Activation of epithelial and myoepithelial cells in the salivary glands of patients with Sjögren's syndrome: high expression of intercellular adhesion molecule-1 (ICAM.1) in biopsy specimens and cultured cells
E. K. Kapsogeorgou,
Laboratory of Immunology, Department of Pathophysiology, School of Medicine, National University of Athens, Athens, Greece
Dr M.N. Manoussakis, Dpt. of Pathophysiology, School of Medicine, National University of Athens, Mikras Asias 75, Athens 11527, Hellas. E-mail: firstname.lastname@example.org
ICAM.1 (CD54) is a surface protein expressed on epithelial and other nonhematopoietic cells upon activation and is known to play an important role in the stimulation of T cells by the provision of cellular adhesion and costimulatory support. Sjogren's syndrome (SS) is an autoimmune exocrinopathy, which is characterized by chronic lymphocytic infiltration of exocrine glands and aberrant activation of epithelial tissues. To address the contribution of ICAM.1 in the pathogenesis of SS, the expression of this protein was studied by immunohistochemistry and flow cytometry in minor salivary gland (SG) biopsies as well as in cultured SG epithelial cell (SGEC) lines obtained from 18 SS patients and 16 controls. In biopsies from SS patients (but not controls), strong ICAM.1 was expressed by infiltrating mononuclear cells (52%) and by a significant proportion of periacinar myoepithelial cells (18%). In addition, a patchy pattern of moderate ICAM.1 expression was detected in 31% of ductal epithelia of SS patients. These ICAM.1-expressing epithelial and myoepithelial cells were observed throughout glandular tissues and were not confined in areas proximal to lymphoid infiltrates. In support to an intrinsic activation profile of SGEC in SS, long-term cultured non-neoplastic SGEC lines derived from SS patients displayed significantly upregulated spontaneous expression of ICAM.1, compared to controls (P < 0.05). The high expression of ICAM.1 protein by the salivary epithelium of SS patients is likely suggestive of its important role in the pathogenesis of the disorder. Further, our results support a model of intrinsic activation of salivary epithelial and myoepithelial cells in SS, whereby these cells actively participate in the induction and maintenance of lymphocytic infiltrates of patients.
Sjogren's syndrome (SS) is a relatively common autoimmune disease characterized by the dysfunction and destruction of several exocrine glands associated with lymphocytic infiltrates . Several lines of evidence indicate that epithelial cells are implicated in the pathogenesis of the disorder [2,3], most likely by acting as nonclassical antigen-presenting cells . Intercellular adhesion molecule-1 (ICAM.1/CD54) is a surface protein that is expressed on various cell types, constitutively or after activation by cytokines . Along with its ligand Leucocyte Function-Associated antigen-1 (LFA.1, CD11a/CD18), ICAM.1 has been shown to participate in leucocyte adhesion , as well as in the costimulation of T lymphocytes activation . The aberrant expression of ICAM.1 protein in lesional biopsy tissues and the protective capacity of blocking antibodies indicate its crucial role in the pathogenesis of several human and experimental autoimmune disorders [7–10].
The possible role of ICAM.1 in the induction of histopathologic lesions of SS has been previously addressed. Increased ICAM.1 expression has been observed, however, the cellular distribution of this expression has been controversial. Although, this has been primarily attributed to infiltrating mononuclear cells [11–18], limited ductal or acinar glandular ICAM.1 expression has been described in some studies [15–17], whereas increased glandular ICAM.1 expression has been observed in SS patients with tubulointerstitial nephritis . Fortuitous preliminary immunochemical observations had indicated to us that the epithelial structures are also an important source of ICAM.1 expression in minor salivary gland biopsy samples and cultured epithelial cells from patients with SS. To better evaluate this, we sought to re-address the cellular pattern of ICAM.1 expressed in SS lesions by the immunohistochemical analysis of several biopsy specimens obtained from patients with well-defined SS as well as from disease controls. In addition, we aimed to study the processes that influence ICAM.1 expression by SGEC in the lesions of SS patients. For this purpose, non-neoplastic SGEC lines were established from the minor salivary glands of patients and disease controls and the level of ICAM.1 molecules expressed spontaneously or under the influence of T cell-derived cytokines was examined.
