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Clin Exp Immunol. Oct 1999; 118(1): 154–163.
PMCID: PMC1905402

CD4 cytotoxic and dendritic cells in the immunopathologic lesion of Sjögren's syndrome

Abstract

The existence of CD4+ T lymphocytes with cytotoxic activity in minor salivary gland (MSG) biopsies from Sjögren's syndrome (SS) patients was investigated using in situ double immunohistochemistry technique. The presence of dendritic cells (DC) in SS lesions was examined by using single and double immunohistochemistry methods and a panel of different MoAbs to specific cell surface markers (i.e. CD3, CD11c, DRC). Furthermore, the ultrastructural morphology of DC was characterized by electron microscopy (EM). Immunogold labelling technique using the DRC surface marker was also applied. Finally, we investigated the existence of germinal centres (GC) in the salivary gland lesions of SS patients. Seven patients with primary SS and five patients with non-specific sialadenitis were the subjects of this study. Our results indicate the existence of a CD4+ cytotoxic cell population that utilizes perforin-mediated cell destructions as they expressed perforin mRNA. Quantitative analysis of these cells revealed that they comprised approximately 20% of the existing T lymphocytes. We also identified a population of CD4+ T cells that expressed the CD11c activation marker. Furthermore, we observed a distinct cell subtype which expressed the DRC cell surface marker. These cells had the characteristic ultrastructural morphology of DC and were DRC+ when examined by immunoelectron microscopy. Finally, the formation of GC structures in the histopathologic lesions of the salivary glands was observed. The above findings indicate that both CD4+ cytotoxic T lymphocytes (CTL) and DC may be involved in the initiation and perpetuation of SS pathogenesis. Moreover, the formation of GC in the lesions reveals a possible mechanism for in situ differentiation and proliferation of activated B lymphocytes.

Keywords: cytotoxicity, dendritic cells, Sjögren's syndrome

INTRODUCTION

Sjögren's syndrome (SS) is a chronic autoimmune disorder characterized by lymphocytic infiltrations of the salivary and lachrymal glands and B cell hyperreactivity leading to production of circulating autoantibodies [1]. One of the major immunopathologic events observed in minor salivary gland (MSG) biopsies of SS patients is epithelial cell destruction by infiltrating lymphocytes, leading to the subsequent replacement of the salivary gland tissue by mononuclear cells [2]. Previous studies have shown that epithelial cell destruction in MSG biopsies of SS patients is mediated by the mechanism of programmed cell death or apoptosis [3]. Two major pathways have been implicated in this process: (i) interactions between epithelial cells expressing the Fas molecule and surrounding T lymphocytes expressing Fas ligand (FasL); and (ii) perforin–granzyme secretion by cytotoxic T lymphocytes (CTL), leading to subsequent degeneration of epithelial cells [4].

In this study, we examined the phenotype of the cells with cytotoxic activity, as well as the mechanism employed by these CTL for epithelial cell destruction in MSG biopsies of SS patients. CTL activity is considered to be a property mainly of CD8+ class I-specific T cells and natural killer (NK) cells. However, many reports have documented the existence of CD4+ class-II restricted CTL in several murine and human diseases [58]. This population of CD4+ T cells with cytotoxic activity was found to utilize both Fas–FasL and perforin–granzyme-dependent mechanisms for target cell killing [9]. As is well documented, in MSG biopsies of SS patients CD4+ T cells comprise 60–70% of the infiltrating lymphocytes, while CD8+ T cells comprise only 10–20% of the lymphocytic population [10]. This observation prompted us to examine the possible existence of CD4+ T cells with cytotoxic activity in SS lesions. We applied in situ double immunohistochemistry technique and identified CD4+ T lymphocytes which expressed perforin mRNA.

The next step was to investigate which is the cell that participates in the antigen presentation mechanism and thus is responsible for the migration and retention of activated lymphocytes in the immunopathologic lesions. Phenotypic analysis of the cells participating in the pathogenesis of SS has shown that antigen-presenting monocytes and macrophages comprise less than 2% of the existing cells [10]. Recent reports have also documented the participation of epithelial cells of MSG of SS patients in the antigen presentation mechanism, as acinar and ductal cells express HLA-DR and B7 costimulatory molecules [1113]. However, the existence of dendritic cells (DC) in MSG biopsies of SS patients has not been sufficiently investigated [14].

In order to identify the existence and phenotype of DC in MSG biopsies of SS patients, we used single and double immunohistochemistry methods and identified a distinct cell subset phenotypically characterized as DRC+. This cell subset was morphologically and phenotypically characterized using electron microscopy (EM) and immunogold labelling, respectively.

