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The effect of pilocarpine on salivary constituents in patients with chronic graft-versus-host disease
Archives of Oral Biology 46 (2001) 689– 695 www.elsevier.com/locate/archoralbio
The effect of pilocarpine on salivary constituents in patients with chronic graft-versus-host disease Rafael M. Nagler 1*, Arnon Nagler Bone Marrow Transplantation Department and Cancer Biology Research Laboratory, Hadassah Uni6ersity Hospital, Jerusalem, Israel Accepted 27 February 2001
Abstract Chronic graft-versus-host disease (cGVHD) is a complex clinical entity with various target organs, including the salivary glands. Oral pilocarpine (Salagen®), 30 mg/day, can ameliorate cGVHD-induced xerostomia and improve the flow rate from the major salivary glands. The purpose here was to evaluate the effect of this drug at 30 mg/day on salivary biochemical and immunological composition in cGVHD patients. Significantly higher concentrations of salivary sodium (Na), magnesium (Mg), total protein, albumin, epidermal growth factor (EGF) and total IgG, accompanied by a concomitant increase in total IgA which did not reach significance, were observed in cGVHD patients in comparison with controls, in both resting and stimulated conditions (p B 0.05), while salivary potassium, calcium and phosphate were not altered. Two weeks of oral pilocarpine, at 30 mg/day, resulted in normalization of the altered salivary biochemical and immunological composition in the cGVHD patients. Oral pilocarpine was able to reduce and normalise the elevated Na, Mg, total protein, albumin, EGF, IgG and IgA concentrations in both resting and stimulated conditions. The ability of oral pilocarpine to normalise and reverse the salivary biochemical and immunological alterations induced by cGVHD parallels its known stimulatory effect on salivary flow rates. As the biochemical and immunological composition of saliva provides its protective antimicrobial characteristics, the ability of pilocarpine to abrogate cGVHD salivary gland abnormalities may be of clinical significance. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Saliva; Chronic graft-versus-host disease; Pilocarpine; EGF; Electrolytes; Immunoglobulins
1. Introduction Abbre6iations: EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay. * Corresponding author. Present address: Department of Oral and Maxillofacial Surgery, Rambam Medical Center, POB 9602, Haifa 31096, Israel. Tel: + 972-4-6442003; fax: + 972-4-6541295. E-mail address: [email protected] (R.M. Nagler). 1 Dr R.M. Nagler is currently with the Department of Oral and Maxillofacial Surgery, Rambam Medical Center and Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. In addition, he is Director of their Oral Biochemistry Laboratory.
Chronic graft-versus-host disease is a complex clinical entity that may occur after bone marrow transplantation (Sullivan et al., 1981; Slavin and Nagler, 1991; Peszkowski et al., 1996). The disease is mediated by autoreactive T-lymphocytes that infiltrate various target organs and tissues, including the oral mucosa and salivary glands (Nagler, Laufer and Nagler, 1996; Nagler et al., 1996; Nagler et al., 1998; Nagler et al., 1999). In recent years, we and others have demonstrated, both in animal models and in humans, that this Tlymphocyte infiltration in major salivary glands is ac-
0003-9969/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0003-9969(01)00035-8
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companied by concomitant loss of function as expressed by a reduction in salivary flow rate (xerostomia) in both resting and stimulated conditions (Nagler, Nagler, Laufer and Nagler, 1996; Nagler et al., 1998, 1999). Furthermore, this reduction, which causes significant morbidity in patients with chronic graft-versus-host disease, can be ameliorated by routine administration of oral pilocarpine (Salagen®) (Singhal et al., 1995, 1997; Nagler and Nagler, 1999). However, an increase in salivary flow rate may be insufficient in itself and should be accompanied by a restoration of normal salivary composition. The association between the normal composition of saliva and its ability to fulfil various roles, such as defence against microbial and viral infections and immunosurveillance, protection of oral hard and soft tissues and the alimentary mucosa, and chemosensorial, speech and nutritional functions, is well known (Fox et al., 1985; Mandel, 1989). These functions are of great importance to patients with chronic graft-versushost disease, who are heavily immunosuppressed and prone to fulminant infections (Sullivan et al., 1981). The salivary composition of these patients has been little evaluated to date. Our purpose here was to measure the salivary biochemical and immunological composition of patients with chronic graft-versus-host disease before and after the administration of pilocarpine, at 30 mg/day. We also assessed the ability of pilocarpine to ameliorate or normalise the biochemical and immunological alterations found in the saliva of these patients.
and underwent non-T-cell-depleted allogeneic peripheral blood stem-cell transplantation. The mean time from the stem-cell transplantation to the onset of graft-versus-host disease was 5.7 9 1.3 months. Eight healthy, age-matched individuals (five males, three females) served as controls. As 2 weeks of continuous administration of pilocarpine, at 30 mg/day, resulted in a major increase of salivary flow rates, as we and others have previously shown (Singhal et al., 1995, 1997; Nagler and Nagler, 1999), we chose this timepoint for analysing the maximal effect of the drug on salivary composition in this study.