Patients and methods
Antibodies, cytokines and culture media
Purified and/or phycoerythrin-conjugated monoclonal antibodies against human CD54/ICAM.1 (clone HA58) and CD11a/LFA-1 (clone HI111) were purchased from Pharmingen (San Jose, CA, USA). Monoclonal antibodies to human epithelial membrane antigen (clone E29), cytokeratin 19 (clone RCK108), high molecular weight (high-mw) cytokeratins (cytokeratins 1, 5, 10 and 14, clone 34βE12) and alpha-smooth muscle actin (clone 1A4) were obtained from Dako (Glostrup, Denmark). Monoclonal antibodies to human cytokeratins 8 and 18 (clone CAM 5·2) and to human CD3 (clone SK7) were from Becton-Dickinson (San Jose, CA, USA). Human recombinant interferon-gamma-1b (IFNγ, Imukin, specific activity 3 × 104 IU/μg) was from Boehringer (Ingelheim, Germany), human recombinant tumour necrosis factor-alpha (TNFα, specific activity 2 × 104 IU/μg) was from Endogen (Cambridge, MA, USA) and interleukin-1β (IL-1β) was from R & D Systems (Minneapolis, MN, USA). The serum-containing epithelial cell medium (SEM, consisted of a 3 : 1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium [Gibco, NY])  and the serum-free Keratinocyte Basal Medium (KBM, Clonetics, Walkersville, MD, USA)  were applied for the propagation of salivary gland epithelial cells. Both media were supplemented with 0·4 μg/ml hydrocortisone (Upjohn, Kalamazoo, MI, USA), 0·125 units/ml insulin (Novo, Bagsvaerd, Denmark), and 10 ng/ml epidermal growth factor (EGF, Sigma, MO, USA), 2 mm glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin. In addition, SEM was supplemented with 2·5% fetal bovine serum (FBS, Gibco), whereas KBM was supplemented with 25 μg/ml bovine pituitary extract (Sigma). In certain experiments, complete KBM was further supplemented with calcium chloride (Sigma) to a final Ca2+ concentration of 0·80 mm (calcium-supplemented KBM, CS-KBM), which is equivalent to that of SEM.
Minor salivary gland (SG) biopsies
Labial minor SG biopsies were obtained with informed consent from 34 individuals (all women) during their diagnostic evaluation for symptoms and signs indicative for primary or secondary SS (primary SS; SS found alone, secondary SS; SS found in conjunction with another autoimmune disease), as established by the European classification criteria . Accordingly, patients studied (Table 1) included 11 with primary SS and 7 with secondary SS (in five associated with systemic lupus erythematosus [SLE], in one with rheumatoid arthritis and in one with diffuse scleroderma). Control group consisted of 16 patients who did not fulfil the European classification criteria for SS. None of the patients had evidence of lymphoma, essential mixed cryoglobulinemia or infection by hepatitis B virus, hepatitis C virus or human immunodeficiency virus-1. Biopsy specimens were snap frozen or paraffin-embedded for tissue immunohistochemical analyses or placed in culture medium and processed for the development of epithelial cell cultures.