Another important characteristic that is often observed in the immunopathologic lesion of the MSG of SS patients is the formation of focal lymphocytic clusters comprising activated B and T lymphocytes which resemble germinal centres [15]. As is well known, germinal centres (GC) normally arise in primary follicles of secondary lymphoid organs [16]. However, ectopic GC formation has been documented in non-lymphoid organs of many autoimmune diseases such as the thyroid gland of patients with Hashimoto's thyroiditis and rheumatoid arthritis synovial tissue [17,18]. In this study we investigated the existence of GC in MSG biopsies of SS patients using the cell cycle-associated nuclear antigen Ki-67 which discriminates between the characteristic dark and light zones of a mature GC.

PATIENTS AND METHODS

Patients

Seven patients fulfilling the diagnostic criteria for primary SS and five patients with non-specific sialadenitis (control group) were the subjects of this study [19]. MSG biopsies were obtained following the procedure used routinely for diagnosis. Tissues were embedded in OCT compound, snap-frozen in 2-methylbutane, chilled on liquid nitrogen and then stored at −70°C for sectioning. Following haematoxylin–eosin staining, the histopathological grading of the biopsy specimen was performed according to Chilsolms' classification [20].

In situ double staining

MSG biopsies from patients with SS were incubated at 4°C for 3 h with freshly made filtered fixative (DEPC-treated PBS containing 4% paraformaldehyde) and soaked at 4°C overnight in sucrose solution (DEPC–PBS containing 30% sucrose). Cryostat sections (8 μm) were cut and then dried in an oven at 40°C for 3 h. Sections were then post-fixed in 4% paraformaldehyde solution for 5 min, acetylated for 10 min in 0.1 m triethanolamine buffer containing 0.25% acetic anhydride and finally incubated at 37°C for 2 h in hybridization buffer. Digoxigenin (DIG)-labelled oligonucleotide probes were diluted in hybridization buffer and slides were incubated with diluted probes overnight in a humidified chamber at 42°C. Post-hybridization washes were as follows: 2× SSC for 15 min, 1× SSC for 30 min, 0.1× SSC for 30 min, all at 37°C. Hybridized probes were detected by using a sheep anti-DIG alkaline phosphatase antibody and the colour reaction was developed using NBT/BCIP purple-colour substrate for alkaline phosphatase. After the development of an optimal colour reaction slides were washed in TBS and subjected to an alkaline phosphatase anti-alkaline phosphatase (APAAP) immunohistochemical reaction for the detection of the CD4 molecule. Fast-red substrate was used to give a red colour to cells expressing the CD4 antigen in contrast to cells expressing perforin mRNA, which were labelled purple. No counterstaining was applied on sections.

Quantitative analysis

A semiquantitative analysis of the number of cells expressing both the CD4 antigen and perforin mRNA in the MSG was made by examining 10 contiguous fields across the whole section at × 40 magnification. Double-positive cells in each section were counted field by field at × 16 magnification on a television screen connected to a microscope (Image Pro-Plus 2.1 system) by two independent observers. The percentage of cells expressing both CD4 antigen and perforin mRNA in the sections was then determined.

Immunohistochemistry

Serial cryosections (6 μm) were air-dried for 60 min and fixed in acetone at 4°C for 10 min. For detection of the MoAbs against CD4 (T-cell marker; Becton Dickinson, Mountain View, CA), DRC (Dendritic Reticulum Cell; R4/23; Dako) and CD11c (anti-human protein p150, 95; Dako, Glostrup, Denmark), an indirect APAAP technique was applied. Briefly, sections were blocked in 10% non-immune horse serum for 20 min and incubated with primary antibody for 90 min at room temperature. Sections were subsequently incubated with rabbit anti-mouse immunoglobulin (Dako) for 30 min followed by a 30-min incubation with APAAP complex (Dako). In control sections, irrelevant isotype-matched antibodies were applied. Finally, sections were incubated in fast-red substrate (Dako) for 20 min to give a red colour reaction and counterstained with Mayer's haematoxylin (Sigma Chemical Co., St Louis, MO). The immunoperoxidase technique was employed for the identification of the following primary MoAbs: CD3 (T-cell marker; Becton Dickinson), CD4 (T-cell marker; Becton Dickinson). Diaminobenzidine (DAB; Sigma) was applied as chromogen. Finally, sections were counterstained with Mayer's haematoxylin.

