Grynkiewicz equation and custom-written software. course of any possible interactions. Down-regulation of. Solutions the secretory process could, for example, ...
Rheumatology 2000;39:1226–1233
An investigation of interactions between the immune system and stimulus–secretion coupling in mouse submandibular acinar cells. A possible mechanism to account for reduced salivary flow rates associated with the onset of Sjo¨gren’s syndrome L. J. Dawson, S. E. Christmas1 and P. M. Smith Department of Clinical Dental Sciences, The University of Liverpool, Liverpool and 1Department of Immunology, The University of Liverpool, Liverpool, UK Abstract Objectives. To determine whether chronic exposure to lymphocyte-derived cytokines could inhibit the fluid secretory mechanism in salivary gland acinar cells and so account for the loss of gland function seen in the early stages of Sjo¨gren’s syndrome. Methods. Mouse submandibular acinar cells maintained in primary culture were exposed to a profile of cytokines produced by concanavalin A-activated splenic lymphocytes in vitro for periods up to 72 h. Agonist-evoked changes in intracellular Ca2+ were determined microfluorimetrically in both control and cytokine-treated cells. Results. Acinar cells maintained in primary culture in the presence of cytokines for up to 72 h were able to mobilize intracellular calcium in response to stimulus by acetylcholine in an identical fashion to those maintained in primary culture in the absence of cytokines. Acute application of the conditioned medium produced by the activated lymphocytes had an antisecretory effect on acetylcholine-evoked Ca2+ mobilization, which was found to be mediated by cholinesterase rather than by cytokines. Conclusion. Neither chronic nor acute exposure to the profile of cytokines released by concanavalin A-activated splenic lymphocytes interfered in any way with the second messenger cascade and fluid and electrolyte secretion in acinar cells. Our data suggest an alternative hypothesis, in which elevated levels of cholinesterase can metabolize acetylcholine released within the salivary glands and thus prevent fluid secretion. K : Sjo¨gren’s syndrome, Cytokines, Cholinesterase, Intracellular calcium, Signal transduction.
Sjo¨gren’s syndrome is an autoimmune disease of which the main symptom is a reduction in both resting and, usually, stimulated salivary flow rates [1, 2]. The clinical consequences of Sjo¨gren’s syndrome range from difficulty in speaking and eating, oral candidosis and rampant caries to chronic sialadenitis, blindness and B-cell lymphoma [3–5]. The prevalence of Sjo¨gren’s syndrome is similar to that of rheumatoid arthritis [6, 7], with which it may be associated. In contrast to the extensive, multifaceted approach to the pathogenesis of rheumatoid arthritis, which has already yielded a number of therapeutic Submitted 9 November 1999; revised version accepted 1 June 2000. Correspondence to: P. M. Smith, Department of Clinical Dental Sciences, The University of Liverpool, Edwards Building, Daulby Street, Liverpool L69 3GN, UK.
interventions [8], research into Sjo¨gren’s syndrome has been limited and largely restricted to charting the immunological progression of the disease [3]. This follows, at least in part from the traditional view of the disease, in which the symptoms arise as a result of destruction of glandular tissue [3, 9]. However, two observations indicate that this mechanism is perhaps, simplistic. First, recent clinical observations have suggested that xerostomia may precede any substantial destruction of the glandular acinar tissue responsible for normal gland function [10; P. M. Speight, personal communication]. Second, salivary glands have a substantial functional reserve and tolerate some degree of atrophy, for example as a normal consequence of ageing [11] without reduction in flow rates [12–14]. Thus, Sjo¨gren’s syndrome patients may have ample salivary gland acinar tissue and yet still have reduced salivary flow rates.