Table 1. Features of patients with primary and secondary SS and controls included in the study
Mean duration of sicca manifestations (years, ± SE)
2·5 ± 1·1
1·6 ± 0·8
1·1 ± 0·3
Number of SS criteria fulfilled (mean ± SE)
4·3 ± 0·2
3·2 ± 0·2
1·0 ± 0·0
Biopsy focus score (mean ± SE)
2·1 ± 0·2
1·9 ± 0·4
0·1 ± 0·0
Anti-Ro(SSA) positive (%)
Anti-La(SSB) positive (%)
Development and culture of SG epithelial cell lines
Primary SG epithelial cell (SGEC) cultures were established from minor SG biopsies (one lobule per patient) by explant outgrowth technique as previously described . SGEC cultures were maintained as previously . The epithelial origin of cultured SGEC lines was routinely verified by morphology as well as uniform and consistent staining with monoclonal antibodies to epithelial membrane antigen and the various cytokeratins, and absence of surface T and B cell markers. Cultured cells were not apparently of myoepithelial origin, as it was illustrated by lack of reactivity to alpha-smooth muscle actin monoclonal antibody. For immunocytochemical studies, SGEC lines (at second to third passage) were cultured in parallel onto 8-or 16-well multichamber slides (Nunc, IL, USA) for five days, at a density of 1·5 × 104 or 0·75 × 104 cells/well, respectively. Subconfluent cultures treated for 24–72 h with or without IFNγ (500 IU/ml), TNFα (200 IU/ml) or IL-1β (5 ng/ml) were also similarly prepared.
Immunohistochemistry and immunocytochemistry
For immunohistochemical analyses, biopsy specimens were sectioned (4-μm) and mounted on aminoalkylsilane (Sigma). Slides with tissue samples or multichamber slides with cultured SGEC lines were fixed in methanol-acetone solution (1 : 1, 5 min at −20°C) and immediately analysed. In addition to fresh-frozen sections, the presence and location of cytokeratin- and alpha-smooth muscle actin-positive myoepithelial cells was also investigated in paraffin-embedded biopsy specimens that were subjected to citrate-treatment prior to immunohistochemistry. Monoclonal mouse antibodies to specific antigens and isotype controls were used in an indirect avidin-biotin immunoperoxidase technique, as previously described . Staining of biopsy sections or slides with cultured cells was evaluated blindly by three independent investigators (EKK, IDD, MNM), unaware of the diagnosis or antibody used. In biopsy specimens, the staining of different cellular elements by each antibody was recorded. Positive cells were counted in each of 10 different fields across the whole section at 100× magnification and expressed as percentage of infiltrating mononuclear cells, acinar epithelial, ductal epithelial, fibroblasts or myoepithelial cells. Myoepithelial cells present in SG tissues were identified by characteristic spindle-shape morphology, basal abluminal location around ducts and acini and strong immunoreactivity for cytokeratins and smooth muscle actin antigen [21–24]. Myoepithelial cells were distinguished from fibroblastoid cells, which were not expressing the above antigens. The coexpression of ICAM.1 with other antigens was determined by immunostaining of adjacent sections. The staining intensity (SI) of cultured cells was scored on an arbitrary scale from 0 to 3+ (0: absent, 1+ : weak, 2+ : definite, 3+ : strong staining). The results were analysed by unpaired two-tailed Student's t-test and chi-square test with Yate's correction, as well as by the nonparametric Wilcoxon and Spearman rank correlation coefficient tests, where appropriate.
Flow cytometric analysis
The expression of cell surface ICAM.1 protein on cultured SGEC (5 × 104 cells) was analysed with a FACSCalibur flow cytometer using the CellQuest software (Becton-Dickinson) and phycoerythrin-conjugated monoclonal antibody to ICAM.1 (Pharmingen) or an isotype control (Becton-Dickinson) by standard procedures and with appropriate forward and side scatter adjustments for epithelial cells.
Cellular expression of ICAM.1 and LFA.1 proteins in minor SG biopsies
Immunohistochemical stainings for ICAM.1 and LFA.1 proteins were performed in 17 minor SG biopsies, including 9 obtained from patients with SS (six with primary SS and three with secondary SS associated with SLE) and 8 derived from disease controls. Control specimens only occasionally displayed ICAM.1 expression, which involved a limited number of scanty resident mononuclear cells and a few strongly positive fibroblastoid cells around certain ducts, whereas no evidence of ICAM.1 expression by ductal or acinar epithelial cells was found (Figs 1g and 2).
