Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product.
The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin. (+info)
Expression of B7 costimulatory molecules by salivary gland epithelial cells in patients with Sjogren's syndrome.
OBJECTIVE: To investigate the expression of B7 costimulatory molecules in the lymphoepithelial lesions of salivary gland (SG) biopsy tissues and in SG epithelial cell lines derived from patients with Sjogren's syndrome (SS). METHODS: B7.1 and B7.2 protein expression was studied by immunohistochemistry in minor SGs obtained from 11 patients with SS and 10 disease control patients with nonspecific sialadenitis and in cultured SG epithelial cell lines obtained from minor SGs from 15 SS patients and 15 control patients. B7.1 and B7.2 messenger RNA (mRNA) expression by SG epithelial cell lines was examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In biopsy tissues from SS patients, but not control patients, ductal and acinar epithelial cells showed increased expression of both B7.1 and B7.2. Intense spontaneous B7.1 protein expression (as well as HLA-ABC, but not B7.2 or HLA-DR) was also found in 73% of SG epithelial cell lines from SS patients versus 13% of those from control patients (P < 0.01). Interferon-y treatment induced, or up-regulated, B7.1, B7.2, and HLA-DR expression in all SG epithelial cell lines tested. B7.1 and B7.2 expression by SG epithelial cell lines was also verified at the mRNA level by RT-PCR. CONCLUSION: Human SG epithelia are intrinsically capable of expressing B7 proteins upon activation. In SS patients, the expression of B7 molecules by SG epithelial tissues and by SG epithelial cell lines indicates the activated status of SG epithelial cells in this disorder and, possibly, their capacity for presenting antigens to T cells. (+info)
Analysis of V(H)-D-J(H) gene transcripts in B cells infiltrating the salivary glands and lymph node tissues of patients with Sjogren's syndrome.
OBJECTIVE: In patients with Sjogren's syndrome (SS), B lymphocytes have been found to infiltrate salivary glands, resulting in sialadenitis and keratoconjunctivitis. The disease is frequently associated with benign and neoplastic lymphoproliferation. The present study was undertaken to investigate whether clonal B cell expansion takes place in lymphocytic infiltrations of salivary glands under (auto- [?]) antigen stimulation, by analyzing in more detail the variable part (V(H)-D-J(H)) of the immunoglobulin heavy chain genes expressed in these B cells. METHODS: Biopsies of the labial salivary glands and lymph nodes were performed on 2 female patients with SS. The Ig gene rearrangements in these tissues were amplified by reverse transcriptase-polymerase chain reaction using specific primers. RESULTS: A total of 94 V(H)-D-J(H) transcripts were cloned and sequenced. Our data suggest a polyclonal origin of the B cell infiltrates. In 92 of the transcripts, V(H) genes were modified by somatic mutation. Further analysis showed counterselection for replacement mutations within the framework regions, suggesting that those B cells were stimulated and selected for functional expression of a surface Ig. In labial salivary glands from both patients, clonally related B cells became evident. Members of 1 particular clone were found in both the lip and lymph node material. CONCLUSION: These data provide evidence, on the nucleotide sequence level, that an antigen-triggered clonal B cell expansion takes place in the salivary glands of patients with SS who do not have histologic evidence of developing lymphoma. It may be speculated that those B cell clones expand during disease progression, resulting in lymphomagenesis. (+info)
CD4 cytotoxic and dendritic cells in the immunopathologic lesion of Sjogren's syndrome.
The existence of CD4+ T lymphocytes with cytotoxic activity in minor salivary gland (MSG) biopsies from Sjogren'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. (+info)
Study of quantitative oral radioactivity in salivary gland scintigraphy and determination of the clinical stage of Sjogren's syndrome.
In this study, the oral radioactivity seen in salivary gland scintigraphy, which was established entirely on the basis of radioactive saliva secreted by the parotid and submandibular glands, was evaluated quantitatively in healthy volunteers and in patients with Sjogren's syndrome. METHODS: Salivary gland scintigraphy and labial biopsy were performed on 70 patients with Sjogren's syndrome. After intravenous administration of 99mTc-sodium pertechnetate, dynamic scintigraphy was performed and time-activity curves for the oral cavity and four major salivary glands were generated. Lemon juice stimulation was delivered at 40 min. The prestimulatory oral activity index, poststimulatory oral activity index, and time interval between the vascular perfusion peak and the prestimulated maximum oral activity point were calculated to quantify the oral activity. Other glandular functional parameters-namely, maximum accumulation (MA), maximum secretion, secretion velocity, time at maximum count, time interval from stimulation to minimum count, and uptake ratio (UR) of the parotid and submandibular glands-were also calculated. Salivary gland scintigraphy was also performed on 21 healthy subjects with no evidence of salivary gland malfunction. RESULTS: Histopathologic grade 1 or 2 was found in 29 patients and grade 3 or 4 was found in 41 patients, and they were regarded as being in the early and advanced stages of Sjogren's syndrome, respectively. After overall analysis, all of the oral activity indices and the MA and UR of the submandibular gland clearly decreased as clinical severity progressed, and statistically significant differences were observed. CONCLUSION: New oral activity indices correlated with the stage of Sjogren's syndrome, and these quantitative oral indices together with certain glandular parameters (mainly MA and UR of the submandibular gland) were found to be sensitive enough to distinguish the disease severity of Sjogren's syndrome. (+info)
Functional expression of a costimulatory B7.2 (CD86) protein on human salivary gland epithelial cells that interacts with the CD28 receptor, but has reduced binding to CTLA4.
