Salivary glands of primary Sj?gren's syndrome patients express factors vital for plasma cell survival.

7.1 MB·English

by  Szyszko, Ewa A

Szyszkoetal.ArthritisResearch&Therapy2011,13:R2
http://arthritis-research.com/content/13/1/R2
RESEARCH ARTICLE Open Access
’
Salivary glands of primary Sjögren s syndrome
patients express factors vital for plasma cell
survival
Ewa A Szyszko1,2*, Karl A Brokstad1, Gunnvor Øijordsbakken2, Malin V Jonsson3, Roland Jonsson1,4,
Kathrine Skarstein2
Abstract
Introduction: The presence of circulating Ro/SSA and La/SSB autoantibodies has become an important marker in
the classification criteria for primary Sjögren’s syndrome (pSS). Plasma cells producing these autoantibodies are
mainly high affinity plasma cells originating from germinal centre reactions. When exposed to the right
microenvironment these autoimmune plasma cells become long-lived and resistant to immunosuppressive
treatment. Since autoimmune plasma cells have been detected in the salivary glands of SS patients, we wanted to
investigate if the glandular microenvironment is suitable for plasma cell survival and if glandular residing plasma
cells are the long-lived plasma cell subset.
Methods: Single, double and triple immunohistochemistry as well as immunofluorescence staining was performed
on minor salivary gland tissue retrieved from pSS, chronically inflamed and normal subjects.
Results: We detected significant numbers of CD138+, non-proliferating, Bcl-2 expressing plasma cells in the salivary
glands of pSS patients with high focus score (FS). Furthermore, we demonstrated that CXCL12 and interleukin (IL)-6
survival factors were highly expressed in pSS salivary gland epithelium and by focal mononuclear infiltrating cells.
Notably, adipocytes when present in the salivary gland tissue were an important source of CXCL12. We clearly
demonstrate that plasma cells are localised in close proximity to CXCL12 and IL-6 expressing cells and thus that
the environment of salivary glands with high FS provide factors vital for plasma cell survival.
Conclusions: Plasma cells residing in the salivary glands of pSS patients with high FS showed phenotypic
characteristics of the long-lived plasma cell subtype. Furthermore, the pSS salivary gland microenvironment
provided niches rich in factors vital for plasma cell survival.
Introduction autoantibodies in sera and saliva of SS patients [4-6]
Sjögren’s syndrome (SS) is a heterogeneous autoimmune raise questions about the origin and contribution of sali-
disease characterized by focal mononuclear cell infiltra- vary gland plasma cells to the pathogenesis of SS.
tion in the exocrine glands and high serum titres of Ro/ In addition to plasma cells, the focal infiltrates in sali-
SSA, La/SSB and rheumatoid factor (RF) autoantibodies. vary glands of SS patients consist of T-cells, B-cells,
Plasma cells producing these autoantibodies are primar- macrophages, follicular dendritic cells, dendritic cells
ily class-switched, somatically mutated IgG plasma cells and plasmacytoid dendritic cells [7-10]. In approxi-
that origin from germinal centers (GCs) reactions [1]. mately one-fourth of patients with primary SS (pSS), the
However, detection of autoreactive plasma cells in the accumulating cells form structures that resemble GCs as
inflamed salivary glands [2,3] and presence of IgA seen in secondary lymphoid organs [11-15]. Together
with the fact that both Ro and La antigens have been
detected in the salivary glands of SS patients [16,17]
*Correspondence:[email protected] there exists a possibility that autoimmune plasma cells
1BroegelmannResearchLaboratory,TheGadeInstitute,UniversityofBergen,
are produced at the site of inflammation. Another
TheLaboratoryBuilding,Bergen,N-5021,Norway
Fulllistofauthorinformationisavailableattheendofthearticle
©2011Szyszkoetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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possibility is that the glandular microenvironment com- this study. All biopsies had been performed between
prises factors necessary for prolonged plasma cell survi- 2004 and 2007 at the Department of Otolaryngology/
val and that autoantibodies are being produced by Head and Neck Surgery at Haukeland University Hospi-
plasma cells independently of activation and differentia- tal in Bergen. All tissue sections had been previously
tion of new B-cells. This phenomenon has been shown evaluated by an oral pathologist and the focus score
for the bone marrow residing plasma cells that in the (FS) (the number of focal mononuclear cell infiltrates
presence of particular survival signals produce circulat- with >50 mononuclear cells per 4 mm2) was deter-
ing immunoglobulins in an antigen independent fashion mined. All patients had FS ≥1 and were divided in two
[18-24]. The bone marrow subset of plasma cells is groups; patients with given FS equal to 1 (FS = 1) and
often referred to as a long-lived plasma cell subset. patients with FS equal to or more than 2 (FS ≥2).