SS biopsy specimens exhibited significantly higher ICAM.1 expression compared to controls (Figs 1a–f and 2). This was largely attributed to the strongly positive T cell infiltrations, as identified by positive anti-CD3 staining in adjacent sections (data not shown). In these specimens, salivary gland myoepithelial cells (and to a lesser extent periductal fibroblastoid cells) were also a remarkable source of strong ICAM.1 expression (Fig. 1d,e). This was particularly evident in specimens derived from patients with primary SS, where the proportions of ICAM.1-positive myoepithelial cells (mean ± standard error; 26·0 ± 9·1) and fibroblasts (8·0 ± 2·3) were significantly higher to those with secondary SS (3·5 ± 2·0, P < 0·01 and 0·4 ± 0·2, P < 0·02, respectively) (Fig. 2). In SS tissues, ICAM.1-expressing myoepithelial cells were primarily detected in periacinar locations and frequently distantly from lymphoid infiltrates (Fig. 1d). In addition, a significantly high prevalence of ICAM.1-expressing ductal (but not acinar) epithelial cells was observed in SS specimens, but not controls (P < 0·001, Fig. 1a,c–h). Such ICAM.1 expression was mostly patchy and quite often involved an increased proportion of total ductal cells (i.e. 6/9 of SS specimens manifesting positive staining of > 25% of ductal cells) (Fig. 2). However, its staining intensity was weaker compared to that of infiltrating mononuclear and myoepithelial cells, and thus, it could be easily overlooked in regions adjacent to lymphoid infiltrates. In general, there was no apparent relationship between the localization of mononuclear cell infiltrates and ICAM.1 expression by ductal epithelial or myoepithelial cells, as the latter was observed throughout glandular tissues and was not confined in areas proximal to lymphoid infiltrates. In fact, no evidence of ICAM.1 expression was noticed in certain ducts surrounded by heavy lymphocytic infiltrates (data not shown). Furthermore, no correlation was found between the intensity or the cellular distribution of ICAM.1 expression and the biopsy focus score values (data not shown).
Staining with anti-LFA.1 antibody revealed positive cells solely among infiltrating or resident mononuclear cells present in SS and control biopsies, respectively (Fig. 1b,f,h). In all cases the intensity of LFA.1 expressed by mononuclear cells was comparably high to that of ICAM.1 (Fig. 1b,f). Compared to the resident mononuclear cells of controls, the inflammatory lymphocytes in the salivary glands of SS patients displayed significantly higher proportions of LFA.1-expressing cells (mean ± standard error; 29·3 ± 3·6 versus 53·4 ± 7·6, respectively, P < 0·05).
Constitutive and cytokine-induced expression of ICAM.1 protein by long-term cultured SGEC lines
In preliminary experiments, the expression of ICAM.1 was found essentially unchanged through long-term cultivation of established cell lines (data not shown). In addition, no significant differences in the morphology, the overall survival or proliferation rates were observed between established SGEC lines from patients with SS and controls. In this study, 19 SGEC lines grown in parallel in the standard SEM or KBM culture media were selected for the assessment of ICAM.1 expression by immunocytochemistry and included 11 derived from patients with SS and eight from control individuals. The majority of these cell lines were found to express constitutively low or moderately high levels of ICAM.1 protein (Fig. 3), but not LFA.1 (data not shown). This was particularly apparent in SGEC lines cultured in SEM (Fig. 3a,b), whereas cultivation in the serum-free low calcium-containing KBM culture medium was associated with significantly diminished spontaneous ICAM.1 expression (Fig. 3c). In KBM-based cultures, the down-regulation of spontaneous ICAM.1 expression occurred in the vast majority of cells with the exception of a small ICAM.1-positive subpopulation of SGEC (approximately 5–10% of total cultured cells), mostly distinguishable by their large and flattened appearance (Fig. 3c). The differential expression of ICAM.1 in SEM-and in KBM-based cultures was also verified by flow cytometry analysis of selected cell lines derived from SS and controls applied (data not shown). In SEM-based cultures, the spontaneous expression of ICAM.1 was intense (i.e. SI score ≥2+) in 15 of 19 SGEC lines tested and involved all cell lines derived from primary SS patients (11/11), compared to half (4/8) of controls (P < 