2.2. Sali6ary analysis The salivary gland study included sialochemical analysis at rest and in stimulated conditions at 3 days before the start of oral pilocarpine (Salagen®; Boehringer Ingleheim Pharmaceuticals), 30 mg/day, and at 2 weeks after the beginning of this administration. Saliva was collected as described by Nagler et al. (1996) in a quiet room during the morning, between 09:00 and 12:00, at least 1 h after eating. For the resting collection, the patients were asked to collect saliva in their mouths and to spit into a wide testtube for 10 min. For stimulated saliva, their tongues were swabbed every 30 s with 2% citric acid and the saliva was collected for 5 min. The resting and stimulated salivary flow rates were estimated by measuring the volume of saliva collected during 10 and 5 min, respectively. Whole resting and stimulated saliva samples were collected by the spitting method, centrifuged and kept at 4°C until analysed.
2. Materials and methods
2.3. Sialochemical analysis 2.1. Patients and study design Six consenting patients (four males, two females), median age 22 years (range 18 –39 years), each with chronic graft-versus-host disease diagnosed according to the Seattle criteria (Sullivan et al., 1981), were entered into the study. Four had limited, moderate, and two had extensive, severe chronic graft-versus-host disease. Organ involvement included salivary glands, mucosa, skin and eyes in all six patients, and liver in one. The underlying diagnosis was acute myelogenous leukaemia in three patients, acute lymphoblastic leukaemia in one, chronic myelogenous leukaemia in one and non-Hodgkin lymphoma in one. Three patients were in relapse from allogeneic peripheral blood stem-cell transplantation, while the other three were in remission (acute myelogenous leukaemia, two patients) or chronic (chronic myelogenous leukaemia) phases. All patients were conditioned with total body irradiation, 1200 cGy (200 cGy twice daily for 3 days), and cyclophosphamide, 60 mg/kg for 2 days,
The concentrations of the following salivary variables were analysed: sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), phosphate (P), total protein, albumin, IgA and IgG, and EGF. The concentrations of Na and K were measured by flame photometry; P was measured spectophotometrically, and Ca and Mg were measured by atomic absorption as previously described (Lowry et al., 1951; Baum et al., 1984; Ben-Aryeh et al., 1993; Ben-Aryeh et al., 1996). Total protein was measured by the method of Lowry et al. (1951), and EGF by Quantikine EGF solid-phase ELISA (enzyme-linked immunosorbent assay) (RandD Systems, Minneapolis MN, USA) (Nexo et al., 1992). Total IgA, IgG and albumin were measured by the radial immunodiffusion method of Mancini et al. (1965), using an Oxford viewer for measuring the diameters of the precipitation rings; the square diameter of the ring formed is related quantitatively to the concentration of the salivary variable analysed in this method.
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2.4. Statistical analysis The salivary data were collected and calculated, and means and SEM were computed. Results obtained before and after immunotherapy were analysed with the t-test for paired differences (level of significance p5 0.05).
3. Results
3.1. Sali6ary electrolytes The salivary concentrations of Na, K, Ca, Mg and P in the resting condition in the patients with untreated chronic graft-versus-host disease were 39.4 9 11.1, 28.6 94.7, 1.3 90.12, 0.65 90.25 and 8.2 92.0 mmol, respectively (n=6) (Table 1). The salivary concentrations of Na and Mg were higher by 454% (p B0.01) and 113% (pB0.05), respectively, in comparison to controls, while the concentrations of K, Ca and P were similar to the controls (Table 2). Following 2 weeks of oral pilocarpine, the Na and Mg concentrations in the patients with chronic graft-versus-host disease were significantly reduced, by 54% (pB0.05) and 50% (pB 0.05), respectively, in comparison to pretreatment values, approximating normal values; there was a minimal effect on K, Ca and P concentrations, which were 19.8 91.7, 1.12 90.1 and 6.96 90.66 mmol, respectively (n=6) (Table 1). In stimulated conditions, the salivary concentrations of Na, K, Ca, Mg and P in the patients with untreated chronic graft-versus-host disease were 33.2 96.7, 25.2 95.2, 1.05 90.1, 0.45 90.1 and 5.13 91.06 mmol, respectively (n=6) (Table 1). The salivary concentra-
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tions of Na and Mg were higher by 395% (pB 0.01) and 109% (pB 0.05), in comparison with controls, while the concentrations of K, Ca and P were similar to the controls (Table 2). Following 2 weeks of oral pilocarpine, the Mg concentration in the patients with chronic graft-versus-host disease was significantly reduced, by 66% (pB 0.05), and there was a trend towards a reduction in Na as well, from 33.2 9 6.7 to 25.69 8.4 mmol, in comparison to pretreatment values, approximating normal values. There were no significant changes in K, Ca and P concentrations, which were 18.89 2.7, 1.25 9 0.2 and 4.8 9 0.63 mmol, respectively (n =6) (Table 1).
3.2. Sali6ary EGF Pretreatment concentrations of EGF were 3.7 9 0.6 and 4.7 9 0.79 ng/ml in the patients with chronic graftversus-host disease in resting and stimulated conditions, respectively, and 3.6 9 0.4 and 2.8 90.3 ng/ml, respectively, in the controls. Following the administration of oral pilocarpine, the resting and stimulated salivary concentrations of EGF were reduced significantly, by 52% (pB 0.01) and 64% (pB 0.05), respectively, in comparison to pretreatment values (Fig. 1A).