For identification of GC, the polyclonal antibody against the Ki-67 (Dako) antigen was used. Immunoperoxidase technique was applied on paraffin-embedded tissue sections while positive staining was detected using DAB as chromogen. Finally, sections were counterstained as described above.

For double-staining experiments, the CD4+ T lymphocytes were visualized using the immunoperoxidase technique (brown colour reaction), while the DRC+ cells were visualized using the APAAP technique (red colour reaction).

Transmission electron microscopy

MSG biopsies were cut in 1-mm3 pieces and fixed in 2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer pH 7.4 and post-fixed in 2% aqueous osmium tetroxide. Tissues were dehydrated in a graded series of ethanol solutions, embedded in Epon-Araldite resin mixture and polymerized at 60°C for 48 h. The areas of lymphocytic infiltrations were localized using phase contrast microscopy. Subsequently, ultrathin sections on these areas were cut and stained with lead citrate and uranyl acetate. Morphological observation of the sections was performed using a Philips EM300 electron microscope.

Evaluation of the presence of cells with dendritic morphology in ultrathin sections was performed by two independent observers who identified the existence of these cells in several resin blocks from each MSG biopsy specimen tested. Seven different resin blocks from each salivary gland tissue sample were examined. In order to estimate the percentage of cells with dendritic morphology in relation to the total cell number, we examined a total number of 28 different grids from each tissue sample. Cell counts were performed using a digital image analysis system on low EM magnification.

Immunogold staining

Immunoreagents

The following MoAbs were used: anti-DRC (Dendritic Reticulum Cell, R4/23; Dako) and anti-CD3 (T-cell marker; Becton Dickinson). Anti-CD3 MoAb was used as a positive control for the immunogold staining procedure. A 5-nm colloidal gold-labelled goat anti-mouse antibody (Biocell, Cardiff, UK) was employed to detect the MoAbs.

Method

A pre-embedding immunogold labelling technique was applied as described by Manara et al. [21]. Briefly, MSG biopsies were cut in small pieces (< 0.5 mm3) and prefixed in 2% paraformaldehyde in PBS pH 7.4 for 20 min. The samples were transferred to 0.1% Tween-20 in PBS for 30 min and subsequently incubated in blocking solution containing 10% horse serum and 1% bovine serum albumin (BSA) for 30 min. Specimens were incubated with the MoAbs overnight at 4°C. Afterwards, tissue samples were washed in PBS for 1 h and incubated with 5-nm gold-labelled goat anti-mouse antibody (1:10 dilution in 1% BSA, TBS pH 8.2) for 60 min. Specimens were then subjected to the silver enhancement procedure. Tissue samples were then washed in PBS for 1 h and fixed in 1% glutaraldehyde in PBS. They were then osmicated in 1% OsO4 in PBS, dehydraded in ethanol and embedded in Epon–Araldite resin mixture. Ultrathin sections were selected on uncoated grids and examined unstained in a Philips EM-300 electron microscope.

The immunolabelling specificity was checked by (i) substituting the primary antibody with PBS–1% BSA; and (ii) replacing the MoAbs with non-reactive IgG of the same isotype.

RESULTS

CD4 cytotoxic cells expressing perforin mRNA in MSG biopsies of patients with SS

The in situ double-staining experiments revealed that 20% of the CD4+ cells expressed perforin mRNA in the immunopathologic lesions of MSG of SS patients (Fig. 1a,b).

Fig. 1
(a) In situ double immunohistochemistry technique on a cryostat section from a minor salivary gland biopsy of a Sjögren's syndrome (SS) patient using the anti-sense probe of perforin mRNA. CD4+ T lymphocytes (red colour) which express perforin ...

Existence and localization of cells with dendritic morphology in MSG biopsies of SS patients

The expression of the CD11c+ marker that is present on DC, mainly those of the mature phenotype, was examined [21]. Using serial sections it was observed that the CD11c+ molecule was mainly expressed by CD4+ lymphocytes in the infiltrates of the salivary gland tissue of SS patients (Fig. 2a,b). Since the CD11c molecule could not solely identify cells of the DC lineage the next step of our study was to investigate the expression of the dendritic reticulum cell (DRC) molecule, which is a cell surface marker expressed by follicular DC as well as by some B lymphocytes. We thus identified a cell subset which expressed the DRC molecule. For localization of the DRC+ cells observed in the MSG biopsies of SS patients double-labelling experiments were performed. DRC+ cells (red colour) were located mainly in periacinar and periductal areas (arrows) near CD4+ (brown colour) large lymphocytic infiltrations (Fig. 3a).