The importance of long-lived plasma cells in autoim- Hematoxylin and eosin (H&E) stained formalin fixed
munity has been brought to light after observations and paraffin embedded minor salivary gland tissue sec-
made during clinical studies utilizing the B-cell deplet- tions were further screened for the presence of germ-
ing monoclonal antibody rituximab in systemic lupus inal center (GC) like structures. Individuals that were
erythematosus. As an outcome, anti-CD20-treated auto- evaluated for pSS, but did not fulfill the AECC criteria
immune patients showed drastic reductions in B-cell served as non-pSS tissue controls and were divided
numbers, but unchanged serum levels of autoantibodies into chronically inflamed (CIG) (small focal infiltrates
[25,26]. Thus, it has been proposed that at least some with less than 50 cells; chronically inflamed tissue not
portion of these autoantibodies is being produced not sufficient for SS) and normal glands (NG) (no inflam-
by newly generated cells but by pre-existing long-lived mation in the salivary gland tissue). Medical records
plasma cells, unaffected by treatment. were obtained from patients’ charts at the Department
In order to survive, long-lived plasma cells need con- of Rheumatology, Haukeland University Hospital and
tact with particular factors present in their environment. information was collected regarding routine laboratory
In the bone marrow these survival signals are provided assessments including RF detection, antinuclear antibo-
by the so-called survival niches generated by bone mar- dies (ANA), anti-Ro/SSA, anti-La/SSB, and serum
row stroma [22,23,27-29]. Interestingly, many of the sur- immunoglobulin levels (IgG, IgA and IgM). The char-
vival molecules in particular cytokines and chemokines, acteristics of patients and subjects included in the
are also important modulators of the immune responses study are shown in Table 1. The study was approved
that occur within the inflamed tissues. In the salivary by the Committee of Ethics at the University of Bergen
glands of SS patients, the mononuclear cell infiltrates (145/96-44.96 and 242.06). All studied subjects gave
are one of the hallmarks of the disease. However, it is their informed consent.
the glandular microenvironment and stromal cells of the
glands that are responsible for accumulation, location Primary antibodies
and retention of the inflammatory cells [30]. The following primary anti-human antibodies were used
In the present study we aimed to investigate: I) If the in this study: mouse monoclonal CD138 (1:00) (clone
microenvironment of the salivary glands of pSS patients MI15, Dako A/S, Glostrup, Denmark), rabbit polyclonal
provides factors necessary for plasma cell survival and if IgA (1:50000) (Dako A/S), rabbit polyclonal IgG
plasma cells are indeed in close contact with these fac- (1:30000) (Dako A/S), mouse monoclonal Ki-67 (1:150)
tors; II) If plasma cells present in minor salivary glands (clone MIB-1, Dako A/S), rabbit monoclonal Bcl-2
of patients with pSS are potential candidates for the (1:100) (clone E17, Abcam plc., Cambridge, UK), mouse
long-lived plasma cell subset; III) If the glandular micro- monoclonal CXCL12 (1:250) (clone 79014.111, R&D
environment of pSS is disease specific and thus differs Systems, Minneapolis, MN USA), rabbit polyclonal IL-6
from the glandular microenvironment in non-SS tissue; (1:500) (Abcam plc) and rat anti-PNAd carbohydrated
IV) If GC-like structures have an impact on the type of epitope (clone MECA-79, BD Bioscience, Trondheim,
microenvironment seen in minor salivary glands; and Norway). Isotype and concentration matched antibodies
finally V) If the survival molecules contribute to the (mouse IgG1 and rabbit Ig fraction, Dako A/S) were
migration of plasma cells into inflamed salivary glands. used as negative controls.