1226
© 2000 British Society for Rheumatology
Stimulus–secretion coupling and Sjo¨ gren’s syndrome
An alternative 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 [15]. Glandular atrophy is a wellknown consequence of loss of function [16–19] and it is possible that the atrophy of salivary glands in Sjo¨ gren’s syndrome is the result of immune-mediated inhibition rather than the cause of it. Cytokines are a possible mediator of interaction between the initial stages of the immune response and fluid secretion. The agonist-evoked changes in the intracellular concentration of Ca2+ that drive the secretory process [20, 21] can easily be measured in isolated salivary gland acinar cells in vitro using microfluorimetric techniques, and the effect of cytokines on fluid secretion can be assessed by measuring their impact on the signal transduction cascade. In Sjo¨ gren’s syndrome, acinar cells are chronically exposed to a range of cytokines released by infiltrating lymphocytes, including interleukin (IL)-2, IL-6, IL-1a, tumour necrosis factor a ( TNF-a) and interferon-c (IFN-c) [22]. At present, we do not know which cytokine or combination of cytokines might interfere with stimulus–secretion coupling, nor do we know the time course of any possible interactions. Down-regulation of the secretory process could, for example, result from a reduction in receptor density occurring over a period of hours or days. Therefore, we have developed an in vitro system in which isolated mouse submandibular acinar cells can be exposed to cytokines for up to 72 h. We have employed the well-established technique of activating splenic lymphocytes using concanavalin A [23–26 ] and harvesting the cytokines produced by the cells. This process yields a cytokine profile that includes IL-2, IL-3, IL-4, IL-6 and INF-c [24, 26 ], a profile similar to that present in vivo in Sjo¨ gren’s syndrome. Thus we can mimic in vitro the conditions that apply in vivo and then assess the effects of these conditions on the ability of cells to respond to secretory agonists.
Materials and methods Cell preparation Adult male CD1 mice were killed by cervical dislocation and submandibular acinar cells were isolated by collagenase ( Worthington Diagnostic, Lakewood, NJ, USA) digestion in extracellular media containing 1 m Ca2+ as described previously [27]. Following dispersal, cells were suspended in serum-free 50:50 Dulbecco’s MEM:F12 medium containing antibiotics and antimycotics (Life Technologies, Paisley, UK ) and placed on circular glass coverslips (22 mm diameter) coated with a thin (#1 mm) layer of a basement membrane matrix (Matrigel; Becton Dickinson, Bedford, MA, USA) [28]. Survival rates for cells cultured on uncoated coverslips were poor compared with cells placed on Matrigel. Each coverslip was placed in one well of a six-well plate and covered with either control or conditioned medium and cultured for up to 72 h at 37°C with 5% carbon dioxide.
1227
Cells were removed from culture immediately before an experiment and loaded with fura-2 by incubation for 20 min in the presence of 2 m of cell-permeable fura-2 acetoxymethylester (Fura-2 AM, Molecular Probes, Eugene, OR, USA). Experimental The glass coverslips formed the base of a perfusion chamber that was placed on the stage of an inverted microscope (Nikon Diaphot). All experiments were carried out at 24 ± 2°C. Measurements were made using 1000× magnification on single cells, either completely isolated or part of a small (2–8) cell clump. Cells were superfused continuously at 0.5 ml/min from one of several parallel superfusion pipettes. Microfluorimetry The ratio of UV light emitted at 510 nm following excitation at 340 nm to that emitted following excitation at 380 nm was measured using a Cairn spectrophotometer (excitation was at 96 Hz; data were averaged online and collected at 4 Hz.). Intracellular Ca2+ activity ([Ca2+] ) was calculated from this ratio using the i Grynkiewicz equation and custom-written software. Solutions The bathing solution contained (in m) 140 NaCl, 4.7 KCl, 1.13 MgCl , 1.2 CaCl , 10 glucose, buffered to 2 2 pH 7.2 with 10 m HEPES. Solutions containing more than one agent, for example acetylcholine (ACh) and conditioned medium, were prepared approximately 15 min before the start of the experiment. Molecular weight determination Conditioned medium was first filtered through 0.2 m Acrodisks and then centrifuged through Microsept centrifugal concentrators for 1 h at 3200g and at 10°C. Conditioned medium The conditioned medium was prepared using methods based on those of Fidler et al. (1976) [23]. In brief, adult male CD1 mice were killed by cervical dislocation. The spleen was removed, broken apart using needles and filtered through sterile gauze. The extracted lymphocytes were then suspended to an average density of 4–6 × 106/ml in DMEM/F12 medium and cultured in the presence of 2.5 mg/ml concavalin A (Type IV, Sigma, Poole, UK ) for 72 h at 37°C with 5% carbon dioxide. Lymphocytes were removed from the conditioned medium by centrifugation. Conditioned medium prepared by these methods has been shown to contain IL-2 (#3 ng/ml ), IL-3 (#35 ng/ml ), IL-4 (#10 ng/ml ), IL-6 (#50 pg/ml ) and IFN-c (#50 U/ml ) [26 ].
