Apoptosis in labial salivary glands from Sjogren’s syndrome (SS)patients: comparison with human T lymphotropic virus-I (HTLV-I)-seronegative

and -seropositive SS patients

H. NAKAMURA, T. KOJI*, M. TOMINAGA, A. KAWAKAMI, K. MIGITA, Y. KAWABE, T. NAKAMURA,S. SHIRABE & K. EGUCHI First Department of Internal Medicine and*Department of Histology and Cell Biology,

Nagasaki University School of Medicine, Nagasaki, Japan

(Accepted for publication 29 June 1998)

SUMMARY

Apoptosis is a type of cell death that occurs during morphogenesis and development of the immunesystem. One of the mechanisms is mediated through the Fas and Fas ligand (FasL) pathway. Todetermine the possible involvement of Fas and its ligand in salivary gland destruction, we analysed theappearance of nuclei with DNA fragmentation by using nick end labelling (TUNEL) and the expressionof Fas and FasL by immunohistochemistry in labial salivary glands. Furthermore, we compared thefeatures of apoptosis in labial salivary glands between HTLV-I¹ and HTLV-Iþ SS. When the frozensections of 10 primary SS patients in the absence of anti-HTLV-I antibody were examined, severalapoptotic cells were found in the acinar and ductal epithelial cells as well as infiltrated mononuclearcells. Both Fas and FasL were detected in the infiltrated mononuclear cells. Acinar epithelial cells,which are surrounded by FasLþ mononuclear cells, were also double-positive with Fas and FasL,although the expression of FasL was localized at their apical border, suggesting that apoptosis ofmononuclear cells was achieved by activation-induced mechanisms through Fas/FasL pathways, andthat of acinar epithelial cells was mediated by FasL derived from either acinar epithelial cellsthemselves or infiltrated mononuclear cells. Interestingly, Fas expression in ductal epithelial cellswas localized around the lumen side of the ducts, indicating that FasL secreted from acinar epithelialcells may induce Fas-mediated apoptosis of ductal epithelial cells. We also studied the labial salivaryglands from nine SS patients with anti-HTLV-I antibodies. There was no significant difference in theoccurrence of apoptotic cells or in the expression of Fas and FasL between HTLV-Iþ and HTLV-I¹ SSpatients. It was of note that neither the expression of Fas and FasL nor the presence of apoptotic cellswere determined in labial salivary glands from subjects without SS. These findings indicate that Fas-mediated apoptosis in salivary glands could be involved in the pathological manifestations of SS,irrespective of HTLV-I seropositivity.

Keywords apoptosis Sjo¨gren’s syndrome Fas Fas ligand

INTRODUCTION

SS is an autoimmune disorder characterized by lymphocyte infil-tration into salivary and lachrymal glands with concomitant tissuedestruction. Patients typically have dry mouth (xerostomia) anddry eyes (kerato-conjunctivitis sicca) [1]. Immunohistochemicalstudies have shown that the salivary glands are predominantlyinfiltrated by CD4þ cells at an early stage of SS, and then B cellsinto the glandular tissue, along with the appearance of serum

autoantibodies [2]. However, relatively little is known about themechanism underlying the tissue destruction in patients with SS.

Apoptosis is involved in a variety of physiological situations,including immunity, embryogenesis and carcinogenesis [3].Recent reports have demonstrated that apoptosis is tightly con-trolled by the expression of a set of genes [4]. Among them, Fas(CD95/APO-1) has been identified as a possible death factortransducing apoptotic signals into cells [5]. More recently FasL,which belongs to the tumour necrosis factor (TNF) protein family,has been cloned [3]. Upon binding and cross-linking of FasL toFas, apoptotic signals are delivered into the cells, leading toapoptosis. FasL protein was originally found in T cells [4],

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Correspondence: Katsumi Eguchi MD, Professor, First Departmentof Internal Medicine, Nagasaki University School of Medicine, 1-7-1Sakamoto, Nagasaki City, Nagasaki 852, Japan.

where diverse stimuli induced FasL gene expression [6,7], and it isbelieved that the expression of FasL is responsible for the killingactivity of CD4þ T cells and in part of CD8þ T cells.