3.3. Sali6ary total protein and albumin The total protein represents all the various components with a protein structure in saliva; albumin is serum-borne, not produced by the salivary glands, and often, when found in whole saliva, is due to pathological leakage through damaged oral and perioral barriers. In resting conditions, the salivary total protein and albumin concentrations of the patients with untreated
Table 1 Salivary electrolyte concentrations before and after administration of pilocarpine hydrochloride Electrolyte (mmole)
Pretreatment
Post-treatment
Mean
SEM
Range
Mean
SEM
Range
Resting condition Sodium Potassium Calcium Magnesium Phosphate
39.4 28.6 1.3 0.65 8.2
11.1 4.7 0.12 0.25 2.0
13.5–72.0 18.0–46.0 0.97–1.55 0.15–1.25 1.0–16.0
19.6a 19.8 1.12 0.3a 6.96
5.2 1.7 0.1 0.1 0.66
6.3–40.0 14.0–24.0 0.7–1.47 0.05–0.8 5.2–9.26
Stimulated condition Sodium Potassium Calcium Magnesium Phosphate
33.2 25.1 1.05 0.45 5.13
6.7 5.2 0.1 0.1 1.06
9.0–54.0 5.2–39.0 0.57–1.27 0.1–1.0 1.03–8.33
25.6 18.8 1.2 0.15a 4.8
8.4 2.7 0.2 0.05 0.63
7.3–65.0 9.1–27.0 0.45–1.62 0.05–0.85 3.0–6.5
a
pB0.05, n = 6
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Table 2 Salivary composition profile in healthy individuals (n = 8) Electrolyte (mmol)
Sodium Potassium Calcium Magnesium Phosphate
Resting
Stimulated
Mean
SEM
Range
Mean
SEM
Range
7.1 24.2 1.0 0.3 7.2
3.2 5.8 0.17 0.065 1.13
2–15 14–38 0.5–2.5 0.1–0.5 2.66–10.0
6.7 23.4 0.9 0.21 6.4
3.0 1.9 0.2 0.07 0.66
3–25 12–29 0.5–2.0 0.05–0.45 3.66–9.33
chronic graft-versus-host disease were 340 9 65 mg/dl and 2.0 ×10 − 3 90.23×10 − 3 mmol, respectively, while in stimulated conditions these concentrations were 211 919 mg/dl and 1.61 ×10 − 3 90.19×10 − 3 mmol, respectively (n=6) (Fig. 1B,C). The salivary concentrations of total protein and albumin in stimulated conditions were higher by 58% (pB 0.05) and 115% (p B0.05), respectively, in comparison with controls. Following pilocarpine therapy, the salivary concentrations of total protein and albumin in both resting and stimulated conditions were reduced significantly, by 34%–40% (pB0.05) and 60% –70% (p B0.05), respectively, in comparison with pretreatment values, reaching normal values (n=6) (Fig. 1B,C).
3.4. Sali6ary immunoglobulins Salivary concentrations of total IgA and IgG in the patients with chronic graft-versus-host disease in resting and stimulated conditions were 9.93 ×10 − 4 92.4× 10 − 4 and 13.8 ×10 − 4 92.46 x 10 − 4 mmol and 4.46 × 10 − 4 90.53×10 − 4 mmol and 6.2 ×10 − 4 90.93× 10 − 4 mmol, respectively. IgA concentrations were higher than, but not statistically different from, controls: 5.26 × 10 − 4 92.53×10 − 4 mmol and 2.73 × 10 − 4 91.06×10 − 4 mmol in resting and stimulated conditions, respectively. IgG concentrations, however, were 182% –190% (pB0.05) higher than controls in resting and stimulated conditions, respectively. Following pilocarpine therapy, there was normalization of the IgA and IgG in both resting and stimulated conditions, in comparison with pretreatment values (n= 6) (Fig. 1D, E).
4. Discussion The oral mucosa and salivary glands are severely affected in chronic graft-versus-host disease (Nagler et al., 1996). Evaluating the biochemical and immunological changes in the saliva of these patients may enhance our understanding of the pathological changes in the glands and of the effect they might have on the oral
mucosa, whose integrity is partly dependent on normal salivary composition. Our data show that the biochemical and immunological composition of saliva is altered in patients with chronic graft-versus-host disease. There were significantly higher concentrations in the patients than controls, in both resting and stimulated conditions, of Na by 395% –454% (pB 0.01); of Mg by 109%–113% (p B0.05); and of EGF, total protein, albumin and IgG by up to 190% (p B0.05). These altered concentrations may compromise quantitatively the functional capacity of the saliva, as this is related directly to the normal composition (Fox et al., 1985; Mandel, 1989). In this event, the saliva may not be able to play its pivotal part in nutritional function, nor in preserving oral homeostasis, maintaining mucosal integrity and protection against foreign proteins and infections, controlling the formation of periodontal calculus and sustaining its anticariogenic function (Fox et al., 1985; Mandel, 1989). Furthermore, the altered salivary composition may act synergistically with the compromised salivary flow rate in these patients, which is reduced by 55% –90% in humans and in animal models (Nagler, Laufer and Nagler, 1996; Nagler et al., 1996, 1998). The profound increase in Na concentration is in agreement with two studies that demonstrated an increase in Na concentrations in the secretions of minor salivary glands and the whole saliva of patients with chronic graft-versus-host disease (Izutsu, Sullivan et al., 1983; Izutsu, Schubert et al., 1983). The observed threeto four-fold increase in Na concentration could be accounted for by disease-induced and lymphocyte infiltration-mediated damage to the Na-reabsorbing salivary ductal cells (Izutsu, Sullivan et al., 1983; Izutsu, Schubert et al., 1983). A similar increase in salivary Na is found in patients with Sjo¨ gren’s syndrome, the cause of which autoimmune disease is similar and is based on the role played by infiltrating autoreactive lymphocytes (Chisholm and Mason, 1968; Mandel and Baurmash, 1976). Concentrations of the other three electrolytes analysed, K, Ca and P, were similar in patients and controls. Izutsu, Sullivan et al. (1983) report that the
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Fig. 1. Mean resting and stimulated salivary concentrations of EGF (A), total protein (B), albumin (c), IgA (D) and IgG (E) of the six patients with chronic graft-versus-host disease analysed before (white bars) and after (crossed bars) pilocarpine hydrochloride, 30 mg/day. *pB 0.05; **pB 0.0.