Materials and methods Immunohistochemistry
Patients and controls Single-staining
Lower labial minor salivary gland biopsies from 21 con- Formalin fixed, paraffin embedded minor salivary glands
secutive patients fulfilling the American-European were cut (4 to 6 μm) on a Leica serial microtome (Leica
consensus group criteria (AECC) of pSS [31] and Instruments GmbH, Nussloch, Germany) and placed on
®
10 individuals not fulfilling the pSS criteria were used in SuperFrost Plus microscope slides (Thermo Scientific,
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Table 1Information onpatients andsubjects included in the study
Patient/SubjectNr. Focusscore GC+/- Age Gender Salivarysecretion Ro/SSA La/SSB ANA RF IgG IgA IgM
pSS 1 1 - 48 F 2.0 - - 0.36 <16 10.0 2.3 1.3
pSS 2 1 - 50 F 1.6 nt nt nt nt nt nt nt
pSS 3 1 + 72 F 1.9 nt nt nt nt nt nt nt
pSS 4 1 - 50 F 1.1 - - 0.57 <11 10.1 1.2 0.9
pSS 5 1 - 48 F 0.2 - - 0.68 <11 10.7 0.8 0.9
pSS 6 1 + 62 F 1.7 + - 2.20 <11 13.9 1.8 3.0
pSS 7 1 - 48 F 0.7 + - 6.91 nt 19.4 4.1 0.5
pSS 8 1 - 37 F 0.5 - - 4.55 <11 15.9 3.1 1.7
pSS 9 1 - 68 F 0.8 - - 2.00 <16 9.8 2.2 0.6
pSS 10 4 - 54 F 0.1 + - 4.77 32 15.1 4.4 0.9
pSS 11 2 - 58 F 2.5 - - 0.60 nt** 12.4 1.5 1.3
pSS 12 3 + 40 F 0.2 + + nt nt nt nt nt
pSS 13 4 + 52 F 1.0 - - 0.17 nt** 12.4 2.7 1.1
pSS 14 12 + 46 F 0 - - 0.66 nt** 11.9 2.4 1.7
pSS 15 4 - 72 M 0.2 + + 6.05 nt 16.6 3.7 0.9
pSS 16 4 + 60 F 0.1 - - 8.05 <11 8.0 1.4 2.1
pSS 17 4 - 56 F 1.2 - - 0.29 <11 7.1 0.9 1.5
pSS 18 2 - 56 F 0.5 - - -* <11 9.1 0.7 1.8
pSS 19 2 - 50 F 0.8 + + 1.27 nt** nt nt nt
pSS 20 4 - 58 M 1.5 + + 9.72 <16 22.1 1.7 0.9
pSS 21 6 + 59 F 4.6 + + +* 224** 19.8 1.5 1.4
CIG 22 0 - 46 F 0.9 - - 0.39 <11 9.0 2.0 0.7
CIG 23 0 - 66 F 4.5 + - 0.77 <11 19.2 6.4 0.5
CIG 24 0 - 49 F 0.8 - - 0.1 <11 9.9 1.8 1.0
CIG 25 0 - 70 F 0.9 - - 0.39 nt nt nt nt
CIG 26 0 - 54 F 1.0 - - 0.12 <11 14.1 2.0 0.5
NG 27 0 - 54 F 0.2 - - 0.28 <11 8.8 1.1 0.4
NG 28 0 - 58 F 1.0 - - 0.45 <11 9.6 1.6 0.8
NG 29 0 - 54 F 2.1 - - 0.41 nt nt nt nt
NG 30 0 - 55 F 0 nt nt nt nt nt nt nt
CIG,chronicallyinflamedglands;NG,normalglands;pSS,primarySjögren’ssyndrome.Focusscore:thenumberoffocalinfiltratesofmorethan50mononuclear
cellsper4mm2.GC+/-:presenceorabsenceofgerminalcenterlikestructuresbasedonmorphologyoftheinfiltrates.Age,ageatthetimeofSSdiagnosis.
Gender,Female/Male.Salivasecretion,Meanun-stimulatedwholesalivasecretionper15minutes,normalvalues≤1.5ml/15minutes.Ro-SSAandLa-SSB
autoantibodiesinserum,positiveornegative.ANA,Antinuclearantibodiesinserum:negative<0.99,greyzone1.00to1.65andpositive>1.65.*From2009
measureaspositiveornegative.RF,rheumatoidfactorinserum:<11or<16negative,32uncertain.**Patientswithhighlypositivecycliccitrullinatedpeptide
antibody(CCP).Immunoglobulinlevelsinserum,normalvalues:IgG6.0to15.3g/L,IgA1.0to4.1g/LandIgM0.5to2.5g/L.nt,nottested.