The presence of apoptosis and the expression of Fas/FasL havebeen determined in the involved organs from human chronicinflammatory diseases such as the synovium from rheumatoidarthritis (RA) patients [7] and the colon from ulcerative colitis(UC) patients [8]. In addition, Konget al. have recently reportedthe expression of Fas/FasL in the salivary glands of patients withprimary Sjogren’s syndrome (pSS) [9]. These results suggest theimportance of Fas-mediated apoptosis regarding the pathologicalmanifestations in human chronic inflammatory diseases.

In the present study, we initially examine the appearance ofapoptotic cells, and the expression of Fas/FasL in the salivaryglands from patients with SS in the absence of HTLV-I antibody.Next, we compare both Fas/FasL expression and the features ofapoptosis in labial salivary glands between HTLV-I¹ andHTLV-I þ SS patients.

PATIENTS AND METHODS

PatientsWe compared 10 patients (one man and nine women) aged59·86 9·4 years (mean6 s.d.; range 45–75 years) with SS in theabsence of anti-HTLV-I antibody and nine patients with SS in thepresence of anti-HTLV-I antibody. The latter group consisted ofsix patients with HTLV-I-associated myelopathy (HAM) and threepatients without HAM. The six HAM patients, who were diag-nosed by criteria proposed by Osameet al. [10], were a man andfive women aged 64·36 16 years (range 32–73 years). Threepatients who were seropositive for HTLV-I, but had no clinicalmanifestations of HAM, were women aged 61·36 11·5 years(range 48–68 years). Patients with secondary SS complicatingother autoimmune diseases, such as systemic lupus erythematosus(SLE), RA and progressive systemic sclerosis, were not included inthis study. All the HTLV-I¹ and HTLV-Iþ patients fulfilled thepreliminary criteria for diagnosis of SS as defined by the EuropeanCommunity [11]. Five patients who have sicca symptoms butcannot to be diagnosed as SS by the European Community(histologically including no focus) were also examined in thisstudy. Informed consent was obtained from all participatingpatients and the study was conducted in accordance with humanexperimentation guidelines of our institution.

Serological testsSera were screened for antibodies to HTLV-I using ELISA (Eitest-ATL kit; Eisai, Tokyo, Japan) and the particle agglutination assay(Serodia-ATL kit; Fuji Rebio, Tokyo, Japan). Anti-HTLV-I anti-body was confirmed using a commercially available immunoblotkit (Problot-HTLV-I; Fuji Rebio). In this test kit anti-HTLV-Iantibody reacted with HTLV-I antigens including three gag pro-teins (p19, p24 and p53) and one env protein (gp46). According tothe criteria proposed by the World Health Organization, it wasthought that reaction with both HTLV-I gp46 and more than one ofthe gag proteins scored positive.

Biopsy of labial salivary glandsMinor labial salivary glands were obtained from the mucosa of thelower lip at 0·5–1 cm lateral from the midline under local anaes-thesia. Tissues were fixed in 4% paraformaldehyde (PFA) in PBSpH 7·4 immediately after biopsy and were immersed in 10, 15 and

20% sucrose, successively. These specimens were frozen in liquidnitrogen and stored at –808C.

Anti-Fas and anti-Fas L antibodiesTo generate anti-Fas antibody, Fas D, corresponding to theextracellular domain (Fas D; amino acids 104–114) of humanFas, was selected [12]. To generate anti-FasL antibody, syntheticpeptides corresponding to the intracellular domain (P5; aminoacids 41–55) of rat FasL were selected [13]. Antisera againsthuman Fas and rat FasL synthetic oligopeptides were obtainedfrom rabbits immunized eight times with 0·19 mg peptide con-jugated with keyhole limpet haemocyanin (KLH) together withFreund’s complete adjuvant (FCA). Titration of antisera wasperformed by an ELISA, with peptides used for immunization asantigen. When the antibody titre reached a plateau, blood wastotally removed, and the serum was separated. These anti-Fasand FasL antisera reacted specifically with human Fas and FasL,respectively [8].

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Fig. 1. Nick end labelling of anti-HTLV-I antibody-negative SS patients(original mag.×200). (a) Staining of labial salivary gland by terminal d-UTP nick end labelling (TUNEL), which indicated DNA fragmentationinsitu. Positive staining was found in one ductal cell (arrow). (b) Positivestaining by TUNEL was found in a ductal cell (arrow) and some lympho-cytes out of massive infiltrated lymphocytes (arrowhead).

