Hydroxychloroquine therapy in patients with primary Sjögren's syndrome may improve salivary gland hypofunction by inhibition of glandular cholinesterase
L. J. Dawson1,
V. L. Caulfield1,
J. B. Stanbury1,
A. E. Field1,
S. E. Christmas2 and
P. M. Smith1
1 Clinical Dental Sciences and 2 Immunology, University of Liverpool, Liverpool, UK.
Correspondence to: L. J. Dawson. E-mail: ldawson{at}liv.ac.uk
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Abstract
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Objective. To determine whether (i) cholinesterase activity is increased in the saliva of patients with primary Sjögren's syndrome (pSS), (ii) increased levels of cholinesterase of lymphocyte origin could interfere with the secretory activity of submandibular acinar cells, and (iii) hydroxychloroquine at therapeutic doses could interfere with cholinesterase activity.
Methods. The Ellman method was used to determine the levels of salivary cholinesterase activity and the Ki of both chloroquine and hydroxychloroquine for serum cholinesterase. The ability of lymphocyte cholinesterase to inhibit the acetylcholine (ACh)-evoked rise in [Ca2+]i in mouse submandibular acinar cells was determined using fura-2 microfluorimetry.
Results. Patients with pSS had significantly higher levels of cholinesterase activity in both their unstimulated (P<0.05) and stimulated saliva (P<0.0001) compared with control subjects. Lymphocyte cholinesterase was capable of inhibiting the ACh-evoked rise in [Ca2+]i. The in vitro Ki for hydroxychloroquine inhibition of cholinesterase was 0.38 ± 1.4 µM.
Conclusion. These data suggest that increased levels of cholinesterase present in the salivary glands of patients with pSS may contribute to glandular hypofunction and provide evidence that the therapeutic enhancement of salivary secretion in patients with pSS by hydroxychloroquine may be mediated by inhibition of glandular cholinesterase activity, although further in vivo investigation is needed.
KEY WORDS: Sjögren's syndrome, Cholinesterase, Salivary acinar cells, Calcium, Hydroxychloroquine
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Introduction
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Primary Sjögren's syndrome (pSS) is an organ-specific autoimmune exocrinopathy associated with lymphocytic invasion of the salivary glands combined with severe salivary gland hypofunction (SGH) [1]. The clinical consequences of the SGH have a severe impact on the quality of life of the sufferer [24]. However, currently there are no reliable treatments.
The mechanisms responsible for fluid secretion by salivary acinar cells are well understood. The initiating event is the binding of the parasympathetic neurotransmitter acetylcholine (ACh) to cell surface muscarinic type-3 receptors (M3R) [5, 6]. Once released, ACh must diffuse for approximately 100 nm in order to reach the acinar cell M3R [7]. This diffusion path, from the terminal axon to the acinar cell, makes ACh vulnerable to degradation by cholinesterases. Previously, we have speculated that increased levels of cholinesterase within the salivary glands may exacerbate the SGH seen in patients with pSS [8]. The source of the increased glandular cholinesterase levels could be serum, as a result of inflammatory exudate [9] in response to glandular infection and/or from the infiltrating T lymphocytes, which dramatically increase expression of acetylcholinesterase (AChE) on their surface following activation [10, 11]. Any increase in the amount of cholinesterase activity within the salivary gland could be mirrored by an increase in the saliva.
Hydroxychloroquine is a recognized therapy for patients with pSS, and data from the majority of studies have indicated that hydroxychloroquine therapy results in an improvement in immunological markers of disease, including IgG [1216], ESR [1216], IL-6 [15], ANA [14] and RF [14, 17]. However, despite an improvement in immunological disease markers, only two out of the five studies [1216] in which glandular function has been assessed have reported a complementary improvement in salivary flow rate [12, 14]. Currently, the mechanism of action of hydroxychloroquine leading to an improvement in the immune profiles of patients with pSS is unknown, although interference with the mechanisms involved in antigen processing has been suggested [18]. However, it is well established that other antimalarials, such as chloroquine, are potent inhibitors of cholinesterase activity [19, 20]. Therefore, inhibition of cholinesterase activity in the salivary glands is a potential mechanism to account for the improvement of SGH seen in patients with pSS taking hydroxychloroquine.
