SUMMARYWe evaluated possible modes of epithelial cell destruction and restoration in minor salivary gland biopsies from patients with SS. Minor salivary gland biopsies from 10 primary Sjögren's syndrome (pSS) patients and eight control individuals were evaluated by immunohistochemical staining for the expression of apoptosis-related molecules, substances released by activated cytotoxic T cells, as well as proteins involved in epithelial cell repair. The results were analysed by computer screen analysis and they were expressed as average percentages. Apoptosis-promoting molecules, Fas antigen and Fas ligand were observed in ductal and acinar epithelial cells as well as in infiltrating mononuclear cells of minor salivary glands from SS patients in comparison with control biopsies. Bax protein, which acts as a death-promoter message, was expressed in the ductal and acinar epithelial cells and in mononuclear infiltrating cells of SS patients compared with control individuals, while Bcl-2, an inhibitor of apoptosis, was primarily found in the lymphocytic infiltrates. In situ DNA fragmentation assay (TUNEL) revealed that epithelial cells were apoptotic in patients with SS compared with control subjects. Immunohistochemical staining for perforin and granzyme B, released from granules of activated cytotoxic lymphocytes, revealed their presence in lymphocytic infiltrates of patients with SS compared with control biopsies. pS2, a member of the trefoil protein family which functions as promoter of epithelial cell repair and cell proliferation, was expressed in epithelial cells in biopsies from SS patients. These studies suggest that the functional epithelium of minor salivary glands in patients with SS appears to be influenced by both intrinsic and extrinsic mechanisms of destruction, while a defensive mechanism of epithelial restoration seems to be active.

Objective. Many studies have shown that the microanatomic organization of infiltrating leukocytes in the salivary gland lesions of patients with Sjögren'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 transcriptasepolymerase 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.

Objective. To investigate the expression of B7 costimulatory molecules in the lymphoepithelial lesions of salivary gland (SG) biopsy tissues and in SG epithe-lial cell lines derived from patients with Sjögren'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-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 epithe-lial 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.

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.

Previous studies established that in the rat, the uterus can accept a developing blastocyst for implantation only during a limited period of time on day 5 of gestation, termed the receptive phase. Our previous studies showed that the expression of calcitonin, a peptide hormone that regulates calcium homeostasis, is induced in rat uterus between days 3-5 of gestation and is switched off once the implantation process has progressed to day 6. In the present study, we analyze in detail how the expression of calcitonin messenger RNA (mRNA) in the uterus is regulated by the steroid hormones progesterone and estrogen and explore the possibility that calcitonin may serve as a potential marker of uterine receptivity. We demonstrate by in situ hybridization that calcitonin mRNA is synthesized specifically in the glandular epithelial cells between days 3-5 of pregnancy. Interestingly, calcitonin synthesis is also induced in these cells during pseudopregnancy, indicating that this peptide hormone is produced in the endometrium in response to maternal, rather than embryonic, signals. We also demonstrate that calcitonin mRNA expression during pseudopregnancy, like that in normal pregnancy, is under progesterone regulation. We further examined the steroid hormone regulation of uterine calcitonin expression in a delayed implantation model. In pregnant rats in which implantation is blocked upon removal of both ovaries on day 4 of gestation, continued administration of progesterone sustains calcitonin expression in the uterus for several days in the absence of estrogen. Administration of estrogen, which allows delayed implantation, also rapidly reduces calcitonin expression, indicating a role for this steroid hormone in turning off calcitonin gene expression. In gene transfection studies, expression of the progesterone receptor B isoform in cultured endometrial cells induces RNA synthesis from a reporter gene containing a 1.3-kb calcitonin promoter fragment in a hormone-dependent manner. As expected, mifepristone-complexed progesterone receptor B isoform fails to activate the calcitonin promoter. Progesterone acting through its nuclear receptor therefore regulates the expression of the calcitonin gene at the level of transcription. Finally, using RIA we investigated whether calcitonin is secreted from its glandular site of synthesis at the time of implantation by analyzing uterine flushings obtained from pregnant rats. We report the detection of a significant amount of calcitonin in the luminal secretions collected on day 4 and a lower amount on day 5 of gestation, whereas similar samples collected from animals on either day 3 or 6 of gestation did not contain detectable amounts of this peptide hormone. A transient burst of calcitonin secretion into the uterine lumen therefore occurs immediately preceding implantation. Based on these results, we propose that calcitonin is a measurable marker that forecasts the receptive state of rat endometrium during blastocyst implantation.