Rheumatology Research Group, The University of Liverpool, Department of Medicine, 3rd Floor UCD Building, Liverpool L69 3GA, UK
Sjögren's syndrome is one of a number of organ-specific, autoimmune diseases, which include rheumatoid arthritis. The classification of Sjögren's syndrome into primary and secondary forms has now been widely accepted, but the debilitating effects of xerostomia and xeropthalmia may go unrecognized for long periods of time despite a gradual increase in symptoms. The clinical consequences of Sjögren's syndrome range from difficulty in speaking and eating, oral candidosis, and rampant caries to chronic sialadenitis, blindness and B-cell lymphoma [1, 2]. Treatment of the disease, at the present time, is palliative and mainly restricted to ameliorating the effects of a dry mouth and dry eyes [3]. Data from epidemiological studies suggest that the prevalence of Sjögren's syndrome may approach, or even exceed [4], that of rheumatoid arthritis, which affects between 1 and 3% of the general population [5].
Research into Sjögren's syndrome has been limited and largely restricted to charting the immunological progress of the disease [1]. This is in contrast to the extensive, multifaceted approach to the pathogenesis of rheumatoid arthritis, which has already yielded a number of therapeutic interventions [6]. Research into Sjögren's syndrome has been a neglected area of research, mainly because of the traditional view of the disease that symptoms arise as a result of destruction of glandular tissue [1, 7]. A further barrier to investigation is the lack of agreement among investigators as to the criteria for the diagnosis of the disease. It is difficult to evaluate laboratory or clinical research data when as few as 15% of patients characterized as Sjögren's syndrome sufferers by one set of criteria match those set out elsewhere [810].
Until recently, it has been widely assumed that the loss of salivary function seen is Sjögren's syndrome occurred as a direct consequence of salivary gland atrophy. This would appear to be a straightforward mechanism to account for the symptoms of the disease. However, two observations indicate that this mechanism is perhaps too simplistic. First, recent clinical observations have suggested that xerostomia may precede any substantial destruction of the glandular acinar tissue responsible for normal gland function [1013] (Speight, personal communication). Second, salivary gland atrophy is a normal consequence of ageing [14] that does not in itself lead to a reduction in flow rates [1517]. Studies of age-related changes in the salivary glands indicate that these tissues have a substantial functional reserve that can preserve normal flow rates in the elderly. Thus, Sjögren's syndrome patients may have ample salivary gland acinar tissue and yet still have reduced salivary flow rates.
These findings contest the view that the loss of function in Sjögren's syndrome, at least in the early stages of the disease, is as a consequence of gland atrophy. The most reasonable hypothesis, based on the data available to date, is that some component of the immune response specifically inhibits fluid secretion from the salivary gland acinar cells. Glandular atrophy is a well-known consequence of loss of function and it is possible that the atrophy of salivary glands in Sjögren's syndrome is the result of immune-mediated inhibition rather than the cause of it.
Further research into Sjögren's syndrome must, therefore, be directed at identifying a product of the immune response that is capable of suppressing acinar cell secretory function. Initial experiments may be carried out using an in vitro animal model for the disease. Most of the previous work into Sjögren's syndrome using animal models has centred around the immunological aspects of the disease with the goal of reproducing as many of the Sjögren's syndrome symptoms as possible [18]. This approach may help to clarify which aspects of the immune system are involved in the disease, but it does not necessarily help in determining the mechanisms that link the immune response to salivary hypofunction. Creating a perfect animal model for Sjögren's syndrome would not by itself tell us why the immune response prevents the glands from working.
We propose an alternative approach. Rather than attempting to reproduce Sjögren's syndrome in vivo we believe that it may be possible to reproduce the symptoms in vitro. In short, take normal, healthy, fluid secreting acinar cells, expose them to the products of the immune system and determine whether or not they are then capable of fluid secretion. Thus, as we have a much more precise control over the environment in vitro as compared with in vivo, it should be relatively straightforward to determine which factor or factors are responsible for the symptoms of the disease.
In order to create such an in vitro model it must be possible to:
All of these requirements are now attainable.