Walldorf, Germany). Sections were deparaffinised in performed at room temperature (RT) and Tris-buffered
xylene and rehydrated through graded ethanol series saline (TBS) (pH 7.6 with 0.1% Tween) was used for 10
and distilled water. After heat-induced epitope retrieval minutes between every step. Sections were counter-
(HIER) with citrate buffer (Dako Target Retrieval Solu- stained with Haematoxylin (Dako S3301) for three min-
tion, pH 6.0, S1699), endogenous peroxidase activity was utes, dehydrated and mounted in Eukitt (O. Kindler
blocked with 0.03% Dako Peroxidase Block for five GmbH&Co,Freiburg,Germany).
minutes. Sections were further incubated with primary Double-staining
antibody (CD138, Bcl-2, IgA, IgG, IL-6 or PNAd) for 60 Expression of Ki-67/CXCL12/IL-6 and CD138 was
minutes, followed by incubation with horse radish per- detected by double-staining. Sections were pre-treated
oxidase (HRP)-conjugated anti-mouse or anti-rabbit as described above and Dako Dual Block was used for
EnVision+ (Dako) for 30 minutes. Except for PNAd, dia- 10 minutes to block endogenous peroxidase and endo-
minobenzidine (DAB) was used as chromogen for genous AP. The first primary antibody was incubated
10 minutes in development of all antibodies. Alkaline for one hour at RT followed by the procedure described
phosphatase (AP) and Liquid Permanent Red (LPR, in single staining. After development of the first primary
K0640, Dako, Glostrup, Denmark). were used in the antibody with DAB, sections were washed with water
development of PNAd staining. All incubations were for 5 minutes, with TBS for 10 minutes and treated
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with Doublestain Block (Dako) for 3 minutes. Second close proximity to the acinar or ductal epithelium) and
primary antibody was incubated first for 60 minutes at mononuclear cells in focal infiltrates were analysed.
RT and than over night at 4°C. Binding of the second Cells were counted using a grid and a 10× or a 40×
primary antibody was detected by AP polymer and LPR objective. Cells were considered positive if 50% or more
for seven minutes. Between every step sections were of the cell membrane, nucleus or cytoplasm were posi-
washed with TBS for 10 minutes. Sections were coun- tively stained. Intensity of staining was not evaluated in
terstained with Hematoxylin, dehydrated and mounted this study. For the analysis of double staining, cells were
in Eukitt. considered in contact when more than 10% of the cell
Triple-staining membranes from both cells were in contact with each
Expression of CD138, PNAd and CXCL12 was detected other. Counted areas were randomly selected and
by triple-staining. Sections were pre-treated as described between three and four minor salivary glands were ana-
aboveandincubatedwithDakoDualBlockfor10minutes lysed for each patient or subject. Except for CD138
to block endogenous peroxidase and endogenous alka- expression, the data are presented as the number of
line phosphatase. Avidin was used for 15 minutes, TBS positive cells/mm2 of salivary gland tissue. For the
for 15 minutes and Biotin for 15 minutes (Blocking Kit CD138 expression in the focal infiltrates the data are
Avidin/Biotin, Vector Laboratories, Burlingame, CA, presented as the percentage of CD138 positive cells of
USA) to block endogenous biotin. CD138 was incubated the total number of cells.
as the first primary antibody. The procedure for single
staining was followed. Second primary antibody PNAd Statistical analysis
was incubated over night at 4°C, followed by 30 minutes Statistical analyses were performed using SPSS version
incubation with biotinylated rabbit -anti-rat (Dako, 15.0 software (IBM, Chicago, IL, USA). Normal distribu-
E0468) antibody diluted 1:200 in 3% bovine serum albu- tions of the results were tested by Kolmogorov-Smirnov.
min (BSA). Development of PNAd was performed by All data were normally distributed and thus statistic sig-
using Vector ABC Kit for 30 minutes and LPR for nificance was tested by Student t-test and presented as
7 minutes. The third primary antibody CXCL12 was mean ± standard error of mean (SEM). Differences were
incubated over night at 4°C followed by incubation with considered significant when P < 0.05.