The aims of this study were to determine: (i) whether the levels of cholinesterase are increased in the saliva of patients with pSS, (ii) whether cholinesterase expressed on the surface of activated T lymphocytes can interfere with ACh-evoked [Ca2+]i in salivary acinar cells, and (iii) whether therapeutic doses of hydroxychloroquine could reduce cholinesterase activity sufficiently to increase salivary acinar cell function.
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Materials and methods
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Saliva collection
Following local ethical approval (LM-99-5) and informed consent, stimulated saliva was collected from nine patients (seven female and two male; mean age 60 yr) with pSS (who fulfilled all current criteria [2123]) and nine healthy controls (seven male and two female; mean age 40 yr). Subjects chewed paraffin wax over a 5-min interval and expectorated all of the saliva entering their mouth into a preweighed vessel under standardized conditions. All collections were made at the same time of day and subjects had refrained from smoking, eating drinking or tooth-brushing for at least 3 h prior to collection. All subjects underwent a periodontal examination [24] to ensure there was no periodontal disease, which has been shown to increase salivary cholinesterase activity [25]. Salivary samples were centrifuged at 2430 g and 4°C for 15 min to remove debris and stored at 20°C until assayed. Cholinesterase is known to be stable for extended periods of time under these conditions [26, 27].
Salivary cholinesterase determination
The levels of cholinesterase within salivary samples were determined spectrophotometrically using a modified form of the Ellman method [28]. Briefly, each cuvette contained 1.5 ml of phosphate buffer (0.1 M, pH 8), 20 µl of substrate (0.075 M acetylthiocholine iodide), 50 µl of reagent [DTNB: 39.6 mg of 5,5-dithiobis-2-nitrobenzoic acid dissolved in 10 ml pH 7.0 phosphate buffer (0.1 M)] and 50 µl of test saliva or control enzyme. The control solution comprised 1.5 ml + 50 µl of buffer, substrate and DTNB solution. All reactions were performed at 24 ± 2°C and cholinesterase activity was determined kinetically by monitoring the rate of formation of a coloured product at 412 nM. Control butyrylcholinesterase (BChE) was purchased from Sigma (C1057). One enzyme unit is defined as the amount of enzyme that hydrolysed 1 µmol of butrylthiocholine iodide in 1 min.
Lymphocyte cholinesterase determination
The method was essentially the same as above with the following changes. The cuvette contained 1.5 ml activated T lymphocytes (1 x 106 cells/ml) in phosphate buffer (0.1 M, pH 8), 20 µl of substrate (0.075 M acetylthiocholine iodide) and 50 µl of reagent (DTNB as defined above).
Mouse acinar cell collection
Adult male CD1 mice were killed by cervical dislocation and submandibular acinar cells were isolated by collagenase (Worthington Diagnostic, USA) digestion in extracellular media containing 1 mM Ca2+ as described previously [29].
Human acinar cell collection
Following relevant ethical committee approval (EC 38.02) and informed consent, small samples of human submandibular glands were obtained from patients undergoing salivary gland removal for other reasons. Subjects were screened so as to avoid collecting material from patients who had previously undergone therapeutic irradiation to the head and neck or were taking xerogenic drugs.
Mouse and human acinar cell preparation
Following dispersal, cells were suspended in serum-free 50:50 Dulbecco's MEM:F12 medium containing antibiotics and antimycotics (Life Technologies, UK), and placed onto circular glass coverslips (22 mm diameter) coated with a thin (
1 mm) layer of a basement membrane matrix (Matrigel; Becton Dickinson, UK) as described previously [8]. Each coverslip was placed into one well of a six-well plate and covered with serum-free 50:50 Dulbecco's MEM:F12 medium containing antibiotics and antimycotics (Life Technologies) and cultured for up to 48 h at 37°C with 5% CO2. Cells were removed from culture immediately before an experiment and loaded with Fura-2 by incubation for 20 min in the presence of 2 µM of cell-permeable fura-2 acetoxymethylester (Fura-2 AM; Molecular Probes).
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 1000x magnification on single cells, either completely isolated or part of a small clump of cells (two to eight cells). Cells were superfused continuously at 0.5 ml/min.