Results In order to test the effects of chronic exposure of acinar cells to cytokines, the acinar cells must be maintained in primary culture. Although previous studies have reported on the morphology of acinar cells maintained
1228
L. J. Dawson et al.
in culture [29], there are few previous data on any changes in stimulus–secretion coupling in cultured acinar cells. We have extensive baseline data for agonistevoked changes in [Ca2+] measured in acutely isolated i acinar cells [27, 30, 31]. Figure 1A shows an example of these data. ACh was applied over three orders of magnitude (50–5000 n) and the data indicate the time course and magnitude of the change in [Ca2+] evoked i at each agonist concentration. Figure 1B shows the steady-state increase in [Ca2+] plotted against agonist i dose. These data represent the mean of 30 experiments. An identical protocol was applied to cells that had been maintained in culture for 24, 48 and 72 h. These data (Fig. 2) indicate that primary culture for up to 72 h has itself no measurable impact on the ACh-evoked change in [Ca2+] over three orders of magnitude of ACh i concentration. The data in Fig. 3 were the result of a series of experiments identical to those shown in Fig. 2 using cells which had been exposed chronically to the cytokinecontaining conditioned medium. Conditioned medium was not present during the experiment itself. As may be seen, there was no significant difference in the response to ACh at 50, 500 or 5000 n following 24, 48 or 72 h incubation in the cytokine-containing conditioned medium. F. 2. Time course of the change in [Ca2+] evoked by i 50–5000 n ACh in mouse submandibular acinar cells maintained in primary culture for (A) 24 h, (B) 48 h and (C ) 72 h.
F. 1. (A) Time course of the change in [Ca2+] evoked by i 50–5000 n ACh measured in acutely isolated mouse submandibular acinar cells. (B) Change in [Ca2+] plotted against i ACh concentration measured in acutely isolated mouse submandibular acinar cells. Each point is the average of 30 experiments. The bars indicate the standard error of the mean.
The data in Fig. 4 show the average increase in [Ca2+] i (plateau–baseline) for up to 30 experiments following stimulation with 500 n ACh in acutely isolated cells and cells that had been maintained in primary culture both in the presence and absence of conditioned medium. We found that neither primary culture itself nor primary culture in the presence of conditioned medium caused any significant alteration in the average increase in [Ca2+] evoked by 500 n ACh. The same i was true of the averaged response to 50 and 5000 n ACh (data not shown). The data shown in Figs 1–4 were obtained using an extracellular medium containing 1 m Ca2+, therefore these data show a Ca2+ signal resulting from both Ca2+ release from intracellular stores and Ca2+ influx across the plasma membrane. These two components of the Ca2+ signal may be separated by performing experiments in the absence of extracellular Ca2+. Such data are shown in Fig. 5, which shows a transient increase in [Ca2+] evoked by ACh in the absence of extracellular i Ca2+ and regeneration of the Ca2+ signal when Ca2+ was readmitted to the extracellular bathing solution in the continued presence of ACh. The data in Fig. 5 were taken from cells maintained in culture for 24 h in the absence ( Fig. 5A) and presence (Fig. 5B) of conditioned medium. Comparison of Fig. 5A and B shows no effect of conditioned medium on either the initial transient response, which depends largely on Ca2+ release from
Stimulus–secretion coupling and Sjo¨ gren’s syndrome
1229
F. 5. Time course of the change in [Ca2+] evoked by 500 n i ACh in the absence of extracellular Ca2+ ([Ca2+] 0 m) in o mouse submandibular acinar cells maintained in primary culture for 24 h in (A) the absence and (B) the presence of conditioned medium. F. 3. Time course of the change in [Ca2+] evoked by i 50–5000 n ACh in mouse submandibular acinar cells maintained in primary culture in conditioned medium for (A) 24 h, (B) 48 h and (C ) 72 h.