ImmunohistochemistryThe location of Fas and FasL was examined using the polyclonalantibodies described above. Immunohistochemistry was performedas reported previously [13]. Briefly, prefixed frozen sections wereimmersed in 3% H2O2 in methanol to block endogenous perox-idase activity, and were preincubated with 500mg/ml goat IgG and

1% bovine serum albumin (BSA) in PBS for 1 h at room tempera-ture to block non-specific binding of antibodies. Then the sectionswere reacted with the primary antibodies (1:200–1:400) dilutedwith 1% BSA in PBS for 2 h at room temperature in a humidifiedatmosphere. After washing in PBS with 0·075% Brij (Sigma,St Louis, MO) four times for 15 min each time, the sections were

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Fig. 2. Localization of Fas and FasL in labial salivary gland of a patient with HTLV-I¹ SS (original mag.×200). (a,b) Expression of Fas inseronegative SS patients (arrow). In (a) Fas is expressed on acinus and in (b) on ducts. (c,d) Expression of FasL in seronegative SS patients(arrowhead). There were two positive lymphocytes in (c) and some of the lymphocytes in lymphoid aggregates in (d). (e) FasL expression ofacinar epithelium (arrowheads).

incubated with horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG (1:100) diluted with 1% BSA in PBS for 1 h at roomtemperature, and washed as described above. The sites of HRPwere visualized by DAB and H2O2. Finally, the sections werecounterstained with methyl green.

Terminal deoxy (d)-UTP nick end labellingTo analyse DNA fragmentation in histological sections, terminaldeoxy (d)-UTP nick end labelling (TUNEL) was carried outaccording to the method of Gavrieliet al. [14] with a slightmodification. Briefly, tissue sections were digested with 1mg/mlproteinase K for 7·5 min at 378C, and the slides were then washedin PBS three times for 5 min each time. The sections were rinsedonce with distilled deionized water and incubated with TdT buffer(0·2M Tris–HCl pH 6·6, 40 mM potassium cacodylate, and 5 mM

cobalt chloride) alone. Then, the TdT buffer containing 0·3 U/mlTdT (Boehringer, Mannheim, Germany) and 10 mM biotinylated16-dUTP (Boehringer Mannheim) was added to the sections, andthe slides were incubated in a humidified atmosphere at 378C for60 min. The reaction was terminated by transferring the slides to50 mM Tris–HCl pH 7·5, and washed with 50 mM Tris–HCl pH 7·5four times for 10 min each time at room temperature. Endogenous

peroxidase was inactivated by immersing the sections in 0·3%H2O2 in methanol for 15 min at room temperature and washedthree times with PBS for 5 min each time. After incubation with5% BSA in PBS for 1 h at room temperature to block non-specificbinding, the sections were incubated with HRP-labelled goat anti-biotin (1:100) diluted with 5% BSA in PBS for 2 h at roomtemperature and washed in PBS. The sites of HRP were visualizedby H2O2 and DAB for 10 min

Statistical analysisStatistical analysis was performed using unpairedt-test.P< 0·05was considered statistically significant.

RESULTS

In situ detection of DNA fragmentation in the labial salivaryglands from HTLV-I¹ SS patientsTo determine whether nuclei with fragmented DNA are present inthe labial salivary glands from SS, TUNEL was performed in thefrozen sections of 10 patients with SS. The typical TUNEL stainingof a labial salivary tissue section in shown in Fig. 1. Severalapoptotic cells showing condensed nuclei were found in the ductalepithelial cells (Fig. 1a). In some tissue sections, infiltrated mono-nuclear cells surrounding the ducts and acini were also positive toTUNEL staining (Fig. 1b).

Immunohistochemical detection of Fas and FasL in the labialsalivary glands from patients with HTLV-I¹ SSTo clarify an involvement of the Fas system in the induction ofapoptosis in the labial salivary glands from SS patients, theexpression of Fas and FasL was determined immunohistochemicallyin the tissue sections from 10 HTLV-I¹ SS patients. The represen-tative staining results are shown in Fig. 2; acinar cells were clearlystained with the rabbit anti-Fas antibody (Fig. 2a). In some speci-mens, Fas was expressed in the ductal epithelial cells (Fig. 2b) andmononuclear cells in the lymphoid aggregates. When a sequentialsection of labial salivary tissues to that used in Fig. 2a or 2b wasstained with anti-FasL antibody, the staining for FasL was found inthe infiltrated mononuclear cells (Fig. 2c and 2d, respectively). Asshown in Fig. 2d, the mononuclear cells expressing FasL werepresent around both acinar and ductal Fasþ epithelial cells. Further-more, some acinar cells as well as the infiltrated mononuclear cellsexpressed FasL (Fig. 2e). No staining was found with normal rabbitserum, instead of the specific antibody or after omission of theprimary antibody (data not shown).