0·05) (Table 2). Mean SI scores (± standard error) for ICAM.1 were also significantly higher in SS cultures compared to controls (SS: 2·5 ± 0·2, Controls: 1·4 ± 0·4, P < 0·05). The down-regulation of ICAM.1 on SGEC lines cultivated in KBM was associated with the acquisition of morphological features indicative of less differentiated epithelial cells (i.e. round, without cell-to-cell contact) (Fig. 3c). The rather low calcium content in KBM (0·15 mm) has likely a role in these phenotypic changes [19,25,26]. To examine this, the expression of ICAM.1 was studied in parallel SGEC cultures, which were set up in standard KBM medium and in calcium-supplemented KBM (CS-KBM). Upon transfer to CS-KBM, SGEC acquired morphological features largely identical to those of cells cultivated in SEM (i.e. elongated and flattened appearance and increased intercellular contact) (Fig. 3e,f). Under these conditions, cell lines derived from SS patients regained significant amounts of ICAM.1 (in 4/5 cell lines tested, with maximally restored expression being observed in more than half of cells in each cell line), whereas all five control cultures remained unchanged (Fig. 3e,f). As assessed by flow cytometric analysis, treatment of SGEC lines with IFNγ resulted in the induction of significant ICAM.1 expression (approximately 300-fold, compared to untreated cells, Fig. 3d and data not shown). ICAM-1 was less efficiently induced by TNFα and only marginally by IL-1β (approximately 20–25-fold and 2-fold induction, respectively, data not shown). The degree of ICAM.1 upregulation by the above cytokines was not found to differ between SGEC lines derived from SS patients and those obtained from controls, as well as between the various culture media applied (data not shown).
Table 2. Constitutive ICAM.1 expression in cultured salivary gland epithelial cell (SGEC) lines from SS patients and controls
SGEC lines (No. tested)
No. of positive cell lines ICAM.1 staining intensity score
Results are expressed as staining intensity score on an arbitrary scale from 0 to 3 + (0: absent, 1 + : weak, 2 + : definite, 3 + : strong staining).
Controls (n = 8)
SS (n = 11)
The role of ICAM.1 in the pathogenesis of Sjogren's syndrome has been previously investigated [11–18]. Although in all previous reports, the mononuclear infiltrates appeared the principal cellular source of ICAM.1 in tissues from SS patients, ductal or acinar glandular epithelial expression has been reported in some of the studies [15–18]. We presently sought to re-examine the issue, as directed by our preliminary findings from the assessment of salivary gland biopsy tissues and cultured SGEC lines of SS patients, which demonstrated significant ICAM.1 expression on epithelial cells.
We were able to reconfirm the high expression of ICAM.1 and LFA.1 proteins by the infiltrating mononuclear cells in SS lesions and also to verify considerable ICAM.1 expression on the glandular structures themselves in the salivary tissues of patients, but not controls. Besides mononuclear infiltrating cells, strong immunostaining originated primarily from myoepithelial cells, whereas a patchy pattern of moderate epithelial ICAM.1 expression was also evident on a significant proportion of the ductal structures of patients. Most certainly, the strikingly strong ICAM.1 expression by myoepithelial cells of SS patients requires particular attention. To our knowledge, the phenotypic characteristics of myoepithelial cells present in SS glands have not been previously studied. Myoepithelial cells are ectoderm-derived cells that express both epithelial and stromal cell features and are normally found in various exocrine glands, including salivary glands . These cells are located between the basal lamina and the abluminal aspect of acinar and ductal cells and are mainly thought as part of a contractile apparatus that supports the expulsion of saliva . However, evidence indicates that myoepithelial cells may serve a more complex role, including the preservation of structural integrity of the glands and the modulation of glandular secretory functions [23,24]. The significance of myoepithelial cells in the pathophysiology of SS is unclear, however, our data likely suggest the implication of these cells in the induction of lymphocytic infiltrates of patients. In fact, by virtue of their abluminal location, myoepithelial cells represent the foremost glandular cell lining to deliver localization and activation signals to immune cells.