F. 4. Change in [Ca2+] evoked by 500 n ACh in acutely i isolated mouse submandibular cells compared with cells maintained in primary culture for 24, 48 and 72 h in the presence and absence of conditioned medium. The number of observations is shown above the error bar, which indicates the standard error of the mean.
intracellular stores, or on the regeneration of the Ca2+ signal subsequent to Ca2+ readmission, which depends on Ca2+ influx [27]. The data in Fig. 5 also provide an example of the oscillatory pattern of response to ACh seen in 10–15% of experiments. These oscillations are similar to those reported previously in mouse submandibular cells [27, 31] and typically show no immediate
requirement for extracellular Ca2+. Oscillatory changes in [Ca2+] were observed in acutely isolated cells and in i cells maintained in primary culture both in the presence and absence of cytokines. Acetylcholine-evoked Ca2+ mobilization depends on an inositol 1,4,5-trisphosphate [Ins(1,4,5)P ]-mediated 3 Ca2+ release process. Ca2+ mobilization from intracellular stores and Ca2+ influx may be accomplished in a largely Ins(1,4,5,)P -independent fashion using the ses3 quiterpene lactone thapsigargin [32]. Thapsigargin acts by inhibiting the endoplasmic reticulum ( ER) Ca2+ ATPase, which is required to sequester Ca2+ actively within the ER. When the ER Ca2+ ATPase is inhibited, Ca2+ leaks from the intracellular stores and is extruded from the cells by the plasma membrane Ca2+ ATPase, which is not affected by thapsigargin. We have shown previously that the time-course and magnitude of the thapsigargin response is primarily dependent on the rate of Ca2+ extrusion from the cell [30]. Figure 6 shows that chronic exposure to cytokine-containing conditioned medium did not alter the response to thapsigargin. Taken together, our data show that chronic exposure to cytokine-conditioned medium for up to 72 h did not significantly alter the time course, magnitude or pattern of Ca2+ mobilization in response to subsequent stimulation with ACh or thapsigargin. We also tested the effects of acute exposure to conditioned medium on ACh-evoked Ca2+ signalling. Figure 7A shows that the addition of conditioned medium (25% v/v) abolished the ACh-evoked increase in [Ca2+] . The lack of response to ACh in the presence i
1230
L. J. Dawson et al.
F. 6. Time course of the change in [Ca2+] evoked by 2 m i thapsigargin in mouse submandibular acinar cells maintained in primary culture for 24 h in (A) the absence and (B) the presence of conditioned medium.
lymphocytes. Nevertheless, as shown in Fig. 7B, this conditioned medium was also capable of inhibiting AChevoked Ca2+ mobilization. The data in Fig. 8 show that the active ingredient in the conditioned medium was greater than 30 kDa in mass (Fig. 8A) and that it could be inactivated by heating to 70°C (Fig. 8B). The data in Fig. 8C show that the inhibition caused by conditioned medium could itself be reversed by eserine, a cholinesterase inhibitor, thus identifying the active constituent in conditioned medium as a cholinesterase. Additional confirmation of this is shown in Fig. 8D, which shows that conditioned medium did not reverse [Ca2+] mobilization evoked by carbachol, a cholinesteri ase-resistant muscarinic agonist. Cholinesterases capable of metabolizing ACh are present in serum, activated lymphocytes and erythrocytes [33]. The lymphocytes used to generate the conditioned medium were spun down and washed to remove serum; however, the final preparation did contain erythrocytes. Release of cholinesterase from erythrocytes or lymphocytes could account for the cholinesterase activity of the conditioned medium. Commercially available mouse serum (Sigma) contains 352 IU/l of butyrylcholinesterase (measured by the Department of Clinical Chemistry, Royal Liverpool University Hospital ). Figure 9 shows that serum diluted 1/100 with extracellular medium was sufficient to abolish ACh-evoked Ca2+ mobilization. Serum diluted 1/100 showed the same pattern of heat inactivation and inability to reverse carbachol-evoked changes in [Ca2+] ( Fig. 9) as conditioned medium. i
Discussion
F. 7. Time course of the change in [Ca2+] produced by i 500 n ACh in acutely isolated mouse submandibular acinar cells showing the inhibition caused the addition of conditioned medium derived from (A) CD1 mice (control ) and (B) SCID mice. Where indicated on the trace, perfusion with a solution containing 500 n ACh plus 25% v/v control medium was replaced by perfusion with a solution containing 500 n ACh plus 25% v/v conditioned medium.
