Oestrogen receptors in cultured epithelial cells from salivary glands of Sjögren's syndrome patients

E. Kassi, P. Moutsatsou, C. E. Sekeris, H. M. Moutsopoulos1 and M. N. Manoussakis1

Department of Biological Chemistry and
1 Department of Pathophysiology, University of Athens, Medical School, 75 Mikras Asias, Goudi 11527, Athens, Greece

SIR, Sjögren's syndrome (SS), or autoimmune epithelitis, is a chronic autoimmune disorder characterized by lymphoepithelial lesions of the salivary, lachrymal and other exocrine glands [1]. The disorder has a strong predilection for women in their fourth or fifth decade of life. Epithelial cells appear to play an important role in the initiation and maintenance of autoimmune lesions in SS patients, as indicated by the analysis of tissues and long-term cultured non-neoplastic salivary gland epithelial cell (SGEC) lines [1]. Recent studies from our laboratory have indicated the operation of intrinsic activation processes in the glandular epithelia of SS patients, as illustrated by the aberrant expression of various immunoregulatory protein molecules [2].

Oestrogens are known to modulate immunological responses and to regulate tissue apoptosis [3]. These hormones exert their effects via oestrogen receptors {alpha} (ER{alpha}) and ß (ERß) and their interaction with other transcription factors that modulate gene expression [4]. Splicing variants of ER{alpha} that lack specific functional domains (exons 5 or 7) have been shown to interfere in the transcription activity of the wild-type ER{alpha}, and are thus important regulators of the oestrogenic effect [5]. Moreover, the role of ER{alpha} variants has been addressed in systemic lupus erythematosus (SLE) [6].

Just as they are in other autoimmune diseases with strong female preponderance, oestrogens are thought to play an important role in the pathogenesis of SS [1]. The detection of mRNA transcripts of ER{alpha} in salivary glands has been documented previously [7]. However, evidence implicating oestrogen receptor expression in epithelial cells is lacking, as only total salivary gland tissues have been tested and no studies have used isolated epithelial cells [7]. Therefore, the presence of the oestrogen receptor and the specific role of its expression by the cellular target of the autoimmune reactions in SS remains to be explored. For this purpose, we investigated whether established cultures of non-neoplastic SGEC lines [2] are capable of ER{alpha} expression, thus rendering this in vitro system suitable for the study of the role of oestrogens in SS patients.

Minor salivary gland biopsies were obtained from individuals undergoing diagnostic evaluation for primary SS according to the European–American SS classification criteria [1]. Control samples were derived from patients who did not fulfil the above criteria and had no histopathological evidence of SS. Primary SGEC cultures were established from minor salivary gland biopsies by the explant outgrowth technique, as described previously [2]. Total RNA extracted from three SGEC lines from SS patients and three samples from controls were subjected to reverse transcription and amplification by the polymerase chain reaction (PCR) using five pairs of primers specific to the human ER{alpha} gene coding region, as described [6]. The PCR products corresponding to wild-type and deletion variants of ER{alpha} were further identified by sequence analysis as described previously [6]. The analysis had revealed for the first time that SGEC samples derived from SS patients and healthy individuals uniformly express mRNA transcripts of full-length wild-type ER{alpha} as well as of the deletion variants {Delta}ER2, {Delta}ER5 and {Delta}ER7 (Fig. 1Go). No appreciable qualitative differences could be established in the profile of ER{alpha} or its deletion variants between SS patients and controls. Our findings support the inherent capacity of SGEC to respond to oestrogens and suggest that, in a manner similar to other cells, regulatory influences by deletion mutants are also likely to operate in this particular type of epithelial cells. This realization supports the use of cultured non-neoplastic SGEC lines in the in vitro functional study of the effect of oestrogens on these epithelia.



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FIG. 1. Detection of wild-type ER{alpha} (WTER{alpha}) and exon deletion variants of ER{alpha} ({Delta}ER2, {Delta}ER5 and {Delta}ER7, with deletion of exons 2, 5 and 7, respectively), by reverse transcription–PCR analysis using different sets of primers, designed to amplify five overlapping fragments of the entire ER{alpha} cDNA. Representative analyses are shown and include SGEC samples obtained from controls (1c, 2c, 3c, 4c, 5c) and SS patients (1p, 2p, 3p, 4p, 5p). The nature of the faint band x remains to be determined. Bl, blank; M1, DNA marker (100 bp DNA ladder); M2, marker ({Phi}X 174 DNA, Hae III).

 
The definition of the role of oestrogen in the growth, differentiation and function of SGEC appears mandatory for the understanding of the pathogenesis of SS as well as for the design of therapeutic intervention in patients with this disease. The pathogenesis of many autoimmune disorders, including SS, is thought to involve the action of sex hormones and their receptors, presumably through regulatory influences on the cells of the immune system [6, 8, 9]. Oestrogens, in particular, seem implicated in autoimmunity as enhancers of the immune response and are capable of inducing cell-surface expression of autoantigens [9]. However, the mechanisms of sex steroid action on the target cells of autoimmune processes, such as the salivary epithelia in patients with SS, need to be delineated. Furthermore, recent data support the variation of oestrogen receptor expression with age in certain types of cells [10]. In this context, further studies are needed to evaluate quantitatively the expression of oestrogen receptors in SGEC in relation to age. Taking into consideration the immunoenhancing effects of oestrogens [8, 9], hormone replacement therapy for the vitality of salivary tissues and functions in SS patients needs to be carefully addressed.

Notes

Correspondence to: P. Moutsatsou, Department of Biological Chemistry, 75 Mikras Asias Street, Goudi 11527, Athens, Greece. E-mail: pmoutsatsou{at}med.uoa.gr Back

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Accepted 27 January 2003