OBJECTIVE To localise and characterise follicular dendritic cells (FDC) present in autoimmune lesions of primary Sjogren syndrome.
METHODS Cryostat sections of labial salivary glands from 15 patients with primary Sjogren syndrome were examined by an indirect immunoperoxidase technique and monoclonal antibodies to a panel of dendritic cell markers. Tonsils from two controls were also examined for the same markers.
RESULTS FDC were localised in the centre of 75% of lymphoid focal structures in labial salivary gland biopsies. FDC in labial salivary glands of patients with primary Sjogren syndrome expressed CD35, CD11c, and CD106 (VCAM-1) in a pattern similar to FDC in tonsils, but they did not express either CD14 or CD11b. This indicates that they may not be of myeloid origin, while FDC in tonsillar lymphoid follicles strongly expressed both CD14 and CD11b. FDC in labial salivary glands of patients also lacked VLA-2α and VLA-3α, which were expressed by FDC in tonsils.
CONCLUSIONS The characteristic phenotype and origin of these cells may be of importance in the immune responses involved in Sjogren syndrome and the retention of infiltrating lymphocytes in the glands.
- Sjogren syndrome
- dendritic cell markers
- vascular cell adhesion molecule-1
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Primary Sjogren syndrome is a chronic inflammatory disorder associated with lymphocytic infiltration of exocrine glands.1-3 The infiltrate is composed mainly of T lymphocytes,2 3 though in some labial salivary biopsies non-T/non-B lymphocytes predominate.4 However, CD16 natural killer (NK) lymphocytes are rarely seen.5 The lymphocytic infiltration is progressive6 and the infiltrating lymphocytes are retained and persist in the exocrine glands.5 7 Several autoantibodies have been shown to be associated with Sjogren syndrome (SS) (including rheumatoid factor, anti-SS-A, and anti-SS-B).2 The disease is not associated with any cellular proliferative responses to self antigens.8 Hence there is no explanation as to what keeps the infiltrating lymphocytes in the glands. Several viruses have been proposed as triggers of the autoimmune response.7 9 The mechanisms involved in the retention of infiltrating lymphocytes in the glands may involve changes in the pattern or density of adhesion molecule expression.5 Another possibility is the presence of follicular dendritic cells (FDC) which may functionally maintain the lymphocytes in lymphoid follicles, as seen in the salivary or lacrimal glands of these patients. The aim of this study was to compare the phenotypic characteristics of FDC in labial salivary glands of patients with primary Sjogren syndrome and those seen in tonsillar lymphoid follicles, as an example of organised peripheral lymphoid tissue. This may increase our understanding of Sjogren syndrome.
The origins of dendritic cells are not completely clear.10 11 Dendritic cells can form clusters with T or B lymphocytes.10 11 They may stimulate autologous and allogeneic mixed leucocyte reactions.10 11 They are powerful accessory cells and can induce the expression of activation antigens by lymphocytes.10 11 Dendritic cells are situated in close proximity to lymphocytes in lymphoid organs. This rendered the determination of their antigenic phenotype difficult.10 11There is no accepted classification of dendritic cells, and most classifications describe a phenotypic and functional overlap between different types of dendritic cell.10-13 CD35 (DRC-1) may be expressed on all dendritic cells in lymphoid tissues.12Schriever et al isolated unclustered FDC and phenotypically characterised these cells.10 They reported that FDC express several complement receptors (CR), namely CR1 (CD35), CR2 (CD21), CR3 (CD11b), and the myeloid/monocytic cell marker CD14. Another group showed that isolated FDC lack the expression of CD35.11 This may be due to the isolation of a different subpopulation of dendritic cells from the same lymphoid organ resulting from variable origins of the dendritic cells,14 variable stages of maturation, or functional alteration of these cells in different physiological or pathological situations.15
VLA-2α and VLA-3α are integrin molecules that may be expressed by FDC10 11 16 and they have a common β chain CD29.5 The ligands for VLA-2α and VLA-3α are laminin, collagen, and fibronectin.
Fifteen patients with primary Sjogren syndrome fulfilling at least five (a minimum of four is required) of the European Community criteria1 were the subjects of this study. Patients ranged in age from 24 to 87 years, with a mean of 50.4 years. There were 14 females and one male. A positive Schirmer test was demonstrated in 11 patients (73%) and a positive Rose Bengal test in 14 (93%). Abnormal unstimulated salivary flow rates (< 0.1 ml min-1) were found in 12 patients (80%). Rheumatoid factor was detected in 12 patients (80%), anti-nuclear antibodies (ANA) in 11 (73%), and anti-SS-A and/or anti-SS-B in nine (60%). Serum anti-ds-DNA was not detected in any of the patients. All labial salivary gland biopsies of patients with primary Sjogren syndrome included in this study had two or more lymphoid focal structures per 4 mm2 (grade IV).17
Ethics committee approval was obtained. All subjects gave informed consent to their participation in the study. Two tonsils were obtained from two female patients undergoing routine tonsillectomy at Prince of Wales Hospital, Sydney.
LABIAL SALIVARY GLAND BIOPSY
The technique used for obtaining labial salivary glands from patients was based on previously described methods.9 Labial salivary gland and tonsil specimens were transferred in normal saline (0.9% NaCl) to be used for immunohistology. The biopsies were embedded in OCT compound (Bayer, Miles Laboratories), snap frozen, and cryostat sections 4 microns thick were cut. The sections were fixed in cold (2-4°C) acetone for five minutes followed by phosphate buffered saline (PBS) for five minutes (repeated twice in PBS). Sections were stored at −70°C.