of conditioned medium was observed both in acutely isolated cells and in cells that had been maintained in primary culture. Superfusion with a 1/4 dilution of control medium or medium used to culture fibroblasts or submandibular acinar cells was by itself without effect; furthermore, these media did not inhibit the AChdependent increase in [Ca2+] (data not shown). We i were able to determine that the inhibitory action of conditioned medium was not mediated by lymphocytederived cytokines by preparing conditioned medium from the spleen cells of SCID mice, which lack functional
Salivary gland secretion is controlled by sympathetic and parasympathetic nerves. Release of acetylcholine by parasympathetic nerves and binding of the neurotransmitter to muscarinic receptors on the acinar cells triggers a 2nd messenger cascade that leads ultimately to increased [Ca2+] , Cl− channel activation and electroi lyte and fluid secretion. During disease states, such as Sjo¨ gren’s syndrome, this orderly process is interrupted, as evidenced by a lack of saliva. Sjo¨ gren’s syndrome is typified, in its early stages, by salivary gland inflammation and focal lymphocytic infiltration. It is clear that the immune response is associated with a loss of salivary gland function [34], but the mechanism by which this occurs is unclear [35]. One aspect of the early stages of the disease aetiology that is clear is the release of chemokines and cytokines by epithelial cells [35] and the infiltration of lymphocytes [3] into the interstices of the gland. Cytokines function as intercellular messengers, and we tested the hypothesis that they might carry a signal from the infiltrating lymphocytes to the acinar cells that down-regulate some aspect of the process of Ca2+ mobilization and thus give rise to the inhibition of secretion observed in Sjo¨ gren’s syndrome. We examined several aspects of Ca2+ mobilization that might be altered by chronic exposure to cytokines: Ca2+ release from intracellular stores; Ca2+ influx; Ca2+ extrusion; and the complex interaction of Ca2+ release
Stimulus–secretion coupling and Sjo¨ gren’s syndrome
1231
solution containing 500 n ACh plus 25% v/v conditioned medium plus 1 m eserine. (D) Time course of the change in [Ca2+] produced by 2000 n carbachol in acutely isolated i mouse submandibular acinar cells, showing no inhibition by acute application of conditioned medium. Where indicated on the trace, perfusion with a solution containing 2000 n carbachol plus 25% v/v control medium was replaced by perfusion with a solution containing 2000 n carbachol plus 25% v/v conditioned medium.
F. 8. (A) Time course of the change in [Ca2+] produced by i 500 n ACh in acutely isolated mouse submandibular acinar cells, showing that the inhibitory agent in conditioned medium was removed by centrifugal filtration of molecules smaller than 30 kDa. Where indicated on the trace, perfusion with a solution containing 500 n ACh plus 25% v/v control medium was replaced first by perfusion with a solution containing 500 n ACh plus 25% v/v conditioned medium, then by a solution containing 500 n ACh plus 25% v/v conditioned medium filtered to leave only molecules larger than 30 kDa and then by a solution containing 500 n ACh plus 25% v/v conditioned medium filtered to leave only molecules smaller than 30 kDa. (B) Time course of the change in [Ca2+] i produced by 500 n ACh in acutely isolated mouse submandibular acinar cells, showing that the inhibitory agent in conditioned medium was removed by heating to 70°C for 20 min. Where indicated on the trace, perfusion with a solution containing 500 n ACh plus 25% v/v control medium was replaced first by perfusion with a solution containing 500 n ACh plus 25% v/v conditioned medium, then by a solution containing 500 n ACh plus 25% v/v conditioned medium heated to 70°C for 20 min. (C ) Time course of the change in [Ca2+] produced by 500 n ACh in acutely isolated mouse i submandibular acinar cells, showing inhibition caused by acute application of conditioned medium and reversal of this inhibition by 1 m eserine. Where indicated on the trace, perfusion with a solution containing 500 n ACh plus 25% v/v control medium was replaced by perfusion with a solution containing 500 n ACh plus 25% v/v conditioned medium and then by a
and reuptake, which can give rise to oscillations in [Ca2+] [36, 37]. We found that exposure to conditioned i medium for up to 72 h had no measurable effect on any aspect of Ca2+ mobilization. We can conclude, therefore, that exposure to the range of cytokines found in conditioned medium is unlikely to cause inhibition of fluid secretion in these cells. We were able to show an acute antisecretory action of conditioned medium and to determine that this activity was due to the presence of cholinesterase rather than cytokines in the conditioned medium. When the effects of cholinesterase were removed either by stimulating with carbachol, a cholinesterase-resistant muscarinic agonist, or by using the anticholinesterase agent eserine ( Fig. 8C ), conditioned medium did not inhibit Ca2+ mobilization. Therefore, these data also show, albeit indirectly, that acute application of cytokines also had no antisecretory effect. These data do not support our original hypotheses of cytokine-mediated down-regulation of the acinar cell fluid and electrolyte secretory mechanism. On the contrary, they show that the second messenger pathway remains intact following both acute and chronic exposure to cytokines. However, our data suggest a mechanism by which stimulus–secretion coupling might be interrupted at the first messenger level. Lymphocytes infiltrate the salivary gland as part of an inflammatory
F. 9. Time course of the change in [Ca2+] produced by first i 2000 n carbachol and then 500 n ACh in acutely isolated mouse submandibular acinar cells, showing inhibition of the ACh response but not the carbachol response by acute application of mouse serum. Where indicated on the trace, perfusion with a solution containing 500 n ACh or 2000 n carbachol was replaced by perfusion with a solution containing 500 n ACh or 2000 n carbachol plus 1% v/v mouse serum.