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resting salivary K concentration in patients with chronic graft-versus-host disease is not different from controls. Salivary IgG and EGF are of major importance in defence against infection and maintaining oral mucosal integrity (Fisher, 1988; Deville de Periere and Arancibia, 1988-89; Marti et al., 1989; Gresik, 1994; Farriol and Schwartz, 1995). The significant increase in salivary IgG in patients with chronic graft-versus-host disease is in accord with findings by Izutsu, Sullivan et al. (1983). The significant elevation of EGF, total protein, albumin and IgG, which may result from direct graft-versus-host disease-induced damage to the salivary parenchyma, as previously suggested, could also result from transudation of serum components across the damaged and inflamed oral epithelium and gingiva, as these anatomical sites are known to be affected by the disease (Nagler et al., 1996). Such serum components are concentrated in the saliva, unrelated to their concentrations in the serum. Thus, a multisite mechanism of damage could be responsible, in a differentiated manner, for the observed increase in various components that are secreted from salivary glands or leak from serum into saliva. In any event, a mere ‘concentrating effect’ of a reduced secreted volume due to a decrease of the watery component of the saliva (related to a specific insult to the muscarinic signal-transduction pathway, for example) is excluded, as this might explain an identical increase in the EGF, proteins and immunoglobulins, but not a differentiated one, as observed. A differentiated and multisite mechanism of damage is further supported by the fact that salivary albumin, IgA and IgG are known to be serum-borne (Fox et al., 1985; Mandel, 1989), while most of the salivary EGF is secreted by the submandibular gland (Fisher, 1988; Deville de Periere and Arancibia, 1988-89; Marti et al., 1989; Gresik, 1994). The salivary EGF, more so in resting conditions, is of special importance immunologically and for oral protection. Resting salivary secretion is the dominant condition during most of the day and night and the dominant secreting gland is the submandibular (Mandel, 1989; Nagler and Nagler, 1999). The salivary EGF is a 53-residue/6370-polypeptide hormone (Marti et al., 1989; Farriol and Schwartz, 1995), which has a major role in regulating the mucosal/inflammatory response, preventing epithelial ulceration and promoting wound healing in both the mouth and the digestive tract (Skov Olsen, 1988; Marti et al., 1989; Valdez and Fox, 1991; Farriol and Schwartz, 1995; Sabbadini and Berczi, 1995; Wu-Wang et al., 1995; Yang et al., 1996; Epstein et al., 1997). The chronic graft-versus-host disease-induced, 55% – 90% reduction in salivary flow rate (Nagler, Laufer and Nagler, 1996; Nagler et al., 1996; Nagler et al., 1998) leads to a relatively reduced total excreted salivary EGF, espe-
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cially in resting conditions, in which we found concentrations of EGF similar to controls in the patients with chronic graft-versus-host disease. Furthermore, the reduced flow rate of secreted saliva containing significantly higher concentrations of total protein and albumin results in a highly concentrated and viscous secretion. The EGF contained in such viscous saliva is less available for contacting and affecting the widespread injured mucosa of patients with chronic graftversus-host disease. This may contribute to the severely compromised mucosa of these patients and may act in concert with the previously reported reduced concentration of secretory IgA, the major immunological ‘player’ in the mouth (Izutsu, Sullivan et al., 1983). Regardless of the mechanism responsible for the profound salivary effect of graft-versus-host disease, 2 weeks of continuous administration of pilocarpine (Salagen®), at 30 mg/day, resulted in a major increase of salivary flow rates, as we and others have shown (Singhal et al., 1995, 1997; Nagler and Nagler, 1999). Indeed, we found not only that the effect of the pilocarpine on flow rate was maximal at 2 weeks, returning it to normal (Nagler and Nagler, 1999), but also that all the induced alterations in salivary composition were reversed. These results show that oral pilocarpine not only caused an increase in salivary flow, but also normalised the chronic graft-versus-host disease-induced salivary biochemical and immunological alterations. This reversal of the induced quantitative and qualitative alterations restored the saliva’s normal biochemical, immunological and antimicrobial protective characteristics, which are of vast importance for patients with chronic graft-versus-host disease, in view of the heavy immunosuppression and susceptibility to episodes of overwhelming infection to which they are prone.
Acknowledgements Sincere gratitude is extended to Megapharm Ltd, Hod Hasharon, Israel, which supported the work in part by a research grant. We also thank Mrs S. Gan for statistical analysis and Mrs M. Perlmutter and Ms E. Maor for assistance in the preparation of this paper.