Lymphocyte collection
Following ethical committee approval (LM 98-11) and informed consent, 10 ml of whole blood was collected from healthy volunteers into 30-ml sterile universal flasks containing 30 U/ml preservative-free heparin (Monoparin; CP Pharmaceuticals, Wrexham, UK). Lymphocytes were harvested using standard protocols [30]. Briefly, under sterile conditions 10 ml of the heparinized whole blood was carefully layered onto an equal volume of 1.077 g/l FicollPaque Plus (Amersham, catalogue no. 17-1440-02). The universal flask was sealed and then centrifuged at 990 g for 20 min at room temperature. The monocyte layer was then carefully harvested and resuspended in phosphate-buffered saline (PBS) and centrifuged at 990 g for 10 min for each of three wash cycles.
Preparation of polyclonal T-cell line
Peripheral blood lymphocytes were isolated as described above and suspended in complete RPMI 1640 media (Gibco/BRL) with 10% heat-inactivated fetal calf serum (Life Technologies, catalogue no. 10099/133) at a concentration of 1 x 106 cells/ml. Lymphocytes were activated by the addition of 1 µg/ml phytohaemagglutinin (PHA; Sigma, catalogue no. L9132) and cultured for 72 h at 37°C and 5% CO2. Activated lymphocytes were then collected by centrifugation and washed twice in PBS. Cells were resuspended in serum-free X-Vivo-10 medium (Biowhittaker, catalogue no. 04-380Q). IL-2-dependent T-cell clones were stimulated by the addition of 100 U/ml human recombinant IL-2 (Sigma catalogue no. T3267). The cells were then cultured for 72 h at 37°C and 5% CO2.
Flow cytometry
To determine the percentage of T cells present in the culture, cells were labelled with fluorescein isothiocyanate conjugated anti-CD21 (B-cells), anti-CD56 (NK cells) and anti-CD3 (T-cells) (Dako, Copenhagen) by incubation with the respective antibodies diluted 1:100 for 30 min at 4°C. The percentage make-up of the cell culture was then determined using a flow cytometer (EPICS XLS; Coulter). In all cases the method produced a 99% pure culture of T lymphocytes (data not shown).
Microfluorimetry
The ratio of 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). Where applicable, intracellular Ca2+ activity ([Ca2+]i) was calculated from this ratio using the Grynkiewicz equation and custom-written software.
Cell bathing solutions for microfluorimetry
The bathing solution contained (in mM) 140 NaCl, 4.7 KCl, 1.13 MgCl2, 1.2 CaCl2, 10 glucose buffered to pH 7.2 with 10 mM HEPES. Solutions containing lymphocytes and ACh were prepared 60 min before the start of the experiment.
Cholinesterase inhibition by hydroxychloroquine
Using the Ellman method [28] modified for a microplate reader (MRX TCII; Dynex), the effect of increasing concentrations of hydroxychloroquine on the activity serum cholinesterase (BChE) was determined spectrophotometrically at 412 nM. Each run (n = 10) was performed in triplicate at 37°C. Each test well on the 96-well microplate contained a final volume of 300 µl, comprising (final concentrations) 60 µl phosphate buffer (0.1 M, pH 8.0), 2 µl substrate (0.075 M acetylthiocholine iodide), 10 µl of reagent (DTNB defined as above) and 30 µl containing 10 U BChE [Sigma (C1057)], ± 30 µl 107, 106, 105 or 104 or 103 M hydroxychloroquine (Sanofi-Synthelabo, Newcastle upon Tyne, UK).
Statistics
Where appropriate, data were compared using Student's paired t test.
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Results
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Salivary cholinesterase
The cholinesterase activity in unstimulated whole saliva from controls (n = 9) and patients with pSS (n = 4) was (mean ± S.E.) 9.3 ± 1.2 and 24.8 ± 7.1 U/l respectively. For stimulated whole saliva from controls (n = 9) and subjects with pSS (n = 9), the cholinesterase activity was 10.8 ± 2.1 and 34.3 ± 6.3 U/l respectively. The reduced subject number in the unstimulated pSS group was the result of 5/9 subjects producing insufficient saliva to analyse. Within both groups there was no correlation of salivary cholinesterase activity with subject age or gender (data not shown). When comparing the cholinesterase activity in the unstimulated saliva with that of the stimulated saliva there was no significant difference within the groups. However, when comparing the control group with the pSS group, the cholinesterase activity in both the unstimulated and the stimulated saliva was significantly higher (P < 0.0001 and P<0.05 respectively) (Fig. 1).

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FIG. 1. Cholinesterase activity in pSS patients and control subjects in stimulated (S) (n = 4 and n = 9 respectively) and unstimulated (U) (n = 9 and n = 9 respectively) whole saliva. In both the S and U samples the pSS patients had a significantly higher cholinesterase activity (P < 0.0001 and P<0.05 respectively).