Rabbit/Mouse Link (Dako, K5361) for 30 minutes. Sec-
ondary antibody, conjugated with alkaline phosphatase Results
labelled polymer (EnVision+ Dako, Glostrup, Denmark) Study group
was applied for 30 minutes. Blue Vector (Vector The pSS patients used in this study were divided into
Laboratories, SK-5300) was used in development of two groups according to FS; one group consisted of
CXCL12 for four minutes. Between every step 10 min- patients with FS = 1 and one group consisted of patients
utes rinsing with TBS was used. Sections were counter- with FS ≥2. By morphology, we detected GC-like struc-
stained with Dako Hematoxylin for one second and tures (GC+) in salivary glands of 7/21 (33%) patients
mounted in Eukitt. with pSS. The majority of the GC+ patients were in the
Immunofluorescence staining FS ≥2 group; mean FS in the GC+ was 4.4 as compared
Immunofluorescence was used in detection of Bcl-2 to 2 in the GC- patients (Table 1). Subjects not fulfilling
expressing plasma cells Sections were pre-treated as the criteria of pSS were also divided into two groups;
described above and non-specific binding was inhibited 1) subjects with chronic inflammation which presented
with TBS buffer containing 2% BSA, 10% normal serum as diffuse non-focal infiltration of cellular aggregates
and 0.5% TritonX. Sections were further incubated for with less than 50 cells in the minor salivary gland tissue
one hour at RT with primary antibodies (CD138 and and 2) subjects with normal salivary gland histology;
Bcl-2) followed by incubation for two hours with sec- occasional cellular aggregates but not more than
ondary antibody; AlexaFluor488 (green) (Molecular expected in normal tissue, and absence of focal inflam-
Probes, Invitrogen, Paisley UK) for CD138 and Alexa- mation. The mean unstimulated salivary secretion levels
Fluor 594 (red) (Molecular Probes, Invitrogen, Paisley were lower than 1.5 ml/15 minutes in all study groups
UK) for Bcl-2. Between every step sections were washed (Table 1). Eight pSS patients were positive for Ro/SSA
with TBS for 10 minutes. Sections were counterstained or/and La/SSB. The mean antinuclear antibodies (ANA)
with DAPI and mounted with Moviol. levels in serum were 2.47 in pSS with FS = 1, 3.56 in
Evaluation of staining pSS with FS ≥2, 0.35 in subjects with chronically
The minor salivary gland tissue sections were evaluated inflamed glands and 0.77 for subjects with normal gland
using a light microscope (Leica DMLB, Leica Microsys- histology. Only one pSS patient had high amounts of
tems Wetzlar, Wetzlar, Germany) by two investigators. rheumatoid factor in serum; however, five pSS patients
Both interstitial (small mononuclear cell clusters in were highly positive for cyclic citrullinated peptide
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antibody (CCP). There were no significant differences in plasma cells was observed mostly in the pSS patients
IgG and IgA levels between the groups. The IgM levels with high FS. Similarly to IgA, IgG+ cells clearly
were significantly higher in pSS with FS ≥2 compared to resembled plasma cell morphology (Figure 2B).
chronically inflamed (P < 0.05) and normal (P < 0.05) Approximately the same frequencies of IgA expres-
subjects (Table 1). sing plasma cells were noted in the interstitial areas
of all three groups tested (Figure 2C). However, not
High number of CD138 expressing plasma cells detected significantly, a reduction in IgA+ plasma cells was
in pSS with FS ≥2 noted in the focal infiltrates of pSS patients with FS ≥2
Plasma cells in the salivary gland tissue identified by the (Figure 2D).
expression of CD138 showed both plasma blast and Both pSS groups (FS = 1 and FS ≥2) and subjects with
mature plasma cell morphology. Cells with mature chronically inflamed salivary glands had significantly
plasma cell morphology had characteristic eccentrically higher numbers of interstitial IgG plasma cells com-
placed nucleus, prominent cytoplasm and oval shape, pared with normal tissue. However, pSS patients with
whereas cells with plasma blast morphology were smal- high FS demonstrated the most prominent increase in
ler, spherical and with higher nucleus-to-cytoplasm the interstitial IgG plasma cells (Figure 2E). The number
ratio. of IgG expressing plasma cells in the focal infiltrates was
CD138+ plasma cells were detected in the salivary higher in the pSS with FS ≥2 but due to great variation
gland tissue of all investigated patients. In patients with within the groups, the differences were not significant
FS = 1, CD138 expressing cells were detected intersti- (Figure 2F).
tially as small cell clusters close to ductal and acinar Interestingly, the focal infiltrates of pSS patients with
epithelium and in the periphery of larger infiltrates. In FS = 1 contained similar frequencies of IgA and IgG
the biopsy samples from patients with FS ≥2, CD138 expressing plasma cells, whereas in the focal infiltrates
expression was identified interstitially and both in the of pSS patients with high FS, the numbers of IgG
periphery and within larger focal infiltrates (Figure 1A). expressing plasma cells were three times higher than
Further, we detected CD138+ plasma cells within numbers of IgA positive plasma cells (Figure 2D, F).