1232
L. J. Dawson et al.
response. Cholinesterase, which is present at high concentration in the serum, could pass into the interstices of the salivary gland as part of the inflammatory exudate. Alternatively, cholinesterase is expressed on the surface membrane of activated lymphocytes [38, 39] and could be released into the interstices of the gland under inflammatory conditions [40]. Morphological data indicate that the coupling between nerves and acinar cells is not a tight synapse-like arrangement but rather a more open arrangement in which the neurones form a loose network over the cells [41, 42]. Parts of the neurone are unmyelinated, and acetylcholine released from these regions must diffuse to the acinar cell membrane in order to bind to the muscarinic receptors and trigger Ca2+ mobilization and fluid and electrolyte secretion. Cholinesterase is present in the neurones that innervate the acinar cells and must normally be present in the interstices of the gland in order to break down acetylcholine and terminate the secretory response. We hypothesize that, during inflammation, the cholinesterase concentration within the salivary gland becomes elevated to the point at which acetylcholine released by the parasympathetic neurones is metabolized before it can bind to the muscarinic receptors of the acinar cells. This hypothesis can account for the reduction in salivary flow rate that has been associated with inflammation and lymphocytic infiltration in the early stages of Sjo¨ gren’s syndrome. Furthermore, this hypothesis is consistent with the response of some patients to treatment with pilocarpine [43], another cholinesteraseresistant muscarinic agonist. It is well documented in animal models that a reduction in salivary gland function leads to atrophy of the glands [16–19]. In Sjo¨ gren’s syndrome, prolonged disuse of the gland caused by elevated cholinesterase levels could be the trigger for the widespread atrophy seen as the condition progresses. Whilst these observations and our hypothesis may provide insight into the process that underlies the onset of Sjo¨ gren’s syndrome, there are clearly many other aspects of the immune response, and opportunities for interaction between the immune system and stimulus– secretion coupling, involved as the disease progresses. For example, the antibodies against SS-A and SS-B antigens, which can form the basis of a positive diagnosis for Sjo¨ gren’s syndrome using both the European [2] and the San Diego [1] criteria, and antibodies against muscarinic M receptors, which have also been associ3 ated with Sjo¨ gren’s syndrome [44, 45], may also play a role in either the inhibition of fluid secretion or the process of atrophy (or both) at later stages of the condition. We have demonstrated that it is possible to investigate stimulus–secretion coupling interactions in the immune system using a combination of in vitro techniques. Our intention is to turn these techniques towards events occurring later in disease progression.
Acknowledgements We thank John Stanbury for technical assistance and members of The University of Liverpool Sjo¨ gren’s
Syndrome Research Group, in particular E. A. Field and R. J. Moots for constructive discussions of this work. We would like to acknowledge the support of the Oral and Dental Trust in this work.