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The effect of pilocarpine on salivary constituents in patients with chronic graft-versus-host disease Rafael M. Nagler 1*, Arnon Nagler Bone Marrow Transplantation Department and Cancer Biology Research Laboratory, Hadassah Uni6ersity Hospital, Jerusalem, Israel Accepted 27 February 2001
Abstract Chronic graft-versus-host disease (cGVHD) is a complex clinical entity with various target organs, including the salivary glands. Oral pilocarpine (Salagen®), 30 mg/day, can ameliorate cGVHD-induced xerostomia and improve the flow rate from the major salivary glands. The purpose here was to evaluate the effect of this drug at 30 mg/day on salivary biochemical and immunological composition in cGVHD patients. Significantly higher concentrations of salivary sodium (Na), magnesium (Mg), total protein, albumin, epidermal growth factor (EGF) and total IgG, accompanied by a concomitant increase in total IgA which did not reach significance, were observed in cGVHD patients in comparison with controls, in both resting and stimulated conditions (p B 0.05), while salivary potassium, calcium and phosphate were not altered. Two weeks of oral pilocarpine, at 30 mg/day, resulted in normalization of the altered salivary biochemical and immunological composition in the cGVHD patients. Oral pilocarpine was able to reduce and normalise the elevated Na, Mg, total protein, albumin, EGF, IgG and IgA concentrations in both resting and stimulated conditions. The ability of oral pilocarpine to normalise and reverse the salivary biochemical and immunological alterations induced by cGVHD parallels its known stimulatory effect on salivary flow rates. As the biochemical and immunological composition of saliva provides its protective antimicrobial characteristics, the ability of pilocarpine to abrogate cGVHD salivary gland abnormalities may be of clinical significance. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Saliva; Chronic graft-versus-host disease; Pilocarpine; EGF; Electrolytes; Immunoglobulins
1. Introduction Abbre6iations: EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay. * Corresponding author. Present address: Department of Oral and Maxillofacial Surgery, Rambam Medical Center, POB 9602, Haifa 31096, Israel. Tel: + 972-4-6442003; fax: + 972-4-6541295. E-mail address: [email protected] (R.M. Nagler). 1 Dr R.M. Nagler is currently with the Department of Oral and Maxillofacial Surgery, Rambam Medical Center and Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. In addition, he is Director of their Oral Biochemistry Laboratory.
Chronic graft-versus-host disease is a complex clinical entity that may occur after bone marrow transplantation (Sullivan et al., 1981; Slavin and Nagler, 1991; Peszkowski et al., 1996). The disease is mediated by autoreactive T-lymphocytes that infiltrate various target organs and tissues, including the oral mucosa and salivary glands (Nagler, Laufer and Nagler, 1996; Nagler et al., 1996; Nagler et al., 1998; Nagler et al., 1999). In recent years, we and others have demonstrated, both in animal models and in humans, that this Tlymphocyte infiltration in major salivary glands is ac-
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companied by concomitant loss of function as expressed by a reduction in salivary flow rate (xerostomia) in both resting and stimulated conditions (Nagler, Nagler, Laufer and Nagler, 1996; Nagler et al., 1998, 1999). Furthermore, this reduction, which causes significant morbidity in patients with chronic graft-versus-host disease, can be ameliorated by routine administration of oral pilocarpine (Salagen®) (Singhal et al., 1995, 1997; Nagler and Nagler, 1999). However, an increase in salivary flow rate may be insufficient in itself and should be accompanied by a restoration of normal salivary composition. The association between the normal composition of saliva and its ability to fulfil various roles, such as defence against microbial and viral infections and immunosurveillance, protection of oral hard and soft tissues and the alimentary mucosa, and chemosensorial, speech and nutritional functions, is well known (Fox et al., 1985; Mandel, 1989). These functions are of great importance to patients with chronic graft-versushost disease, who are heavily immunosuppressed and prone to fulminant infections (Sullivan et al., 1981). The salivary composition of these patients has been little evaluated to date. Our purpose here was to measure the salivary biochemical and immunological composition of patients with chronic graft-versus-host disease before and after the administration of pilocarpine, at 30 mg/day. We also assessed the ability of pilocarpine to ameliorate or normalise the biochemical and immunological alterations found in the saliva of these patients.
and underwent non-T-cell-depleted allogeneic peripheral blood stem-cell transplantation. The mean time from the stem-cell transplantation to the onset of graft-versus-host disease was 5.7 9 1.3 months. Eight healthy, age-matched individuals (five males, three females) served as controls. As 2 weeks of continuous administration of pilocarpine, at 30 mg/day, resulted in a major increase of salivary flow rates, as we and others have previously shown (Singhal et al., 1995, 1997; Nagler and Nagler, 1999), we chose this timepoint for analysing the maximal effect of the drug on salivary composition in this study.