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The average paraffin wax-stimulated whole salivary flow rate for the control subjects (n = 9) and the patients with pSS (n = 9) was (mean ± S.E.) 2.3 ± 0.3 and 0.6 ± 0.6 ml/min respectively. The data displayed in Fig. 2 show that there was no correlation between the whole salivary cholinesterase activity with the stimulated salivary flow rate (r2 = 0.16 and r2 = 0.015 for pSS and controls respectively). Furthermore, these data demonstrate that when comparing the levels of cholinesterase activity at the same stimulated salivary flow rate, pSS subjects had significantly higher levels of activity compared with control subjects.

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FIG. 2. Cholinesterase activity as a function of whole salivary flow rate from patients with pSS (n = 9) and controls (n = 9). The data indicate that there is no relationship of cholinesterase activity with flow rate (r2 = 0.16 and 0.015 for pSS and controls respectively).
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Lymphocyte cholinesterase
The AChE activity of our IL-2-dependent T-lymphocyte clones was 1.0 mU/106 cells (data not shown). Therefore, under our experimental conditions, we calculate that the AChE present on the surface of 1 x 106 T lymphocytes would reduce the amount of ACh in a solution containing 500 nM of ACh by approximately 60 nM over a 60-min contact period. Mouse submandibular acinar cells were used as a bioassay to detect the presence of ACh in a solution. The data shown in Fig. 3 are representative of four similar experiments and show a typical mouse submandibular acinar cell response to ACh. Upon addition of agonist there is a rapid rise in the [Ca2+]i which is maintained for the duration of agonist exposure. In the trace shown the Ca2+ response has an oscillatory pattern, which is a common phenomenon in salivary acinar cells [29, 31, 32]. Changing the bathing solution from 500 nM ACh to one containing 500 nM ACh that had previously been exposed to activated T lymphocytes ranging in concentration from 107 to 106 cells/ml resulted in a 56 ± 6 nM fall in the ACh-evoked [Ca2+]i response and a loss of the oscillatory response. However, within the experimental time frame, lower T-lymphocyte concentrations (105 and 104 cells/ml) had no effect on the ACh-evoked [Ca2+]i acinar cell response. In preliminary experiments (data not shown) where the bathing solution was changed from 500 nM ACh to one containing 500 nM ACh that had previously been exposed to 107 activated T lymphocytes/ml and 1 mM eserine (cholinesterase inhibitor), there was no change in the ACh-evoked [Ca2+]i response.

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FIG. 3. Time course of the change in [Ca2+]i in mouse submandibular acinar cells produced by changing the bathing solution from 500 nM ACh to one containing a solution of 500 nM ACh that had previously been incubated with activated human peripheral blood lymphocytes ranging in concentration from 107 to 104 cells/ml for 1 h at room temperature. 1 mM [Ca2+]e was present throughout.
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Hydroxychloroquine and butyrylcholinesterase activity
The data in Fig. 4 show that the in vitro Ki for the inhibitory activity of chloroquine and hydroxychloroquine on BChE was (mean ± S.E.) 0.42 ± 0.9 and 0.38 ± 1.4 µM respectively. Our experimentally determined Ki for chloroquine is comparable to that in earlier reports [33].

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FIG. 4. Effects of the concentration of chloroquine (A) and hydroxychloroquine (B) on the activity of 10 U of butyrylcholinesterase. The Ki values for chloroquine and hydroxychloroquine are 0.42 and 0.38 µM respectively. The data are from three experiments, run in triplicate, best fitted (r2 = 0.99 and r2 = 0.98 for chloroquine and hydroxychloroquine respectively) to a curve corresponding to one-site competition, using Prism software.
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Discussion
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The defining clinical feature of pSS is severe SGH [1]. Data from recent studies have suggested that the cause of the SGH in pSS is likely to be multifactorial; mechanisms that have been implicated include acinar cell muscarinic receptor blockade by antibodies [34, 35], impairment of water movement across acinar cells due to changes in the number of aquaporins [36, 37], reduction in the salivary acinar cell sensitivity to neurotransmitter [31], and cytokine induced down-regulation of neurotransmitter release [38].