References 1. Fox RI, Robinson CA, Curd JG, Kozin F, Howell FV. Sjo¨ gren’s syndrome. Proposed criteria for classification. Arthritis Rheum 1986;29:577–85. 2. Vitali C, Moutsopoulos HM, Bombardieri S. The European Community Study Group on diagnostic criteria for Sjo¨ gren’s syndrome. Sensitivity and specificity of tests for ocular and oral involvement in Sjo¨ gren’s syndrome. Ann Rheum Dis 1994;53:637–47. 3. Venables PJW. Sjo¨ gren’s syndrome. In: Lachmann PJ, Peters K, Rosen FS, Walport MJ, eds. Clinical aspects of immunology. Boston, MA, USA: Blackwell, 1993: 1121–31. 4. Sutcliffe N, Inanc M, Speight P, Isenberg D. Predictors of lymphoma development in primary Sjo¨ gren’s syndrome. Semin Arthritis Rheum 1998;28:80–7. 5. Fox RI. What causes Sjo¨ gren’s syndrome? In: Carsons S, Harris EK, eds. The new Sjo¨ gren’s syndrome handbook. Oxford: Oxford University Press, 1998:11–23. 6. Hazes JM, Silman AJ. Review of UK data on the rheumatic diseases—2. Rheumatoid arthritis. Br J Rheumatol 1990;29:310–2. 7. Price EJ, Venables PJ. The etiopathogenesis of Sjo¨ gren’s syndrome. Semin Arthritis Rheum 1995;25:117–33. 8. Elliott MJ, Maini RN, Feldmann M, Long-Fox A, Charles P, Bijl H, et al. Repeated therapy with monoclonal antibody to tumour necrosis factor alpha (CA2) in patients with rheumatoid arthritis. Lancet 1994;344:1125–7. 9. Fox RI, Saito I. Sjo¨ gren’s syndrome: immunologic and neuroendocrine mechanisms. Adv Exp Med Biol 1994; 350:609–21. 10. Fox RI, Maruyama T. Pathogenesis and treatment of Sjo¨ gren’s syndrome. Curr Opin Rheumatol 1997;9:393–9. 11. Scott J. Qualitative and quantitative observations on the histology of human labial salivary glands obtained post mortem. J Biol Buccale 1980;8:187–200. 12. Heft MW, Baum BJ. Unstimulated and stimulated parotid salivary flow rate in individuals of different ages. J Dent Res 1984;63:1182–5. 13. Ship JA, Baum BJ. Is reduced salivary flow normal in old people? Lancet 1990;15;336:1507. 14. Sreebny LM. Xerostomia: diagnosis, management and clinical complications. In: Edgar WM, O’Mullane DM, eds. Saliva & oral health. London: British Dental Association, 1996:43–6. 15. Humphreys-Beher M, Brayer J, Yamachika S, Peck AB, Jonsson R. An alternative perspective to the immune response in autoimmune exocrinopathy: induction of functional quiescence rather than destructive autoaggression. Scand J Immunol 1999;49:7–10. 16. Scott J, Gunn DL. A comparative quantitative histological investigation of atrophic changes in the major salivary glands of liquid fed rats. Arch Oral Biol 1991;36:855–7. 17. Liu P, Scott J, Smith PM. Intracellular calcium signalling in rat parotid cells that lack secretory vesicles. Biochem J 1998;330:847–52. 18. Scott J, Liu P, Smith PM. Morphological and functional characteristics of acinar atrophy and recovery in the ductligated parotid gland of rat. J Dent Res 1999;78:1711–9.
Stimulus–secretion coupling and Sjo¨ gren’s syndrome
19. Scott J, Berry MR, Gunn DL, Woods K. The effects of a liquid diet on initial and sustained, stimulated parotid salivary secretion and on parotid structure in the rat. Arch Oral Biol 1990;35:509–14. 20. Petersen OH. Stimulus–secretion coupling: cytoplasmic calcium signals and the control of ion channels in exocrine acinar cells. J Physiol (Lond ) 1992;448:1–51. 21. Smith PM. Mechanisms of secretion by salivary glands. In: Edgar WM, O’Mullane DM, eds. Saliva & oral health. London: British Dental Association, 1996:9–26. 22. Shiozawa S, Tanaka Y, Shiozawa K. Single-blinded controlled trial of low-dose oral IFN-alpha for the treatment of xerostomia in patients with Sjo¨ gren’s syndrome. J Interferon Cytokine Res 1998;18:255–62. 23. Fidler IJ, Darnell JH, Budment MB. Tumoricidal properties of mouse macrophages activated with mediators from rat lymphocytes stimulated with concanavalin A. Cancer Res 1976;36:3608–15. 24. Abe E, Ishimi Y, Jin CH, Hong MH, Sato T, Suda T. Granulocyte-macrophage colony-stimulating factor is a major macrophage fusion factor present in conditioned medium of concanavalin A-stimulated spleen cell cultures. J Immunol 1991;147:1810–5. 25. Byrne JA, Butler JL, Cooper MD. Differential activation requirements for virgin and memory T cells. J Immunol 1988;15:141:3249–57. 26. Barrat F, Lesourd B, Boulouis HJ, Thibault D, VincentNaulleau S, Gjata B et al. Sex and parity modulate cytokine production during murine ageing. Clin Exp Immunol 1997;109:562–8. 27. Smith PM, Gallacher DV. Acetylcholine and caffeine evoke repetitive transient Ca2+-activated K+ and Cl− currents in mouse submandibular cells. J Physiol (Lond ) 1992;449:109–20. 28. Hann LE, Kelleher RS, Sullivan DA. Influence of culture conditions on the androgen control of secretory component production by acinar cells from the rat lacrimal gland. Invest Ophthalmol Vis Sci 1991;32:2610–1. 29. Burford-Mason AP, Dardick I, MacKay A. Collagen gel cultures of normal salivary gland: conditions for continued proliferation and maintenance of major cell phenotypes in vitro. Laryngoscope 1994;104:335–40. 30. Smith PM, Reed HE. Amplification of the thapsigarginevoked increase in the cytosolic free Ca2+ concentration by acetylcholine in acutely isolated mouse submandibular acinar cells. Biochem J 1996;317:779–83. 31. Smith PM, Sasaki T, Gallacher DV. Electrolyte and fluid secretion in exocrine acinar cells. In: Takishima T, Shimura S, eds. Lung biology in health and disease. Airway secretion: Physiological basis for the control of
32.