MICROSCOPIC EXAMINATION OF SECTIONS
Sections were examined with light microscopy using a Microphot FX microscope (Nikon, Japan). Labial salivary gland sections were examined for positive staining as indicated by the brown colour developed by the specific immunohistochemical reaction. Follicular dendritic cells were localised as shown by positive staining with CD35 (DRC-1), CD106 (VCAM-1), and their characteristic dendritic morphology. Other cellular elements staining with each antibody were recorded.
EXPRESSION OF DENDRITIC CELL MARKERS BY FDC IN LABIAL SALIVARY GLANDS OF PATIENTS WITH PRIMARY SJOGREN SYNDROME
FDC in labial salivary glands of patients with primary Sjogren syndrome expressed CD35 (DRC 1) to the same extent as in tonsillar sections (figure, A, B, table 2). VLA-2α (CD49b) and VLA-3α (CD49c) were not expressed by follicular dendritic cells in labial salivary glands of patients with Sjogren syndrome (table 2), while FDC (in situ) in lymphoid follicles of tonsils expressed both VLA-2α and VLA-3α strongly, although at a lesser intensity than CD35 or CD11c (figure, C, D, table 2). FDC in labial salivary glands and tonsils expressed CD29 (β-1 integrin), the β chain for VLA-1, -2, -3, -4, -5, and -6 molecules. Acinar and ductal epithelial cells in labial salivary glands of patients with primary Sjogren syndrome expressed both VLA-2α and VLA-3α (figure, D). Similarly, stratified squamous epithelial cells in tonsils expressed both antigens. VCAM-1 was detectable at a variable intensity (from one follicle to another) on FDC in labial salivary glands of patients with primary Sjogren syndrome (figure, H) and in tonsils (table 2). CD11c was strongly expressed by FDC both from labial salivary glands of patients and from tonsils (figure, F, G). Neither CD11b nor CD14 was identifiable on dendritic cells in labial salivary glands of patients with primary Sjogren syndrome. FDC in tonsillar lymphoid follicles expressed CD11b and CD14 (figure, E). Although this study shows the expression of CD35, CD11b, and CD14 on FDC in tonsils, it cannot confirm that all dendritic cells in tonsillar lymphoid follicles expressed these markers. Class I and class II major histocompatibility complex (MHC) expression was demonstrated on FDC of patients with primary Sjogren syndrome and on tonsils (table 2). CD11a (αL integrin), CD18 (β-2 integrin), and their ligand ICAM-1 were also strongly expressed by FDC in labial salivary glands and tonsils (table 2).
Follicular dendritic cells were identified in a considerable proportion of lymphoid follicles present in labial salivary glands of patients with primary Sjogren syndrome. The percentage of lymphoid follicles that have detectable follicular dendritic cells ranged from 29% to 100% of follicles in labial salivary glands. The majority of labial salivary glands contained FDC in more than 50% [mean (SEM) 74.7 (5.9) %] of the lymphoid follicles present. Tonsil sections showed FDC in 99% of lymphoid follicles. FDC were sometimes seen in small lymphoid aggregates not forming follicles. FDC were not seen adjacent to glandular epithelium or epimyoepithelial islands.
In this study we showed that FDC were present in lymphoid follicles in labial salivary glands from patients with primary Sjogren syndrome. The expression of CD11c by these FDC may be important. Blocking CD11c has been shown to decrease the stimulatory effect of dendritic cells on T cell proliferation.18 19 CD11c is expressed by blood dendritic cells18 19 and tonsillar FDC. The lack of expression of the myeloid markers CD14 and CD11b by FDC in labial salivary glands of patients with Sjogren syndrome indicates that these cells are more mature and may have more efficient accessory functions, as shown by blood dendritic cells that do not express these markers.15 19 This may also indicate that the dendritic cells in labial salivary glands of patients with Sjogren syndrome may not be of myeloid origin. Different origins for FDC have been proposed, including myeloid or lymphoid progenitors.10 14 15 The expression of lymphoid markers, such as CD3, or in some foci CD19, by lymphocytes5 in the close proximity of dendritic cells makes definite determination of the expression of these markers by the dendritic cells themselves difficult. Separation of the dendritic cells may allow more satisfactory examination of their phenotypic and functional characteristics, though the small size of the biopsies and small number of dendritic cells present will limit the feasibility of such an in vitro approach. Another interesting finding is the lack of expression of VLA-2α and VLA-3α by FDC in labial salivary glands of patients.10 16 Although FDC in labial salivary glands and tonsils express VCAM-1, blood dendritic cells do not express this molecule.18 If these dendritic cells originated from the blood, they may have acquired VCAM-1 and increased CD11c expression in the local autoimmune lesions in labial salivary glands of patients with Sjogren syndrome.
The results of this study show that dendritic cells are present in labial salivary glands of patients with Sjogren syndrome. They may play an important role in regulating immune responses and the retention of infiltrating lymphocytes in the glands. Although the origin of these dendritic cells is not clear, their phenotypic characteristics suggest that they may have originated from non-myeloid blood dendritic cells. Blood dendritic cells can bind to a number of adhesion molecules18 19 that are expressed on endothelial cells in labial salivary glands of patients with Sjogren syndrome5and this may provide a portal of entry into the glands.
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