Fig. 2
Serial cryostat section from a minor salivary gland biopsy (MSG) of a Sjögren's syndrome (SS) patient using the anti-CD11c (a) and anti-CD4 MoAbs (b). A large population of CD4+ T lymphocytes expressed the activation marker CD11c (× 200). ...
Fig. 3
(a) Double immunohistochemical staining showing CD4+ T lymphocytes (brown colour) and DRC+ cells (arrows) (red colour). DRC+ cells were located in periacinar and periductal areas near a CD4+ large lymphocytic infiltration (× 200). (b) Minor salivary ...

In contrast, immunohistochemical studies in MSG biopsies of patients with non-specific sialadenitis (control group) did not show positive staining for the CD11c molecule (data not shown). Furthermore, it was not possible to identify a similar population of DRC+ cells in the MSG biopsies of the control group (Fig. 3b).

Morphological evaluation of DC by transmission electron microscopy

Since the DRC molecule does not only identify cells of the DC lineage, we further characterized the DRC+ cell subset using EM. The ultrastructural studies in MSG biopsies from patients with SS revealed a population of cells which bore a complex network of cytoplasmic processes (dendrites) and retained large lobulated nuclei with electron dense peripheral heterochromatin. The cytoplasmic processes of these cells contained only few organelles, while characteristic multivesicular bodies could be seen in the perinuclear area (Fig. 4a). Among these morphological features the formation of desmosomes between these cells and lymphocytes was further observed (Fig. 4b). The above data indicate that these cells could be of DC origin, as they had many characteristic features often described for DC [21]. Furthermore, our results indicate that MSG biopsies with a lymphocytic focus score > 1.5 (as classified by Chilsolm's criteria) had lymphoid follicles which contained cells with dendritic morphology. These DC were located among T and B lymphocytes and comprised approximately 2% of the existing mononuclear cells. In contrast, MSG biopsies of SS patients with a lymphocytic focus score between 1.0 and 1.5 had a smaller population of cells with dendritic morphology. Finally, in MSG biopsies with a lymphocytic focus score of 0.6–1.0 DC were not present.

Fig. 4
(a) Transmission electron micrograph of a dendritic cell (DC) with characteristic cytoplasmic processes (arrow), lobulated nucleus with perinuclear condensed heterochromatin and multivesicular bodies (arrowheads) in the cytoplasm. Few cytoplasmic organelles ...

Immunoelectron microscopic characterization of DRC+ cells

DRC+ immunogold-stained cells with characteristic ultrastructural morphology of DC were identified in MSG biopsies of SS patients. The colloidal gold particles were localized mainly on the membrane of cells which resembled DC (Fig. 4c). The DRC+ cells observed in SS lesions were probably DC of the immature phenotype, as they had a round morphology and contained few cytoplasmic processes [22]. DRC+ staining was specific, as adjacent lymphocytes remained unstained (data not shown). Some silver precipitates were observed on the nuclear chromatin and inside the cytoplasm that probably represented a non-specific reaction with metal-linked proteins. Staining with the anti-CD3 MoAb revealed a T lymphocyte with many gold particles on its cell membrane (data not shown). In tissue specimens incubated with non-immune mouse IgG no staining was observed.

Formation of germinal centres in MSG biopsies of patients with SS

Staining with the anti-Ki-67 polyclonal antibody which binds to a nuclear antigen expressed in all stages of the cell cycle except G0 revealed the characteristic dark and light zones of a GC. The dark zones consisted of rapidly dividing cells stained with the–anti-Ki-67 antibody and the light zones comprised non-dividing cells that remained unstained (Fig. 5a). Serial sections revealed that the Ki-67+ cells were CD20+ B lymphocytes (data not shown), while the light zones contained Ki-67CD3+ T cells (Fig. 5b). Secondary follicles containing characteristic GC structures were not similarly identified in MSG biopsies of the control group.

Fig. 5
(a) The classical dark (arrow) and light zones of a mature germinal centre (GC) in a minor salivary gland (MSG) biopsy from a Sjögren's syndrome (SS) patient identified with the anti-Ki-67 MoAb (× 100). (b) Serial section stained with ...

DISCUSSION

In this study an attempt was made to characterize different mechanisms involved in the immunopathology of SS. Our results indicate the presence of a population of CD4+ T cells armed for cytotoxic activity that participate in the induction of apoptosis in MSG biopsies of SS patients. These CD4+ CTL cells comprise 20% of the infiltrating lymphocytes and utilize perforin-mediated cell destruction as they express perforin mRNA.