33. 34.
35.
36. 37.
38. 39. 40. 41.
42. 43.
44.
45.
1233
mucous hypersecretion. New York: Marcel Dekker, 1994:1–36. Kwan CY, Takemura H, Obie JF, Thastrup O, Putney JW Jr. Effects of MeCh, thapsigargin, and La3 + on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells. Am J Physiol 1990;258:C1006–15. Alles GA, Hawes RC. Cholinesterases in the blood of man. J Biol Chem 1940;133:375–87. Ajjan RA, McIntosh RS, Waterman EA, Watson PF, Franklin CD, Yeoman CM et al. Analysis of the T-cell receptor Valpha repertoire and cytokine gene expression in Sjo¨ gren’s syndrome. Br J Rheumatol 1998;37:179–85. Cuello C, Palladinetti P, Tedla N, Di Girolamo N, Lloyd AR, McCluskey PJ et al. Chemokine expression and lymphocyte infiltration in Sjo¨ gren’s syndrome. Br J Rheumatol 1998;37:779–83. Berridge MJ. Elementary and global aspects of Ca2+ signalling. J Physiol (Lond ) 1997;499:291–306. Thorn P, Lawrie AM, Smith PM, Gallacher DV, Petersen OH. Ca2+ oscillations in pancreatic acinar cells. Spatiotemporal relationships and functional implications. Cell Calcium 1993;14:746–57. Szele´ nyi J, Pa´ ldi-Haris P, Holla´ n S. Changes in the cholinergic system of lymphocytes due to mitogenic stimulation. Immunol Lett 1987;16:49–54. Szele´ nyi JG, Bartha E, Holla´ n SR. Acetylcholinesterase activity of lymphocytes: an enzyme characteristic of T-cells. Br J Haematol 1982;50:241–5. Moss DW. Release of membrane-bound enzymes from cells and the generation of isoforms. Clin Chim Acta 1994;226:131–42. Garrett JR. The innervation of normal human submandibular and parotid salivary glands. Demonstrated by cholinesterase histochemistry, catecholamine fluorescence and electron microscopy. Arch Oral Biol 1967;12:1417–36. Young JA, van Lennep EW. The morphology of salivary glands. London: Academic Press, 1978. Vivino FB, Al-Hashimi I, Khan Z, LeVeque FG, Salisbury PL 3rd, Tran-Johnson TK et al. Pilocarpine tablets for the treatment of dry mouth and dry eye symptoms in patients with Sjo¨ gren syndrome: a randomized, placebocontrolled, fixed-dose, multicenter trial. Arch Intern Med 1999;159:174–81. Bacman S, Sterin-Borda L, Camusso JJ, Arana R, Hubscher O, Borda E. Circulating antibodies against rat parotid gland M3 muscarinic receptors in primary Sjo¨ gren’s syndrome. Clin Exp Immunol 1996;104:454–9. Bacman S, Perez Leiros C, Sterin-Borda L, Hubscher O, Arana R, Borda E. Autoantibodies against lacrimal gland M3 muscarinic acetylcholine receptors in patients with primary Sjo¨ gren’s syndrome. Invest Ophthalmol Vis Sci 1998;39:151–6.