Our data showing significantly elevated levels of cholinesterase activity in the saliva of patients with pSS provide strong supporting evidence for the involvement of cholinesterase in the exacerbation of SGH, irrespective of the cause (Fig. 1). The levels of cholinesterase activity found in both the stimulated and unstimulated whole saliva collections from control subjects were comparable to those in previous reports [9, 25, 27]. However, the levels of cholinesterase detected in the whole saliva from the patients with pSS were much higher than previously reported (34.3 ± 6.3 U/l compared with 17.95 ± 2.66 U/l) [9], even when compared with patients with severe periodontal disease, a condition known to increase cholinesterase levels [9]. In agreement with previous reports, we found that the increased cholinesterase activity was not a simple function of reduced salivary flow (Fig. 2) [27, 39]. Increased glandular permeability to albumin has been reported previously in SS [40]. Such an increase in permeability would be expected to result in increased salivary cholinesterase activity. However, under these circumstances cholinesterase would enter the saliva by simple diffusion and therefore activity should be correlated with salivary flow rate. In this and previous studies [27, 39] salivary cholinesterase activity could not be correlated with flow rate. Therefore, currently the mechanism of cholinesterase entry into saliva remains uncertain.
Our data show that, at sufficiently high concentrations, activated T lymphocytes have sufficient AChE activity on their surface to have a potential role in cholinesterase-mediated salivary acinar hypofunction. However, the lymphocytic infiltrate is characteristically perivascular and unless the AChE can separate from the lymphocyte membrane its effect on SGH would be very limited. However, the AChE on the lymphocyte surface is attached via a glycophosphatidylinositol anchor (GPI-linked) [41]. In other cell types the GPI-linked enzymes can be released into the surrounding media by the appropriate triggers [4244]. The same ability has been reported for some lymphocyte GPI-linked enzymes following lymphocyte activation [43]. We did not detect free cholinesterase activity in the media from our activated lymphocyte cultures (data not shown). However, this mechanism leading to the exacerbation of SGH remains highly attractive as it would be site-directed. Further work is needed to determine the relative contributions of AChE present in the total salivary cholinesterase activity we have measured in order to give an indication of the origin of the salivary cholinesterase in pSS.
Hydroxychloroquine is a recognized therapy for patients with pSS. Our data demonstrate that, along with chloroquine [19, 20], hydroxychloroquine is capable of inhibiting cholinesterase activity, the mean in vitro experimentally determined Ki being 0.42 µM for chloroquine and 0.38 µM for hydroxychloroquine (Fig. 4). Hydroxychloroquine is known to have marked pharmacokinetic variability [45]. The levels of hydroxychloroquine determined in whole blood following a single dose range from 1.1 to 2700 µg/l (3 nM to 6 µM) with a mean of 264 µg/l (0.6 µM) [45]. In the present study the mean Ki of hydroxychloroquine for serum cholinesterase was 0.4 µM, which suggests that in many patients the blood levels of hydroxychloroquine should be sufficient to partially inhibit cholinesterase activity, which in turn could lead to an improvement in salivary flow. It is tempting to speculate that the high degree of variability in the steady-state pharmacokinetics of hydroxychloroquine may explain why inconsistency in clinical improvement is found in patients with pSS taking hydroxychloroquine, as it is possible that only those patients who have both a high glandular cholinesterase activity and achieve high blood concentrations of hydroxychloroquine show improvement in salivary flow [12, 14]. Further work is needed to explore this possibility and we intend to determine the in vivo effect of hydroxychloroquine on salivary cholinesterase following 6 months of therapy.
In conclusion, we have provided evidence for a role for cholinesterase in the exacerbation of SGH seen in pSS. If prolonged, increased levels of cholinesterase within the salivary gland parenchyma could also accelerate acinar atrophy, as it is known that cholinesterases have protease activity [46, 47]. In addition, we have speculated on a new mechanism for the action of hydroxychloroquine leading to an improvement in salivary flow rate. Taken together, these findings indicate that further work is needed to re-evaluate the use of hydroxychloroquine in the treatment of pSS and that increased levels of salivary cholinesterase activity could prove to be a useful marker to indicate those patients who would show an improvement in their salivary flow rates if placed on hydroxychloroquine.
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Acknowledgments
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This work was supported by grants from the Royal Liverpool and Broadgreen NHS trust and the British Sjögren's Syndrome Association.
The authors have declared no conflicts of interest.
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Submitted 17 September 2004;
revised version accepted 2 November 2004.