2.2. Sali6ary analysis The salivary gland study included sialochemical analysis at rest and in stimulated conditions at 3 days before the start of oral pilocarpine (Salagen®; Boehringer Ingleheim Pharmaceuticals), 30 mg/day, and at 2 weeks after the beginning of this administration. Saliva was collected as described by Nagler et al. (1996) in a quiet room during the morning, between 09:00 and 12:00, at least 1 h after eating. For the resting collection, the patients were asked to collect saliva in their mouths and to spit into a wide testtube for 10 min. For stimulated saliva, their tongues were swabbed every 30 s with 2% citric acid and the saliva was collected for 5 min. The resting and stimulated salivary flow rates were estimated by measuring the volume of saliva collected during 10 and 5 min, respectively. Whole resting and stimulated saliva samples were collected by the spitting method, centrifuged and kept at 4°C until analysed.
2. Materials and methods
2.3. Sialochemical analysis 2.1. Patients and study design Six consenting patients (four males, two females), median age 22 years (range 18 –39 years), each with chronic graft-versus-host disease diagnosed according to the Seattle criteria (Sullivan et al., 1981), were entered into the study. Four had limited, moderate, and two had extensive, severe chronic graft-versus-host disease. Organ involvement included salivary glands, mucosa, skin and eyes in all six patients, and liver in one. The underlying diagnosis was acute myelogenous leukaemia in three patients, acute lymphoblastic leukaemia in one, chronic myelogenous leukaemia in one and non-Hodgkin lymphoma in one. Three patients were in relapse from allogeneic peripheral blood stem-cell transplantation, while the other three were in remission (acute myelogenous leukaemia, two patients) or chronic (chronic myelogenous leukaemia) phases. All patients were conditioned with total body irradiation, 1200 cGy (200 cGy twice daily for 3 days), and cyclophosphamide, 60 mg/kg for 2 days,
The concentrations of the following salivary variables were analysed: sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), phosphate (P), total protein, albumin, IgA and IgG, and EGF. The concentrations of Na and K were measured by flame photometry; P was measured spectophotometrically, and Ca and Mg were measured by atomic absorption as previously described (Lowry et al., 1951; Baum et al., 1984; Ben-Aryeh et al., 1993; Ben-Aryeh et al., 1996). Total protein was measured by the method of Lowry et al. (1951), and EGF by Quantikine EGF solid-phase ELISA (enzyme-linked immunosorbent assay) (RandD Systems, Minneapolis MN, USA) (Nexo et al., 1992). Total IgA, IgG and albumin were measured by the radial immunodiffusion method of Mancini et al. (1965), using an Oxford viewer for measuring the diameters of the precipitation rings; the square diameter of the ring formed is related quantitatively to the concentration of the salivary variable analysed in this method.
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2.4. Statistical analysis The salivary data were collected and calculated, and means and SEM were computed. Results obtained before and after immunotherapy were analysed with the t-test for paired differences (level of significance p5 0.05).
3. Results
3.1. Sali6ary electrolytes The salivary concentrations of Na, K, Ca, Mg and P in the resting condition in the patients with untreated chronic graft-versus-host disease were 39.4 9 11.1, 28.6 94.7, 1.3 90.12, 0.65 90.25 and 8.2 92.0 mmol, respectively (n=6) (Table 1). The salivary concentrations of Na and Mg were higher by 454% (p B0.01) and 113% (pB0.05), respectively, in comparison to controls, while the concentrations of K, Ca and P were similar to the controls (Table 2). Following 2 weeks of oral pilocarpine, the Na and Mg concentrations in the patients with chronic graft-versus-host disease were significantly reduced, by 54% (pB0.05) and 50% (pB 0.05), respectively, in comparison to pretreatment values, approximating normal values; there was a minimal effect on K, Ca and P concentrations, which were 19.8 91.7, 1.12 90.1 and 6.96 90.66 mmol, respectively (n=6) (Table 1). In stimulated conditions, the salivary concentrations of Na, K, Ca, Mg and P in the patients with untreated chronic graft-versus-host disease were 33.2 96.7, 25.2 95.2, 1.05 90.1, 0.45 90.1 and 5.13 91.06 mmol, respectively (n=6) (Table 1). The salivary concentra-
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tions of Na and Mg were higher by 395% (pB 0.01) and 109% (pB 0.05), in comparison with controls, while the concentrations of K, Ca and P were similar to the controls (Table 2). Following 2 weeks of oral pilocarpine, the Mg concentration in the patients with chronic graft-versus-host disease was significantly reduced, by 66% (pB 0.05), and there was a trend towards a reduction in Na as well, from 33.2 9 6.7 to 25.69 8.4 mmol, in comparison to pretreatment values, approximating normal values. There were no significant changes in K, Ca and P concentrations, which were 18.89 2.7, 1.25 9 0.2 and 4.8 9 0.63 mmol, respectively (n =6) (Table 1).
3.2. Sali6ary EGF Pretreatment concentrations of EGF were 3.7 9 0.6 and 4.7 9 0.79 ng/ml in the patients with chronic graftversus-host disease in resting and stimulated conditions, respectively, and 3.6 9 0.4 and 2.8 90.3 ng/ml, respectively, in the controls. Following the administration of oral pilocarpine, the resting and stimulated salivary concentrations of EGF were reduced significantly, by 52% (pB 0.01) and 64% (pB 0.05), respectively, in comparison to pretreatment values (Fig. 1A).