In situ detection of DNA fragmentation in labial salivary glandsfrom HTLV-Iþ patients with SSTo determine whether there was any difference in the presence ofapoptotic cells between HTLV-I¹ and HTLV-Iþ SS patients, labialsalivary glands from nine HTLV-Iþ patients with SS were alsostudied using the TUNEL method. The representative results areshown in Fig. 3, where the nucleus of a ductal epithelial cell wasstained (Fig. 3a). All nine HTLV-Iþ patients had apoptotic cells inepithelial ducts and/or acini. In some specimens, infiltrated mono-nuclear cells were also positive (Fig. 3b).

Immunohistochemical detection of Fas and FasL in the labialsalivary glands from HTLV-Iþ patients with SSAs shown in Fig. 4, Fas was constitutively expressed in theproliferating ductal epithelial cells (Fig. 4a). In an adjacent section,

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Fig. 3. Nick end labelling of anti-HTLV-I antibody-positive SS patients(original mag.×200). (a) Staining of labial salivary gland by TUNEL,which indicated DNA fragmentationin situ. Positive staining was found ina ductal epithelial cell (arrow). (b) Positive staining by TUNEL was foundin one lymphocyte around the epithelial ducts (arrowhead).

FasL was detected in the infiltrated mononuclear cells surroundingthe epithelial acini and ducts (Fig. 4b). In other specimens,however, infiltrated mononuclear cells as well as ductal epithelialcells were positive for Fas (Fig. 4c), and FasL was seen in theacinar epithelial cells as well as the infiltrated mononuclear cells(Fig. 4d).

The frequency of TUNEL-positive cells in the labial salivaryglands from patients with HTLV-I¹and HTLV-Iþ SSWe counted the number of apoptotic cells in the labial salivarysections from 10 patients with HTLV-I¹ and nine patients withHTLV-I þ SS (Table 1). The percentages of apoptotic cells amongtotal ductal epithelial cells in HTLV-I¹ and HTLV-Iþ patientswere 0·276 0·15% and 0·256 0·25%, respectively. The percen-tages of ducts with apoptotic cells among total ducts in HTLV-I¹

and HTLV-Iþ patients were 14·996 14·50% and 9·046 5·43%,respectively. Therefore, there were no significant differences in thepercentages of apoptotic cells in the labial salivary glands betweenHTLV-I ¹ and HTLV-Iþ patients. In the series of experiments, wealso conducted histological examinations of the labial salivaryglands from subjects without SS, which showed that neitherapoptotic cells nor the expression of Fas and FasL were found inthese patients (Fig. 5 and Table 1).

DISCUSSION

SS is a chronic inflammatory disorder which is histologicallycharacterized by infiltration of activated mononuclear cells and/or ductal destruction in the salivary and lachrymal glands [1].However, little is known about the role of apoptosis in glandulardestruction [9]. Recently, apoptotic cell death, especially Fas-mediated apoptosis, has been reported to be involved in thedevelopment of other chronic inflammatory diseases such as RA[7] and UC [8]. Therefore, we examined both Fas/FasL andapoptotic cell death in the labial salivary glands from SS patients.

Based on our histological examinations, we initially investi-gated the relation of apoptosis found in labial salivary glands in SSpatients, since neither apoptotic cells nor the expression of Fas andFasL were found in those from subjects without SS. We showed inthe present study that infiltrated mononuclear cells expressed bothFas and FasL, and some of the cells underwent apoptosis. Mono-nuclear cells infiltrated in the salivary glands in SS patients havebeen reported to be activated [15], and activated mononuclear cellsin vitro express both Fas and FasL [16]. Since the activation-induced cell death of T cells was achieved by the interactionsbetween Fas and FasL [17,18], apoptosis of infiltrating mono-nuclear cells found in SS patients could be induced through the

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Fig. 4.Localization of Fas and FasL in labial salivary gland of HTLV-Iþ SS (original mag.×200). (a,c) Expression of Fas in seropositive SSpatients. In (a) Fas is expressed on a duct (arrow) and a lymphocyte (arrowhead) and in (c) on ducts (arrow) and lymphocytes (arrowhead).(b,d) Expression of FasL in seropositive SS patients. There are some positive lymphocytes in (b) (arrowhead) and acinar epithelial cells in (d)(arrowheads).

