mechanism, such as redox regulation. These
observations suggest the possibility that intracellular
T W A D F is involved in lymphocyte activation by
certain trans-activators. Future studies will further
elucidate the potential role of TRX/ADF in the pathogenesis of SS.
As previously reported, activation of normal
lymphocytes by mitogens, cytokines, or genotoxic
stresses induces T W A D F expression (22). If EBV
alone is not sufficient to lead to TRWADF expression,
it may be one of many cofactors. It is known that SS
salivary glands express increased levels of cytokines
compared with normal salivary glands (42). Moreover,
it has been reported that EBV can lead to induction of
cytokine expression (43). Therefore, T W A D F expression in SS may result from EBV reactivation and
indirect immune stimulation.
Lymphoproliferation is a characteristic feature
of SS (1). As noted above, we found that SCID mice
reconstituted with mononuclear cells of salivary
glands from SS patients, in contrast to reconstitution
with salivary gland mononuclear cells from patients
with other chronic salivary gland diseases, developed
a lymphoproliferative disease characterized by TRXADF expression by EBV-infected B cells. This result
demonstrates that the lymphoproliferation occurring
in salivary glands of SS patients is reproduced in SCID
mice, and suggests that EBV may play a role in the
lymphoproliferation seen in SS.
A number of properties that may be relevant to
the initiation and perpetuation of the pathogenesis of
SS are now known to be present in the salivary glands
of SS patients (35,44),but not in those of patients with
other salivary gland conditions or in normal salivary
glands. These include intense lymphocytic infiltration,
destruction of epithelial cells, abnormally high levels
of EBV and EBV antigens, production of infectious
EBV in saliva, de novo expression of HLA-DR on cell
surfaces, and ectopic expression of TRWADF. These
observations are consistent with several scenarios in
which EBV plays an important role in the initiation
and/or continuation of an immune system attack in
salivary and lacrimal glands. We thus propose that
TRWADF is a new member of the group of known
factors that are overexpressed in SS, and possibly
induced by EBV infection.
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