3.3. Sali6ary total protein and albumin The total protein represents all the various components with a protein structure in saliva; albumin is serum-borne, not produced by the salivary glands, and often, when found in whole saliva, is due to pathological leakage through damaged oral and perioral barriers. In resting conditions, the salivary total protein and albumin concentrations of the patients with untreated
Table 1 Salivary electrolyte concentrations before and after administration of pilocarpine hydrochloride Electrolyte (mmole)
Pretreatment
Post-treatment
Mean
SEM
Range
Mean
SEM
Range
Resting condition Sodium Potassium Calcium Magnesium Phosphate
39.4 28.6 1.3 0.65 8.2
11.1 4.7 0.12 0.25 2.0
13.5–72.0 18.0–46.0 0.97–1.55 0.15–1.25 1.0–16.0
19.6a 19.8 1.12 0.3a 6.96
5.2 1.7 0.1 0.1 0.66
6.3–40.0 14.0–24.0 0.7–1.47 0.05–0.8 5.2–9.26
Stimulated condition Sodium Potassium Calcium Magnesium Phosphate
33.2 25.1 1.05 0.45 5.13
6.7 5.2 0.1 0.1 1.06
9.0–54.0 5.2–39.0 0.57–1.27 0.1–1.0 1.03–8.33
25.6 18.8 1.2 0.15a 4.8
8.4 2.7 0.2 0.05 0.63
7.3–65.0 9.1–27.0 0.45–1.62 0.05–0.85 3.0–6.5
a
pB0.05, n = 6
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Table 2 Salivary composition profile in healthy individuals (n = 8) Electrolyte (mmol)
Sodium Potassium Calcium Magnesium Phosphate
Resting
Stimulated
Mean
SEM
Range
Mean
SEM
Range
7.1 24.2 1.0 0.3 7.2
3.2 5.8 0.17 0.065 1.13
2–15 14–38 0.5–2.5 0.1–0.5 2.66–10.0
6.7 23.4 0.9 0.21 6.4
3.0 1.9 0.2 0.07 0.66
3–25 12–29 0.5–2.0 0.05–0.45 3.66–9.33
chronic graft-versus-host disease were 340 9 65 mg/dl and 2.0 ×10 − 3 90.23×10 − 3 mmol, respectively, while in stimulated conditions these concentrations were 211 919 mg/dl and 1.61 ×10 − 3 90.19×10 − 3 mmol, respectively (n=6) (Fig. 1B,C). The salivary concentrations of total protein and albumin in stimulated conditions were higher by 58% (pB 0.05) and 115% (p B0.05), respectively, in comparison with controls. Following pilocarpine therapy, the salivary concentrations of total protein and albumin in both resting and stimulated conditions were reduced significantly, by 34%–40% (pB0.05) and 60% –70% (p B0.05), respectively, in comparison with pretreatment values, reaching normal values (n=6) (Fig. 1B,C).
3.4. Sali6ary immunoglobulins Salivary concentrations of total IgA and IgG in the patients with chronic graft-versus-host disease in resting and stimulated conditions were 9.93 ×10 − 4 92.4× 10 − 4 and 13.8 ×10 − 4 92.46 x 10 − 4 mmol and 4.46 × 10 − 4 90.53×10 − 4 mmol and 6.2 ×10 − 4 90.93× 10 − 4 mmol, respectively. IgA concentrations were higher than, but not statistically different from, controls: 5.26 × 10 − 4 92.53×10 − 4 mmol and 2.73 × 10 − 4 91.06×10 − 4 mmol in resting and stimulated conditions, respectively. IgG concentrations, however, were 182% –190% (pB0.05) higher than controls in resting and stimulated conditions, respectively. Following pilocarpine therapy, there was normalization of the IgA and IgG in both resting and stimulated conditions, in comparison with pretreatment values (n= 6) (Fig. 1D, E).
4. Discussion The oral mucosa and salivary glands are severely affected in chronic graft-versus-host disease (Nagler et al., 1996). Evaluating the biochemical and immunological changes in the saliva of these patients may enhance our understanding of the pathological changes in the glands and of the effect they might have on the oral
mucosa, whose integrity is partly dependent on normal salivary composition. Our data show that the biochemical and immunological composition of saliva is altered in patients with chronic graft-versus-host disease. There were significantly higher concentrations in the patients than controls, in both resting and stimulated conditions, of Na by 395% –454% (pB 0.01); of Mg by 109%–113% (p B0.05); and of EGF, total protein, albumin and IgG by up to 190% (p B0.05). These altered concentrations may compromise quantitatively the functional capacity of the saliva, as this is related directly to the normal composition (Fox et al., 1985; Mandel, 1989). In this event, the saliva may not be able to play its pivotal part in nutritional function, nor in preserving oral homeostasis, maintaining mucosal integrity and protection against foreign proteins and infections, controlling the formation of periodontal calculus and sustaining its anticariogenic function (Fox et al., 1985; Mandel, 1989). Furthermore, the altered salivary composition may act synergistically with the compromised salivary flow rate in these patients, which is reduced by 55% –90% in humans and in animal models (Nagler, Laufer and Nagler, 1996; Nagler et al., 1996, 1998). The profound increase in Na concentration is in agreement with two studies that demonstrated an increase in Na concentrations in the secretions of minor salivary glands and the whole saliva of patients with chronic graft-versus-host disease (Izutsu, Sullivan et al., 1983; Izutsu, Schubert et al., 1983). The observed threeto four-fold increase in Na concentration could be accounted for by disease-induced and lymphocyte infiltration-mediated damage to the Na-reabsorbing salivary ductal cells (Izutsu, Sullivan et al., 1983; Izutsu, Schubert et al., 1983). A similar increase in salivary Na is found in patients with Sjo¨ gren’s syndrome, the cause of which autoimmune disease is similar and is based on the role played by infiltrating autoreactive lymphocytes (Chisholm and Mason, 1968; Mandel and Baurmash, 1976). Concentrations of the other three electrolytes analysed, K, Ca and P, were similar in patients and controls. Izutsu, Sullivan et al. (1983) report that the
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Fig. 1. Mean resting and stimulated salivary concentrations of EGF (A), total protein (B), albumin (c), IgA (D) and IgG (E) of the six patients with chronic graft-versus-host disease analysed before (white bars) and after (crossed bars) pilocarpine hydrochloride, 30 mg/day. *pB 0.05; **pB 0.0.