some GC-like structures as described in more detail The presence of GC-like structures appeared to have no
later. impact on the amounts of IgA or IgG expressing plasma
Significantly higher numbers of CD138+ interstitial cells. No correlation was detected when we compared
cells were detected in pSS patients with FS ≥2 compared the increased number of IgG versus IgA plasma cells in
to the other groups tested (Figure 1B). There were no the salivary glands and serum IgG and IgA levels in
significant differences in the amount of CD138+ intersti- patients with pSS.
tial cells between the pSS group with FS = 1, chronic
inflammation and normal glands (Figure 1B). When Accumulation of non-proliferative plasma cells in salivary
looking at focal infiltrates, we found no significant dif- gland tissue
ferences between the percentage of CD138+ cells in pSS Since it has been proposed that the long-lived plasma
patients with FS = 1 and pSS patients with FS ≥2 cells survive without detectable proliferation [21,32], a
(Figure 1C). In our material, the presence of GC-like Ki-67/CD138 double-staining was performed.
structures had no impact on the numbers of detected Ki-67 was generally expressed on mononuclear cells in
CD138 plasma cells. the focal infiltrates of pSS patients with both FS = 1 and
FS ≥2 and in the interstitial cells of all groups included
Up-regulation of IgG but not IgA expressing plasma cells in this study (Figure 3). Frequencies of interstitially Ki-
detected in pSS with FS ≥2 67+ cells were low in the normal tissue, but increased
An analysis of immunoglobulin phenotype showed the significantly in chronically inflamed and in pSS tissue
presence of both IgA and IgG expressing plasma cells in with FS = 1. A remarkable increase of Ki-67+ interstitial
the salivary gland tissue of all patients and subjects cells was observed in the salivary glands of pSS patients
tested (Figure 2). IgA expressing plasma cells were with FS ≥2 (Figure 3A). Only a slight difference was
detected in small cell clusters in close proximity to duc- detected in the expression of Ki-67 by focally infiltrating
tal and acinar epithelium in all groups tested and in the cells in pSS patients with FS = 1 and in pSS patients
periphery of smaller and larger focal infiltrates in pSS with FS ≥2 (Figure 3B). Moreover, GC positivity did not
salivary glands. IgA+ cells clearly resembled plasma cell seem to associate with the numbers of focally infiltrating
morphology (Figure 2A). IgG expressing plasma cells Ki-67+ cells.
were found both inside and in the periphery of large Most of the CD138+ plasma cells did not express
focal infiltrates in pSS patients with FS = 1 and FS ≥2, Ki-67. For the pSS groups with FS = 1 and FS ≥2 we
whereas interstitial accumulation of IgG expressing detected on average four double-positive cells per gland.
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For the group with chronic inflammation we detected
one double-positive cell per gland, whereas no double
positive cells were detected in normal salivary gland tis-
sue. Ki-67+ plasma cells were mainly observed in the
periphery of larger infiltrates with high proliferating
activity and in some small plasma cell aggregates often
in close proximity to vascular structures (Figure 3C).
Bcl-2 expressing plasma cells preferentially associate with
pSS high FS
In order to determine whether the accumulating plasma
cellsintheminorsalivaryglandsofpSSpatientsandcon-
trolsareprimedtosurviveforlongperiodsoftime[33,34]
welookedattheexpressionofanti-apoptoticBcl-2protein.
Bcl-2 was expressed in the cytoplasm of mononuclear
cells both in the focal infiltrates of pSS patients and
interstitially in all study groups (Figure 4A). We
observed significantly higher expression of Bcl-2 in the
interstitial cells of pSS patients with FS ≥2 compared
with the three other groups (Figure 4B). Furthermore,
significantly higher numbers of Bcl-2+ interstitial cells
were found in the pSS tissue with FS = 1 comparing
with normal, but not with chronically inflamed tissue
(Figure 4B). When evaluating focal infiltrates, a dramatic
increase in the levels of Bcl-2 expressing cells was
observed in the pSS with high FS comparing with pSS
with FS = 1 (Figure 4C). GC positivity did not seem to
influence the numbers of Bcl-2 positive cells in the sali-
vary gland tissue included in this study.