B7 molecules expressed on classic APC play a critical role in the regulation of immune responses by providing activation or inhibitory signals to T cells, through the ligation with CD28 or CTLA4 receptors, respectively. We have recently described the expression of B7 molecules by the salivary gland epithelial cells (SGEC) of patients with Sjogren's syndrome (also termed autoimmune epithelitis). The role of such expression needs to be clarified. Thus, in the present study, we sought to address the existence and function of B7.2 proteins on cultured nonneoplastic SGEC lines derived from Sjogren's syndrome patients. The occurrence of B7.2 proteins on SGEC was verified by flow cytometry, immunocytochemistry, immunoprecipitation, and immunoblotting. The assessment of several cell lines in costimulation assays had revealed that the constitutive expression of B7.2 molecules is sufficient to provide costimulatory signals to anti-CD3-stimulated T cells. SGEC-derived costimulation induced IL-2-dependent proliferation of CD4(+) T cells, which was associated with low production of IL-2, but probably also with the secretion of yet undefined autocrine T cell growth factor(s). B7.2 proteins expressed by SGEC were found to display distinctive binding properties denoted by the functional interaction with CD28 receptor and reduced binding to CTLA4. Finally, the detection of a functional soluble form of B7.2 protein in cell-free culture supernatants of both SGEC and EBV-transformed B cell lines is demonstrated. These findings imply a critical role for epithelial cells in the regulation of local immune responses in the salivary glands. (+info)
"Lymphoid" chemokine messenger RNA expression by epithelial cells in the chronic inflammatory lesion of the salivary glands of Sjogren's syndrome patients: possible participation in lymphoid structure formation.
OBJECTIVE: Many studies have shown that the microanatomic organization of infiltrating leukocytes in the salivary gland lesions of patients with Sjogren's syndrome (SS) resembles the structure of lymphoid organs. A newly defined set of chemokines referred to as "lymphoid," which orchestrate leukocyte microenvironmental homing and contribute to the formation of lymphoid structures, provides directional clues. The aim of this study was to investigate the possible existence of "lymphoid" chemokines in the chronic inflammatory lesions of SS patients and thus validate their potential involvement in the disease process. METHODS: Twelve patients with primary SS, 3 patients with secondary SS, 4 patients with other autoimmune disorders, and 4 control individuals were the subjects of this study. Reverse transcriptase-polymerase chain reaction analysis was performed in order to examine the messenger RNA (mRNA) expression of "lymphoid" chemokines. Furthermore, in situ hybridization studies revealed chemokine mRNA localization. Immunohistochemistry was also applied in order to identify the cell types that expressed the chemokine mRNA. RESULTS: STCP-1/monocyte-derived chemokine and TARC mRNA were expressed in the majority of patients with primary and secondary SS, in 2 of 4 patients with other autoimmune disorders, and in 2 of 4 controls. BCA-1, ELC, and PARC mRNA were only detected in patients with primary and secondary SS. SLC mRNA was also detected in 1 non-SS patient. The main cellular sources of chemokine mRNA were ductal epithelial cells and infiltrating mononuclear leukocytes. CONCLUSION: The expression pattern of "lymphoid" chemokine mRNA points further to the role of epithelial cells in the pathogenesis of SS and offers new insight into the potential mechanisms that could be involved in leukocyte attraction and in the in situ formation of secondary lymphoid tissue structures. (+info)
Reversibility of histological and immunohistological abnormalities in sublabial salivary gland biopsy specimens following treatment with corticosteroids in Sjogren's syndrome.
Sjogren's syndrome (SS) is a chronic autoimmune disease characterised by specific lesions in exocrine glands, so sublabial minor salivary gland biopsy (SLGB) plays an important part in its diagnosis. The extent and composition of the lymphocytic infiltrate in SLGB specimens can be considered as target organ specific parameters. They are quantified after histological and immunohistological examination by a focus score (describing the extent of the infiltrate) and IgA% score (describing the composition of the infiltrate), respectively. However, little is known about the factors that contribute to the extent and composition of the infiltrate and whether these features are reversible as repeated SLGBs are rarely performed. A patient with SS is described who underwent SLGBs before and after treatment with high dose corticosteroids. After treatment there was not only clinical improvement, but also improvement in the histological and immunohistological parameters. Although these findings need to be confirmed in further studies, this suggests that histopathological changes may be reversible in SS. Furthermore, it shows that the potential effects of corticosteroid use should be taken into account when interpreting SLGB specimens. When clinical changes do parallel histological changes, repeated SLGBs might offer a marker for disease activity in patients with SS. (+info)