The defining feature of xerostomia is a lack of salivary fluid secretion. The mechanisms of salivary secretion are well understood [19, 20]. Two key elements of the secretory process in acinar cells, increased intracellular Ca2 + activity and increased apical membrane Cl- conductance are amenable to in vitro investigation using microfluorimetry [20] and patch-clamp electrophysiology [21], respectively.
One possible mediator of the interaction between the immune system and fluid secretion are the cytokines. Cytokines are produced by lymphocytes during the initial stages of the immune response and their function is intercell communication. One of the functions of cytokines may be to shut down fluid secretion. Cytokines may be manufactured en masse in cell culture by isolating and activating splenic lymphocytes [22]. This approach has the advantage of producing a cytokine profile similar to that generated in vivo and so should maximize the likelihood of producing a positive result. However, the task of precisely identifying which cytokines or combination of cytokines were most effective would still have to be undertaken. Fortunately, many of the cytokines are commercially available and the cytokine profiles for Sjögren's syndrome-like animal models are available in the literature [2326].
We have recently demonstrated that mouse salivary gland acinar cells may be maintained in primary culture for periods of up to 1 week without altering the capacity of the cells to respond, with increased intracellular Ca2+ activity and ion channel activation, to secretory stimuli [27]. These data indicate that experiments involving a chronic exposure to cytokines are at least feasible and that we will be able to detect any cytokine-dependent change in stimulussecretion coupling.
Although it would seem sensible to develop this in vitro model approach using an animal model, once established it may easily be applied to human tissues. A major advantage of both patch-clamp and microfluorimetric techniques is that they are applied to single cells and therefore only tiny amounts of tissue are required for these experiments. Acinar cells may be obtained from healthy individuals and from patients with Sjögren's syndrome following normal surgical procedures. Tissue obtained for diagnostic purposes may be assessed and later classified as Sjögren's or non-Sjögren's depending on the final diagnosis. Thus, it is possible to investigate the root cause of the disease without in any way interfering with diagnosis or treatment. Once isolated, these tissues may be treated in the same way as those of the animal model.
A further extension to this approach is that the secretory properties of these cells, before and after exposure to immune products, such as cytokines, may be directly compared with the secretory properties of acinar cells obtained from diagnostic labial gland biopsy of patients subsequently shown to be suffering from Sjögren's syndrome. Furthermore, correlation between the degree of secretory competence and a positive diagnosis of Sjögren's syndrome may help to justify the criteria used for diagnosis and thus help resolve differences in diagnosis.
Cytokines are only the first and most obvious candidates for a mediator between the immune system and fluid secretion. Sjögren's syndrome patients generate a wide range of antibodies including rheumatoid factor and antibodies against SS-A and SS-B antigens. The in vitro techniques, described above, may also be applied to investigate any role that these antibodies have in inhibiting fluid secretion. Given that antibodies against muscarinic M3 receptors have been identified in Sjögren's syndrome patients [28] and that activation of the muscarinic M3 receptor stimulates an increase in intracellular Ca2+ and fluid secretion, this is a potentially fruitful area for study.
The programme of research outlined above requires a multidisciplinary approach involving laboratory-based scientists including both immunologists and physiologists and a team of clinicians including oral physicians, ophthalmologists and rheumatologists. Basic research concerning the pathogenesis of Sjögren's syndrome is dependent on clinicians supplying tissue samples, as well as correctly diagnosing and managing patients with this disease. Patients with suspected Sjögren's syndrome must be fully evaluated and have evidence of both clinical and laboratory features of the disease which is sufficient for them to be diagnosed as having Sjögren's syndrome, according to acceptable criteria [8, 9]. A database of clinical and laboratory investigations is a prerequisite of any research programme and in Liverpool we now have a cohort of approximately 60 primary Sjögren's syndrome patients with evidence of systemic autoimmune disease, manifested by autoantibodies and/or minor salivary gland histopathology, characteristic of Sjögren's syndrome.
An understanding of the relationship between the immune response and salivary fluid secretion, especially in the early stages of Sjögren's syndrome, could lead to the development of effective therapeutic interventions for this debilitating and poorly understood disease.
The authors would like to acknowledge the continuing contribution and co-operation of The Liverpool Sjögren's Syndrome Research Group: Roger Bucknall, Luke J. Dawson, E. Anne Field, Susan Higham, S. Kaye, Lesley Longman, Robert J. Moots and Peter M. Smith.
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