Notably, no Bcl-2 negative plasma cells could be
detected in the salivary glands of pSS patients (both
FS = 1 and FS ≥2) (Figure 5A-D). In the chronically
inflamed and normal tissue both Bcl-2+ and Bcl-2-
plasma cells were observed (Figure 5E-H)). Even though
it is difficult to evaluate the intensity of the Bcl-2
expression, a more distinct cytoplasmic Bcl-2 staining
was noted in plasma cells from pSS salivary glands com-
pared to plasma cells from subjects with chronic inflam-
mation and normal salivary gland tissue (Figure 5).
Figure1CD138expressingplasmacellsin salivaryglandsof
pSSpatientsandnon-SScontrols.A)CD138+plasmacells
(brown)inpSSpatientwithFS=1,apSSpatientwithFS=4,ina Plasma cells in pSS salivary glands located in close
subjectwithchronicallyinflamedglands(CIG)andasubjectwith
contact with CXCL12 expressing cells
normalgland(NG)histology.CD138+plasmacellsweredetected
CXCL12 (also known as stromal derived factor 1; SDF-1)
interstitiallyassmallcellclustersclosetoductalandacinar
epitheliuminallpatientsandcontrols.InpSSglandswithFS=1, plays a crucial role in the migration and survival of
plasmacellsweredetectedmainlyintheperipheryoftheinfiltrates, plasma cells [22,23,27]. To investigate the possibility of
whereasinpSSglandswithhighFS,plasmacellsweredetected CXCL12 to facilitate plasma cell survival in the inflamed
bothinperipheryandwithintheinfiltrates.B)CD138+cells
salivary gland tissue, a double-staining with anti-CXCL12
presentedasnumberofinterstitialplasmacellpermm2oras
and anti-CD138 was performed.
C)percentageofCD138positiveplasmacellsinthefocalinfiltrates.
GreybarsrepresentFS=1(n=6),blackbarsrepresentFS≥2(n= CXCL12 staining was detected on acinar and ductal
10),stripedbarsrepresentchronicinflammation(n=5)andwhite epithelial cells in pSS patients and to a lesser extent in
barsrepresentnormalglandhistology(n=5).Resultspresentedas chronic inflammation and normal subjects (Figure 6A).
mean±SEM.StatisticalanalysesdeterminedbyKolmogorov-
CXCL12 was also expressed on the mononuclear cells
SmirnovandStudentt-test(*P<0.05,**P<0.005).
interstitially in pSS and chronic inflammation subjects
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Figure2IgAandIgGexpressingplasmacellsinsalivaryglandsofpSSpatientsandnon-SScontrols.A)IgAandB)IgGpositiveplasma
cellsdetectedinsalivaryglandsofapSSpatientwithFS=1,apSSpatientwithFS=4,inasubjectwithchronicallyinflamedglands(CIG)and
asubjectwithnormalgland(NG)histology.InsertsrepresenthighermagnificationsofIgAorIgGplasmacellsforeachgroup.IgAexpressing
plasmacellsweredetectedinterstitiallyassmallcellclustersclosetoductalandacinarepitheliuminallgroupstestedandintheperipheryof
infiltratesinpSSwithFS=1andFS≥2(A).IgGexpressingplasmacellswerefoundbothinsideandintheperipheryoflargefocalinfiltratesin
pSSpatientswithFS=1andFS≥2,whereasinterstitialaccumulationofIgGexpressingplasmacellswasobservedmostlyinthepSSpatients
withhighFS(B).C)AmountofIgAorE)IgGpositiveinterstitialplasmacellspresentedasnumberofcells/mm2andD)IgAorF)IgGpositive
cellsinthefocalinfiltratespresentedasnumberofpositivecellspermm2.GreybarsrepresentFS=1(n=8),blackbarsrepresentFS≥2(n=7),
stripedbarsrepresentchronicinflammation(n=5)andwhitebarsrepresentnormalglandhistology(n=5).Resultspresentedasmean±SEM.
StatisticalanalysesdeterminedbyKolmogorov-SmirnovandStudentttest(*P<0.05).