Many investigators have described the existence of CD4+ T cells with cytotoxic activity in human diseases [23]. These CD4+ CTL play an important role in terminating immune responses, as they utilize FasL-dependent mechanisms for destruction of activated T lymphocytes [24]. However, CD4+ CTL that employ perforin–granzyme-mediated cell destruction are considered to have a protective role during viral infection rather than an immunomodulatory one [9]. According to the latter hypothesis, in the absence of CD8+ T lymphocytes during an inflammatory immune response, CD4+ CTL which secrete perforin and granzymes develop in the lesion in order to eliminate virus-infected cells and act as the organism's back-up mechanism. In SS, the existence of proviral retroviral sequences in epithelial cells of the salivary glands of SS patients has been identified [25,26], while EM studies revealed intracisternal A-type particles related to HIV in lymphoblastoid cells co-cultured with homogenates of salivary glands of SS patients [27]. Furthermore, HIV-infected patients develop the characteristic symptoms of dry mouth and eyes manifested in SS [28]. From the above data, one could speculate that an infection of epithelial cells by an as yet unidentified virus could be the initiating factor for the development of CD4+ T lymphocytes with cytotoxic activity in SS lesions in order to prevent further spreading of the viral infection, as well as the subsequent T cell over-stimulation and autoantibody production.

Another observation that further characterizes the activation state of CD4+ T cells in SS lesions is that the majority of these cells expressed the CD11c molecule. The CD11c molecule belongs to the β2 subfamily of integrins and participates in cell–cell interactions as well as cell–extracellular matrix adhesion [29]. This molecule is mainly expressed by monocytes, macrophages, activated B lymphocytes and DC. However, it has been found that activated T lymphocytes also express the CD11c molecule in some pathological conditions. The expression of the CD11c molecule by activated T lymphocytes could be associated with their ability to adhere to epithelial cells and also with the induction of CTL activity towards interacting epithelial cells [30]. Our present data demonstrate that the CD11c molecule could be an activation marker of the CD4+ T cell subset, although its exact role in the pathogenic mechanism of SS remains to be further elucidated.

Additionally, the immunophenotypic studies revealed a distinct cell population that expressed the DRC molecule. These cells had the characteristic ultrastructural morphology of DC when examined by EM. This finding was further supported by the immunogold labelling technique that showed the presence of DC with DRC+ gold particles on their cell membrane.

The expression of the DRC molecule by DC indicated that these cells might belong to the follicular DC lineage and comprise an FDC network in the residing lymphoid follicles. Moreover, the formation of desmosome junctions between DC and lymphocytes is a characteristic feature of follicular dendritic cells (FDC) and emphasizes the FDC origin of these cells [21]. This FDC network could also participate in the ectopic formation of GC. In agreement with this hypothesis, the presence of GC in the histopathologic lesions of SS patients was observed. These GC contained characteristic dark zones with Ki-67+/CD20+ B lymphocytes and light zones with Ki-67/CD3+ and few Ki-67+/CD20+ B cells. As is well known, during the GC reaction in lymphoid organs B cells undergo somatic hypermutation of the rearranged immunoglobulin V genes and immunoglobulin class switching. Those cells that express antigen receptors with high affinity towards the stimulating antigen are selected by competition for antigen which is retained on the surface of FDC [31]. The residing FDC within GC of salivary glands from SS patients could retain self antigens on their surface and thus mediate in situ B cell clonal differentiation and activation. In accordance with this, a recent publication documented the existence of clonally differentiated antigen-specific B lymphocytes in large lymphoid follicles resembling GC in MSG biopsies of SS patients [32,33]. The autoantigens, which are possibly retained on the FDC surface, could originate from epithelial cells which die of apoptosis during their interaction with activated T lymphocytes.

Our observations indicate that DC were present in large lymphoid follicles in the inflamed tissue while they were rarely found in lesions of early disease state. This finding is in concert with another report by Aziz et al., who observed the existence of DC cells mainly in MSG biopsies of SS patients with a lymphocytic focus score > 2 [14].

In conclusion, all the above findings indicate that CD4+ CTL and antigen-presenting DC are involved in the induction and perpetuation of SS. However, the exact mechanisms through which these cells interact and contribute to the pathogenesis of this autoimmune disease as well as the initiating aetiopathological factor remain to be further elucidated.

Acknowledgments

The authors are grateful to Dr S. Paikos for obtaining the minor salivary gland biopsies. This work was supported by grant 70/3/2826 PENED, the Greek Ministry of Industry, Energy and Technology.

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