Our study implicates the ICAM.1 protein in the pathogenesis of SS, as indicated by its increased and specific epithelial expression in the salivary glands of patients. Moreover, the quite frequent detection of these ICAM.1-expressing epithelia in the absence of visible lymphoid cell accumulations is apparently indicative of the intrinsic activation of these cells. However, the patchy pattern of ICAM.1 expression by the inflamed epithelia of SS patients most likely suggests that the level of surface expression is probably under complex regulatory microenvironmental influences and may be also temporally related with the inflammatory process itself. In line with this, proinflammatory cytokines such as those produced in the lesions of SS patients , are known to stimulate both the surface expression and the shedding of epithelial ICAM.1 . Furthermore, in various models of autoimmune and nonautoimmune disorders associated with T cell infiltrative lesions, the onset of the injuries has been shown to be preceded by the induction of epithelial ICAM.1, whereas increased expression correlated with early disease and is reduced in later stages [9,29]. Finally, epithelial type-specific regulatory mechanisms might also participate, as implied by the differential ICAM.1 expression observed between acinar epithelial, ductal epithelial and myoepithelial cells. In this context, it may be relevant that the epithelial-specific protein secretory component, which is reportedly over-expressed by the salivary acinar cells of SS patients , may act as an intrinsic inhibitor of ICAM.1 expression .
To further investigate the epithelium-associated processes that operate in patients we assessed the ability of SGEC obtained from SS patients and controls to express ICAM.1 constitutively or after treatment with T cell-derived cytokines, under various culture conditions. Control SGEC lines cultured in the standard serum-containing SEM medium were frequently found to express constitutively low to moderate amounts of ICAM.1 molecules. However, it is noticeable that under the same culture conditions, cell lines obtained from SS patients manifested significantly higher spontaneous expression of ICAM.1 protein compared to controls. This finding, along with the presumed ductal origin of SGEC cultures , is probably in direct agreement with the widespread expression of ICAM.1 by ductal epithelia of patients. To our opinion, as also illustrated for other immune reactivity-related molecules , the upregulated spontaneous expression of ICAM.1 in the SGEC of SS patients is highly indicative of an intrinsic activation operating in these cells. This is further supported by the application of regular (low calcium-containing) and calcium-supplemented formulations of the KBM culture medium. The cultivation of cells in the regular KBM invariably resulted in the down-regulation of spontaneous ICAM.1 expression in SGEC, whereas calcium-supplemented KBM was able to restore the spontaneous ICAM.1 expression only in cell lines derived from SS patients, but not controls. Since ICAM.1 expression has been shown to involve calcium-dependent mechanism(s) , our results most likely suggest that in SGEC derived from SS patients, the above mechanisms of spontaneous ICAM.1 expression are set at a significantly lower threshold of activation, compared to controls. In vitro produced epithelial-derived factors may participate in the upregulated spontaneous ICAM.1 expression by the SGEC of SS patients. These epithelial factors may include the proinflammatory cytokines IL-1 and TNFα, which were found to be efficient inducers of ICAM.1 on SGEC in a manner similar to other epithelial cells [5,28]. In fact, increased in situ epithelial production of these cytokines has been described in the salivary glands of SS patients  and studies are under way to address the association between their autocrine production by SGEC and the spontaneous ICAM.1 expression in the in vitro system.
This study was supported by grants from the Hellenic Secretariat for Research and Technology (PENED/KA70–32776/95ED-1909), the Lillian Voudouri Foundation, the Hellenic State Scholarship Foundation (IKY, to R.F.A.H.) and the Hellenic Association of Immunology (to M.N.M.). We thank Dr S. Paikos for performing the minor salivary gland biopsies and G. Xanthou for her help.