and in focal infiltrates of all pSS patients. No expression In pSS patients and chronic inflammation subjects,
of CXCL12 was detected on the interstitial cells in nor- CD138+ interstitial plasma cells were mainly observed
mal salivary gland tissue (Figure 6A). Additionally, we in close proximity to the CXCL12 expressing ductal and
observed expression of CXCL12 by resident adipocytes acinar epithelium and CXCL12 expressing mononuclear
present in some areas of salivary gland tissue of pSS cells (Figure 6A). In contrast, in normal tissue clusters
patients (Figure 6B). of CD138+ plasma cells were mainly observed in close
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Figure3Proliferatingandnon-proliferatingplasmacellsinsalivaryglandsofpSSpatientsandnon-SScontrols.A)AmountofKi-67+
interstitialcellsandB)Ki-67+cellsinthefocalinfiltratespresentedasnumberofcellspermm2.GreybarsrepresentFS=1(n=8),blackbars
representsFS≥2(n=7),stripedbarsrepresentchronicinflammation(n=5)andwhitebarsrepresentnormalglandhistology(n=5).Results
presentedasmean±SEM.StatisticalanalysesdeterminedbyKolmogorov-SmirnovandStudentttest(*P<0.05,**P<0.005and***P0.001).
C)Double-stainingwithCD138plasmacellmarker(red)andKi-67proliferationmarker(brown)inapSSpatientwithFS=1,inapSSpatient
withFS=4,inasubjectwithchronicallyinflamedglands(CIG)andasubjectwithnormalgland(NG)histology.Insertsrepresenthigher
magnificationsofCD138+Ki-67+plasmacells.Ki-67+plasmacellsweremainlyobservedintheperipheryoflargerinfiltrateswithhigh
proliferatingactivityandinsomesmallplasmacellaggregatesoftenincloseproximitytovascularstructures.
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Figure4Bcl-2expressionininfiltratinglymphocytesofpSSpatientsandnon-SScontrols.A)Bcl-2detectedinsalivaryglandsofapSS
patientwithFS=1,apSSpatientwithFS=4,inasubjectwithchronicallyinflamedglands(CIG)andasubjectwithnormalgland(NG)
histology.Bcl-2wasexpressedinthecytoplasmofmononuclearcellsbothinthefocalinfiltratesofpSSpatientsandinterstitiallyinallstudy
groups.B)AmountofBcl-2+interstitialcellsandC)Bcl-2+cellsinthefocalinfiltratespresentedasnumberofcellspermm2.Greybarsrepresent
FS=1(n=8),blackbarsrepresentFS≥2(n=7),stripedbarsrepresentchronicinflammation(n=5)andwhitebarsrepresentnormalgland
histology(n=5).Resultspresentedasmean±SEM.StatisticalanalysesdeterminedbyKolmogorov-SmirnovandStudentttest(*P<0.05,
**P<0.005and***P<0.001).
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Figure5Bcl-2expressionininfiltratingplasmacellsofpSSpatientsandnon-SScontrols.A)CD138(green)andBcl-2expressingcells
(red)detectedinapSSpatientwithFS=1.DAPI(blue)wasusedfornuclearstainingofsalivaryglandscells.Mergeimageshowsdouble
positive(CD138andBcl-2)cells.B)HighermagnificationsofCD138(green)andBcl-2(red)expressingcellsinapSSpatientwithFS=1.Merge
imagewithCD138+Bcl-2+plasmacells.OnlyBcl-2+plasmacellsweredetectedinthesalivaryglandsofpSSpatientswithFS=1.C)CD138
(green)andBcl-2expressingcells(red)detectedinapSSpatientwithFS=4.DAPI(blue)wasusedfornuclearstainingofsalivaryglandscells.
D)HighermagnificationsofCD138(green)andBcl-2(red)expressingcellsinapSSpatientwithFS=4.MergeimagewithCD138+Bcl-2+
plasmacell.OnlyBcl-2+plasmacellsweredetectedinthesalivaryglandsofpSSpatientswithFS≥2.E)CD138(green)andBcl-2expressingcells
(red)detectedinasubjectwithchronicallyinflamedglands(CIG).DAPI(blue)wasusedfornuclearstainingofsalivaryglandscells.F)Higher
magnificationsofCD138(green)andBcl-2(red)expressingcellsinasubjectwithCIG.MergeimagewithCD138+Bcl-2+andCD138+Bcl-2-
plasmacells.BothBcl-2+andBcl-2-plasmacellsweredetectedinthechronicallyinflamedtissue.G)CD138(green)andBcl-2expressingcells
(red)detectedinasubjectwithnormalgland(NG)histology.DAPI(blue)wasusedfornuclearstainingofsalivaryglandscells.H)Higher
magnificationsofCD138(green)andBcl-2(red)expressingcellsinasubjectwithNGhistology.MergeimagewithCD138+Bcl-2+andCD138+
Bcl-2-plasmacells.BothBcl-2+andBcl-2-plasmacellsweredetectedinthenormaltissue.