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resting salivary K concentration in patients with chronic graft-versus-host disease is not different from controls. Salivary IgG and EGF are of major importance in defence against infection and maintaining oral mucosal integrity (Fisher, 1988; Deville de Periere and Arancibia, 1988-89; Marti et al., 1989; Gresik, 1994; Farriol and Schwartz, 1995). The significant increase in salivary IgG in patients with chronic graft-versus-host disease is in accord with findings by Izutsu, Sullivan et al. (1983). The significant elevation of EGF, total protein, albumin and IgG, which may result from direct graft-versus-host disease-induced damage to the salivary parenchyma, as previously suggested, could also result from transudation of serum components across the damaged and inflamed oral epithelium and gingiva, as these anatomical sites are known to be affected by the disease (Nagler et al., 1996). Such serum components are concentrated in the saliva, unrelated to their concentrations in the serum. Thus, a multisite mechanism of damage could be responsible, in a differentiated manner, for the observed increase in various components that are secreted from salivary glands or leak from serum into saliva. In any event, a mere ‘concentrating effect’ of a reduced secreted volume due to a decrease of the watery component of the saliva (related to a specific insult to the muscarinic signal-transduction pathway, for example) is excluded, as this might explain an identical increase in the EGF, proteins and immunoglobulins, but not a differentiated one, as observed. A differentiated and multisite mechanism of damage is further supported by the fact that salivary albumin, IgA and IgG are known to be serum-borne (Fox et al., 1985; Mandel, 1989), while most of the salivary EGF is secreted by the submandibular gland (Fisher, 1988; Deville de Periere and Arancibia, 1988-89; Marti et al., 1989; Gresik, 1994). The salivary EGF, more so in resting conditions, is of special importance immunologically and for oral protection. Resting salivary secretion is the dominant condition during most of the day and night and the dominant secreting gland is the submandibular (Mandel, 1989; Nagler and Nagler, 1999). The salivary EGF is a 53-residue/6370-polypeptide hormone (Marti et al., 1989; Farriol and Schwartz, 1995), which has a major role in regulating the mucosal/inflammatory response, preventing epithelial ulceration and promoting wound healing in both the mouth and the digestive tract (Skov Olsen, 1988; Marti et al., 1989; Valdez and Fox, 1991; Farriol and Schwartz, 1995; Sabbadini and Berczi, 1995; Wu-Wang et al., 1995; Yang et al., 1996; Epstein et al., 1997). The chronic graft-versus-host disease-induced, 55% – 90% reduction in salivary flow rate (Nagler, Laufer and Nagler, 1996; Nagler et al., 1996; Nagler et al., 1998) leads to a relatively reduced total excreted salivary EGF, espe-
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cially in resting conditions, in which we found concentrations of EGF similar to controls in the patients with chronic graft-versus-host disease. Furthermore, the reduced flow rate of secreted saliva containing significantly higher concentrations of total protein and albumin results in a highly concentrated and viscous secretion. The EGF contained in such viscous saliva is less available for contacting and affecting the widespread injured mucosa of patients with chronic graftversus-host disease. This may contribute to the severely compromised mucosa of these patients and may act in concert with the previously reported reduced concentration of secretory IgA, the major immunological ‘player’ in the mouth (Izutsu, Sullivan et al., 1983). Regardless of the mechanism responsible for the profound salivary effect of graft-versus-host disease, 2 weeks of continuous administration of pilocarpine (Salagen®), at 30 mg/day, resulted in a major increase of salivary flow rates, as we and others have shown (Singhal et al., 1995, 1997; Nagler and Nagler, 1999). Indeed, we found not only that the effect of the pilocarpine on flow rate was maximal at 2 weeks, returning it to normal (Nagler and Nagler, 1999), but also that all the induced alterations in salivary composition were reversed. These results show that oral pilocarpine not only caused an increase in salivary flow, but also normalised the chronic graft-versus-host disease-induced salivary biochemical and immunological alterations. This reversal of the induced quantitative and qualitative alterations restored the saliva’s normal biochemical, immunological and antimicrobial protective characteristics, which are of vast importance for patients with chronic graft-versus-host disease, in view of the heavy immunosuppression and susceptibility to episodes of overwhelming infection to which they are prone.
Acknowledgements Sincere gratitude is extended to Megapharm Ltd, Hod Hasharon, Israel, which supported the work in part by a research grant. We also thank Mrs S. Gan for statistical analysis and Mrs M. Perlmutter and Ms E. Maor for assistance in the preparation of this paper.
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