Fas/FasL system. Other molecules which can induce apoptosis oftarget cells such as granzyme B [19], TIA-1/TIAR [20], and apo-2ligand [21] may also be involved in the apoptotic cell death ofinfiltrated mononuclear cells, since these molecules are expressedin activated mononuclear cells.

Acinar epithelial cells were also double-positive for Fas andFasL. In addition, mononuclear cells surrounding the Fasþ acinarepithelial cells also expressed FasL. These results suggest that

apoptosis of acinar epithelial cells in salivary gland was achievedby FasL derived from either acinar epithelial cells themselves orneighbouring mononuclear cells.

It is of interest that Fas expression of ductal epithelial cells islargely restricted to the apical region of the cells. Therefore, theFasL molecules from the neighbouring mononuclear cells cannotinteract easily with Fas on the ductal epithelial cells. Consideringthat FasL on the acinar epithelial cells was also localized to theapical border of the cells, it is possible to speculate that FasLsecreted from the acinar epithelial cells binds to Fas of the ductalepithelial cells, leading to induction of apoptosis of the latter cells.

The involvement of HTLV-I infection has been suggested inthe development of SS [22,23]. Transgenic mice expressing Taxprotein of HTLV-I develop exocrinopathy affecting lachrymal andsalivary glands, which resembles the features of SS in humans [24].We also recently reported the high prevalence of SS in patientswith HAM [23]. Therefore, we next examined the difference ofFas/FasL expression and apoptosis in labial salivary glandsbetween HTLV-Iþ and HTLV-I¹ SS patients. As shown in Results,we did not find any difference in Fas/FasL expression amonginfiltrated mononuclear cells, acinar epithelial cells, and ductalepithelial cells. Furthermore, there was no significant difference inthe percentages of apoptotic cells in the labial salivary glandsbetween HTLV-Iþ and HTLV-I¹ SS patients. In our previousreport [23], we also did not find statistical differences in serologicalcharacteristics, including anti-SS-A and -SS-B and phenotypicmarkers of infiltrated mononuclear cells, between HTLV-Iþ andHTLV-I ¹ SS patients. These findings may imply that HTLV-Iinfection is one of the many causative agents (Epstein–Barr virus,HIV, hepatitis C virus, etc.) of SS. In other words, cellular andhumoral responses leading to the development of pathologicalmanifestations of SS could be the same, even though the cause,including HTLV-I infection, is different in each case.

In conclusion, this is the first description of Fas-mediatedapoptosis found in salivary glands from patients with SS definedby the presence or absence of HTLV-I antibody. Sequentialanalysis using animal models such as HTLV-I-tax transgenicmice or alymphoid mice is required to investigate further therole of apoptosis seen in SS.

ACKNOWLEDGMENTS

We thank Miss Ayumi Yoshida and Miss Yukiya Matsuo for technical

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Fig. 5.The absence of apoptotic cells (a), the expression of Fas (b) or FasL(c) in labial salivary glands from subjects without SS (original mag.×200).

Table 1. The frequency of terminal d-UTP nick end labelling (TUNEL)-positive cells in labial salivary glands from patients with HTLV-Iþ and

HTLV-I ¹ SS

Apoptotic cells among Ducts with apoptotictotal ductal epithelial cells among total

cells ducts

HTLV-I ¹ SS (n¼ 10)

f0·276 0·15*

f14·996 14·50*

NS NSHTLV-I þ SS (n¼ 9) 0·256 0·25* 9·046 5·43*Normal control (n¼ 5) 0·0306 0·041 1·366 1·86

Data shown as mean6 s.d. (%).P value was calculated by unpairedt-test (*P<0·05versusnormal control).

NS, Not significant.Apoptotic cells were counted per one specimen on a glass slide.

assistance, and Dr F. G. Issa (Department of Medicine, University ofSydney, Sydney, Australia) for the critical reading and editing of themanuscript. Supported in part by a grant-in-aid (05670426) from theMinistry of Education, Science, Sport and Culture, Japan.

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